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New therapeutics that modulate chemokine networks
Abstract
Chemokines are small cytokines that control a wide variety of biological and pathological processes, from immunosurveillance to inflammation, and from viral infection to cancer. The numerous known chemokine receptors have given hope that selective receptor antagonism might be possible, which could allow us to control which cells are recruited and activated at any time and in any place. As chemokine receptors are G-protein-coupled receptors, which are classical targets for the pharmaceutical industry, it is hoped that chemokines could be the first cytokines for which small-molecule receptor antagonists could be developed. Recently, reports of chemokine-receptor antagonists, both in vitro and in animal models of disease, have been published. It is anticipated that this field could produce clinically useful therapies in the next few years.
... Several members of a new class of inhibitors based on blocking human immunodeficiency virus type 1 (HIV-1) entry into target cells are now in, or approaching, human clinical trials (8,52,77,80,85,90,98). These various compounds antagonize different stages in the multistep pathway by which HIV-1 fuses with susceptible cells. ...
... A step in the entry process intermediate between gp120-CD4 attachment and gp41 conformational changes involves a coreceptor for gp120 (21,31,37,85,98). Thus, after gp120 has bound to CD4, it changes conformation to enable it to bind to a coreceptor from the G-protein-coupled receptor superfamily (21,31,37,85,98,107,116). ...
... A step in the entry process intermediate between gp120-CD4 attachment and gp41 conformational changes involves a coreceptor for gp120 (21,31,37,85,98). Thus, after gp120 has bound to CD4, it changes conformation to enable it to bind to a coreceptor from the G-protein-coupled receptor superfamily (21,31,37,85,98,107,116). The most physiologically relevant coreceptors are the chemokine receptors CCR5 or CXCR4, the former used by HIV-1 strains that usually dominate early in infection and the latter used by viruses that sometimes emerge several years later or that are detectable only transiently (21,31,85,99,122). ...
We have described previously the generation of an escape variant of human immunodeficiency virus type 1 (HIV-1), under the selection pressure of AD101, a small molecule inhibitor that binds the CCR5 coreceptor (A. Trkola, S. E. Kuhmann, J. M. Strizki, E. Maxwell, T. Ketas, T. Morgan, P. Pugach, S. X. L. Wojcik, J. Tagat, A. Palani, S. Shapiro, J. W. Clader, S. McCombie, G. R. Reyes, B. M. Baroudy, and J. P. Moore, Proc. Natl. Acad. Sci. USA 99:395-400, 2002). The escape mutant, CC101.19, continued to use CCR5 for entry, but it was at least 20,000-fold more resistant to AD101 than the parental virus, CC1/85. We have now cloned the env genes from the the parental and escape mutant isolates and made chimeric infectious molecular clones that fully recapitulate the phenotypes of the corresponding isolates. Sequence analysis of the evolution of the escape mutants suggested that the most relevant changes were likely to be in the V3 loop of the gp120 glycoprotein. We therefore made a series of mutant viruses and found that full AD101 resistance was conferred by four amino acid changes in V3. Each change individually caused partial resistance when they were introduced into the V3 loop of a CC1/85 clone, but their impact was dependent on the gp120 context in which they were made. We assume that these amino acid changes alter how the HIV-1 Env complex interacts with CCR5. Perhaps unexpectedly, given the complete dependence of the escape mutant on CCR5 for entry, monomeric gp120 proteins expressed from clones of the fully resistant isolate failed to bind to CCR5 on the surface of L1.2-CCR5 cells under conditions where gp120 proteins from the parental virus and a partially AD101-resistant virus bound strongly. Hence, the full impact of the V3 substitutions may only be apparent at the level of the native Env complex.
... [270][271][272][273] Thus, even though targeting the CCL2-CCR2 axis may have context-dependent effects, several CCR2 inhibitors are currently being evaluated for their immunostimulatory activity, mostly in preclinical settings. 274 The majority of these molecules have indeed been tested clinically for the therapy of non-oncological conditions, but their development has come to an impasse. 274 This applies to AZD2423, which has been studied for the treatment of chronic obstructive pulmonary disease and neurotic pain; 275 BMS741672, which has been investigated for safety and efficacy in patients with diabetes or neurotic pain; BMS813160 and CCX140, which have been assessed as therapeutic measures in subjects experiencing diabetic kidney disease; [276][277][278] JNJ17166864, which has been tested as a treatment for allergic rhinitis; MK0812, which has been investigated as a therapy for multiple sclerosis, rheumatoid arthritis and Alzheimer disease; 279 as well as PF04634817, which has been intensively tested in subjects with diabetic kidney disease (source www.clinicaltrials.gov). ...
... 274 The majority of these molecules have indeed been tested clinically for the therapy of non-oncological conditions, but their development has come to an impasse. 274 This applies to AZD2423, which has been studied for the treatment of chronic obstructive pulmonary disease and neurotic pain; 275 BMS741672, which has been investigated for safety and efficacy in patients with diabetes or neurotic pain; BMS813160 and CCX140, which have been assessed as therapeutic measures in subjects experiencing diabetic kidney disease; [276][277][278] JNJ17166864, which has been tested as a treatment for allergic rhinitis; MK0812, which has been investigated as a therapy for multiple sclerosis, rheumatoid arthritis and Alzheimer disease; 279 as well as PF04634817, which has been intensively tested in subjects with diabetic kidney disease (source www.clinicaltrials.gov). Cenicriviroc, a mixed CCR2/CCR5 antagonist, is still being assessed for its activity against HIV-1 infection, primary sclerosing cholangitis, and hepatic disorders, but not as an immunostimulatory agent in cancer patients (source www.clinicaltrials.gov). ...
Progressing malignancies establish robust immunosuppressive networks that operate both systemically and locally. In particular, as tumors escape immunosurveillance, they recruit increasing amounts of myeloid and lymphoid cells that exert pronounced immunosuppressive effects. These cells not only prevent the natural recognition of growing neoplasms by the immune system, but also inhibit anticancer immune responses elicited by chemo-, radio- and immunotherapeutic interventions. Throughout the past decade, multiple strategies have been devised to counteract the accumulation or activation of tumor-infiltrating immunosuppressive cells for therapeutic purposes. Here, we review recent preclinical and clinical advances on the use of small molecules that target the immunological tumor microenvironment for cancer therapy. These agents include inhibitors of indoleamine 2,3-dioxigenase 1 (IDO1), prostaglandin E2, and specific cytokine receptors, as well as modulators of intratumoral purinergic signaling and arginine metabolism.
... The identification of new CCR5-targeting antibodies, chemokines, chemokine analogues, small molecules and peptides, is therefore an important step in the development of new antiviral drugs that target virus entry (Doms, 2000) through mechanisms that differ from those of existing HAART drugs. Several different types of inhibitor for CCR5-mediated HIV-1 entry have now been identified and are in pre-clinical or clinical development as drug candidates (De Clercq, 2002;Horuk, 2003;Kazmierski et al., 2003;LaBranche et al., 2001;Michael & Moore, 1999;Moore & Stevenson, 2000;O'Hara & Olson, 2002;Schwarz & Wells, 2002). In particular we focused our attention on D-Ala-peptide T-amide (DAPTA), or Peptide T, named for its high threonine content (ASTTTNYT). ...
... By interacting with the viral envelope glycoproteins (gp120 or gp41), with CD4 or with co-receptors, these inhibitors block different steps in the complex sequence of events leading to virus-cell fusion (Cocchi et al., 1995;Koenig et al., 1986;Meucci et al., 1998;Michael & Moore, 1999). The HIV-1 co-receptors are particularly attractive targets for entry inhibitors, because they belong to the protein superfamily of Gprotein-coupled receptors (GPCRs) (Horuk et al., 2003;Kazmierski et al., 2003;Schwarz et al., 2002;Seibert & Sakmar, 2004). The present study aimed to determine the effects of DAPTA, a synthetic peptide comprised of eight amino acids of the gp120 V2 region, which is proposed to function as a viral entry inhibitor by selectively targeting the chemokine receptor CCR5 (Polianova et al., 2005;Ruff et al., 2001;Ruff et al., 2003). ...
... The impact of these two disulfide bridges has been studied in a handful of chemokine receptors, where disruption of either bridge was found to reduce receptor surface expression (CXCR4 and CCR5) Chabot et al., 1999) and/or abolish chemokine binding (CXCR2, CCR5, and CCR6) Ai and Liao, 2002;Limatola et al., 2005). This large impact on chemokine interaction is not surprising, as chemokines, due to their large size, primarily interact with the extracellular parts of the receptor (Schwarz and Wells, 2002;Allen et al., 2007), making them critically dependent on correct folding of these regions. ...
... According to the so-called two-step model initially proposed for the C5a receptor (Siciliano et al., 1994) and later suggested for the chemokine system by Pease et al. (1998) and Xanthou et al. (2003), the chemokine initially interacts with the receptor N terminus, followed by an interaction with the extracellular loops. As a last step, the chemokine N terminus initiates receptor activation, either by docking into the main binding crevice or via interaction with the extracellular parts (Schwarz and Wells, 2002). This model was based on investigations of chemokine interactions with chimeric receptors of CXCR1:3 (Xanthou et al., 2003) and CCR1:3 (Pease et al., 1998). ...
In addition to the 7TM receptor-conserved disulfide bridge between transmembrane helix (TM) 3 and extracellular loop (ECL) 2, chemokine receptors contain a disulfide bridge between the N-terminus and what previously was believed to be ECL-3. Recent crystal- and NMR-structures of CXCR4 and CXCR1, combined with structural analysis of all endogenous chemokine receptors indicate that this chemokine receptor-conserved bridge in fact connects the N-terminus to the top of TM-7. By employing chemokine ligands that mainly target extracellular receptor regions and small molecule ligands that predominantly interact with residues in the main binding crevice, we show that the 7TM-conserved bridge is essential for all types of ligand-mediated activation, whereas the chemokine-conserved bridge is dispensable for small-molecule activation in CCR1. However, in striking contrast to previous studies in other chemokine receptors, high affinity CCL3 chemokine binding was maintained in the absence of either bridge. In CCR5, the closest homolog to CCR1, a completely different dependency was observed as neither chemokine activation nor binding was retained in the absence of either bridge. In contrast, both bridges where dispensable for small-molecule activation. This indicates that CCR5 activity is independent of extracellular regions, whereas in CCR1, preserved folding of ECL2 is necessary for activation. These results indicate that conserved structural features in a receptor subgroup, does not necessarily provide specific traits for the whole subgroup, but rather provides unique traits to the single receptors.
... The interactions of chemokines with their cognate receptors are mainly driven by electrostatic interactions between the positively charged chemokines and the correspondingly negatively charged extracellular parts of the receptor (and of glycosaminoglycans) on the cell surface. The receptor N-terminus and most extracellular loops are essential for ligand recognition, as the initial interaction triggers a docking of the chemokine N-terminus into the binding pocket, thereby stabilizing an active receptor conformation (Samson et al., 1997;Wu et al., 1997;Schwarz and Wells, 2002;Jensen et al., 2012;Thiele et al., 2011). The central role of the chemokine N-terminus has been confirmed, as modifications in this region alter the biological properties of the chemokine. ...
... MC148 has the same tertiary structure as endogenous CC-chemokines (thus also CCL1) suggesting an overall similar binding mode, and that the antagonist properties are likely to be explained by the shorter N-terminus of MC148. Thus, in agreement with the overall chemokine/ receptor binding mode (Schwarz and Wells, 2002;Allen et al., 2007), we suggest that the opposite action of CCL1 and MC148 is caused by unique interactions in the main binding crevice exerted by the N-termini of these chemokines. ...
Background and purpose:
Here we present a novel series of CCR8 antagonists based on a naphthalene-sulfonamide structure. This structure differs from the predominant pharmacophore for most small-molecule CC-chemokine receptor antagonists, which in fact activate CCR8, suggesting that CCR8 inhibition requires alternative structural probes.
Experimental approach:
The compounds were tested as inverse agonists and as antagonists against CCL1-induced activity in Gα(i) signalling and chemotaxis. Furthermore, they were assessed by heterologous competition binding against two radiolabelled receptor ligands: the endogenous agonist CCL1 and the virus-encoded antagonist MC148.
Key results:
All compounds were highly potent inverse agonists with EC(50) values from 1.7 to 23 nM. Their potencies as antagonists were more widely spread (EC(50) values from 5.9 to 1572 nM). Some compounds were balanced antagonists/inverse agonists whereas others were predominantly inverse agonists with >100-fold lower potency as antagonists. A correspondingly broad range of affinities, which followed the antagonist potencies, was disclosed by competition with [(125)I]-CCL1 (K(i) 3.4-842 nM), whereas the affinities measured against [(125)I]-MC148 were less widely spread (K(i) 0.37-27 nM), and matched the inverse agonist potencies.
Conclusion and implications:
Despite highly potent and direct effects as inverse agonists, competition-binding experiments against radiolabelled agonist and tests for antagonism revealed a probe-dependent allosteric effect of these compounds. Thus, minor chemical changes affected the ability to modify chemokine binding and action, and divided the compounds into two groups: predominantly inverse agonists and balanced antagonists/inverse agonists. These studies have important implications for the design of new inverse agonists with or without antagonist properties.
... C27/CCR4 exerts strong chemotactic properties, while the C19 peptide weakly activates CCR4 and may be a candidate antagonist of CKLF1 (29). As another GPCR receptor of CKLF1, CCR3 is mainly expressed in eosinophils and can also be detected on the surface of Th2 cells, basophils, and mast cells (30). Mouse model experiments have established that CCR3 plays a critical role in allergic airway inflammation (31). ...
Immunoinflammatory mechanisms have been incrementally found to be involved in the pathogenesis of multiple diseases, with chemokines being the main drivers of immune cell infiltration in the inflammatory response. Chemokine-like factor 1 (CKLF1), a novel chemokine, is highly expressed in the human peripheral blood leukocytes and exerts broad-spectrum chemotactic and pro-proliferative effects by activating multiple downstream signaling pathways upon binding to its functional receptors. Furthermore, the relationship between CKLF1 overexpression and various systemic diseases has been demonstrated in both in vivo and in vitro experiments. In this context, it is promising that clarifying the downstream mechanism of CKLF1 and identifying its upstream regulatory sites can yield new strategies for targeted therapeutics of immunoinflammatory diseases.
... 8 Several chemokine-chemokine receptor axes including CXCR1/2-CXCL1-3/5-8, CXCR3-CXCL9/10/11, CXCR4-CXCL12, and CXCL5/CXCR2, have been confirmed to have effects on protumor or antitumor immune effector cells in lung cancer, hepatocellular carcinoma, and gastric cancer tumors. [9][10][11][12] Hence, studying chemokines can provide a novel understanding of the mechanisms of tumor development, disease evaluation, and even treatment strategies. ...
Objectives
Chemokines have been suggested to play significant roles in the progression of malignant cancers. This study aimed to identify the chemokines related to malignant progression in thyroid carcinoma.
Methods
The mRNA expression levels of 52 chemokines were compared between differentiated thyroid cancer (DTC) samples and normal thyroid tissues from The Cancer Genome Atlas database; survival analysis was then performed on the basis of differentially expressed chemokines. A retrospective study was conducted on the level of differentially expressed chemokines in 76 DTC patients. Functional pathway analysis was performed to explore chemokine-related regulatory mechanisms.
Results
We identified 20 chemokines with differentially expressed mRNA levels through publicly available data. High levels of CCL22 and CCL26 were found to be related with metastasis in clinical DTC samples. High levels of CCL22 were found to be significantly related to poor prognosis in DTC patients. Pathway analyses revealed that cytokines might affect cancer progression through cytokine-cytokine receptor and cytokine-interleukin interactions.
Conclusions
CCL22 and CCL26 could serve as prognostic biomarkers in thyroid carcinoma.
... The integrin b1/a5/JNK/c-JUN signaling pathway in cancer cells is able to upregulate the higher matrix stiffness-induced lysyl oxidase like (LOXL)-2, then subsequently promote production of fibronectin, expression of MMP-9 and C-X-C motif chemokine ligand (CXCL)-12 and recruitment of BMDCs to encourage pre-metastatic niche establishment (95). Chemokines binding to specific receptors on the target cell membrane help to recruit immune cells into the tumor microenvironment, thereby managing immune surveillance, angiogenesis, invasion and metastasis (96). The CXCL-12/C-X-C motif chemokine receptor (CXCR)-4 axis provides a fit microenvironment before breast cancer bone metastasis formation (97). ...
Breast cancer is one of the most common malignancy among women worldwide. Metastasis is mainly responsible for treatment failure and is the cause of most breast cancer deaths. The role of metabolism in the progression and metastasis of breast cancer is gradually being emphasized. However, the regulatory mechanisms that conduce to cancer metastasis by metabolic reprogramming in breast cancer have not been expounded. Breast cancer cells exhibit different metabolic phenotypes depending on their molecular subtypes and metastatic sites. Both intrinsic factors, such as MYC amplification, PIK3CA, and TP53 mutations, and extrinsic factors, such as hypoxia, oxidative stress, and acidosis, contribute to different metabolic reprogramming phenotypes in metastatic breast cancers. Understanding the metabolic mechanisms underlying breast cancer metastasis will provide important clues to develop novel therapeutic approaches for treatment of metastatic breast cancer.
... Chemokines exert chemotactic effects on cells via binding with its receptor. Chemokine family can be divided into the major (CC, CXC) and the minor (C and CX3C) groups based on the N-terminal cysteine residues [49,50]. Among the various chemokine receptors, CXCR4, CCR2, and CCR5 garnered prominent attention in recent years due to their high expression on monocytes/macrophages and closely related leukocytes as well as their crucial roles in CVDs progression [51][52][53][54]. ...
Cardiovascular diseases (CVDs) have been the leading cause of death in United States. While tremendous progress has been made for treating CVDs over the year, the high prevalence and substantial medical costs requires the necessity for novel methods for the early diagnosis and treatment monitoring of CVDs. Macrophages are a promising target due to its crucial role in the progress of CVDs (atherosclerosis, myocardial infarction and inflammatory cardiomyopathies). Positron emission tomography (PET) is a noninvasive imaging technique with high sensitivity and provides quantitive functional information of the macrophages in CVDs. Although 18F-FDG can be taken up by active macrophages, the PET imaging tracer is non-specific and susceptible to blood glucose levels. Thus, developing more specific PET tracers will help us understand the role of macrophages in CVDs. Moreover, macrophage-targeted PET imaging will further improve the diagnosis, treatment monitoring, and outcome prediction for patients with CVDs. In this review, we summarize various targets-based tracers for the PET imaging of macrophages in CVDs and highlight research gaps to advise future directions.
... Chemokines are a family of small cytokine-like molecules that control a wide variety of biological and pathologic processes, such as immune responses, viral infection, and cancer metastasis [69]. By binding to specific receptors on the membrane of the target cells, chemokines can recruit immune cells to the tumor microenvironment, thus regulating immune surveillance, angiogenesis, invasion, and metastasis [70]. To date, approximately 50 chemokines and 20 chemokine receptors have been identified. ...
Distant metastasis to specific target organs is responsible for over 90% of breast cancer-related deaths, but the underlying molecular mechanism is unclear. Mounting evidence suggests that the interplay between breast cancer cells and the target organ microenvironment is the key determinant of organ-specific metastasis of this lethal disease. Here, we highlight new findings and concepts concerning the emerging role of the tumor microenvironment in breast cancer metastasis; we also discuss potential therapeutic intervention strategies aimed at targeting components of the tumor microenvironment.
... An additional challenge is therefore to design therapeutic agents selectively targeting the detrimental effects of chemokines and not their beneficial roles in the normal brain. Thus far, only small-molecule antagonists, peptidederived inhibitors or humanized monoclonal antibodies against chemokines and their receptors have reached clinical trials (242,243). However, innovative approaches such as siRNA nanoparticles and drugs targeting key signaling pathways and transcription factors responsible for the regulation of chemokine and chemokine receptor expression or function may lead to new therapeutic approaches based on the chemokine system (244,245). ...
Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by memory impairment, language deterioration and visuospatial deficits. The central neuropathological hallmarks of AD are neuronal degeneration, loss of synapses, the formation of neurofibrillary tangles, gliosis and amyloid-beta (Aβ) accumulation. Chronic neuroinflammation is thought to play a role in AD pathology, and numerous studies have indicated that microglia-mediated neuroinflammatory responses promote the neurodegeneration observed in AD. However, vascular inflammation and leukocyte accumulation in the AD brain and in transgenic animals with AD-like pathology suggest a role for new inflammation mechanisms in this disease. Notably, recent animal studies have shown that neutrophils migrate into the AD brain and play an unexpected role in the induction of cognitive deficit and neuropathological changes. Furthermore, blocking LFA-1 integrin, which controls leukocyte-endothelial interactions in AD mice, inhibits both Aβ deposition and tau hyperphosphorylation and reduces memory loss. Thus, the emerging role of peripheral leukocytes in the pathogenesis of AD opens new avenues of investigation and may lead to the identification of new therapeutic approaches. This review summarizes our current understanding of the roles of vascular inflammation and circulating leukocytes in AD, focusing on recently discovered neuroinflammation mechanisms. We also discuss a role for adhesion molecules, chemokines and other inflammation mechanisms that may promote brain damage in AD. Given that all current AD therapies are symptomatic, we highlight existing anti-inflammatory treatments as well as novel approaches that may contribute to AD prevention and therapy.
... Several peptide-derived and other small molecule inhibitors of CXCR4-mediated HIV-1 infection have been described in the past few years (De Clercq, 2002;Schwarz and Wells, 2002). Interestingly, several of these molecules, e.g. ...
... The binding of a chemokine to its cognate receptor is initially driven by electrostatic interactions between the overall positively charged chemokine and the negatively charged extracellular surface of the receptor. Then, interactions between the chemokine N-terminus and residues in the main binding pocket of the receptor trigger receptor activation (4)(5)(6). In contrast, small molecule ligands bind deeper in the main binding pocket and constrain the receptors in either active or inactive conformations (7,8). ...
Chemokine receptors play important roles in the immune system and are linked to several human diseases. The initial contact
of chemokines with their receptors depends on highly specified extracellular receptor features. Here we investigate the importance
of conserved extracellular disulfide bridges and aromatic residues in extracellular loop 2 (ECL2) for ligand binding and activation
in the chemokine receptor CCR8. We used IP3 accumulation and radioligand binding experiments to determine the impact of receptor
mutagenesis on both chemokine and small molecule agonist and antagonist binding and action in CCR8. We find that the 7 transmembrane
(7TM) receptor conserved disulfide bridge (7TM bridge) linking transmembrane helix (TM)III and ECL2 is crucial for chemokine
and small molecule action, whereas the chemokine receptor conserved disulfide bridge between the N terminus and TMVII is needed
only for chemokines. Furthermore, we find that two distinct aromatic residues in ECL2, Y184 (Cys+1) and Y187 (Cys+4), are
crucial for binding of the CC chemokines CCL1 (agonist) and MC148 (antagonist), respectively, but not for small molecule binding.
Finally, using in silico modeling, we predict an aromatic cluster of interaction partners for Y187 in TMIV (F171) and TMV
(W194). We show in vitro that these residues are crucial for the binding and action of MC148, thus supporting their participation
in an aromatic cluster with Y187. This aromatic cluster appears to be present in a large number of CC chemokine receptors
and thereby could play a more general role to be exploited in future drug development targeting these receptors.
... The chemokine receptors, therefore, have been proposed as attractive therapeutic targets in lung diseases, giving hope that selective receptor antagonism might allow us to control which cells are recruited and activated (140). Indeed, murine models with deleted chemokine receptor genes as well as studies with neutralizing antibodies and chemokine receptor antagonists have already shown reduction of airway inflammation and disease-like phenotype. ...
Chemokine receptors and their chemokine ligands, key mediators of inflammatory and immune cell trafficking, are involved in the regulation of both physiological and pathological processes in the lung. The discovery that chemokine receptors/chemokines, typically expressed by inflammatory and immune cells, are also expressed in structural lung tissue cells, suggest their role in mediating the restoration of lung tissue structure and functions. Thus, chemokine receptors/chemokines contribute not only to inflammatory and immune responses in the lung, but also play a critical role in the regulation of lung tissue repair, regeneration and remodeling. This review aims to summarize current state-of-the-art on chemokine receptors and their ligands in lung diseases such as chronic obstructive pulmonary disease, asthma/allergy, pulmonary fibrosis, acute lung injury, and lung infection. Furthermore, the therapeutic opportunities of chemokine receptors in aforementioned lung diseases are discussed. The review also aims to delineate the potential contribution of chemokine receptors to the processes leading to repair/regeneration of the lung tissue.
Copyright © 2014, American Journal of Physiology - Lung Cellular and Molecular Physiology.
... In this paper, we report the synthesis of a series of pyridine compounds as potential CXC chemokine receptor type 4 (CXCR4) antagonists. Chemokines and their receptors are involved in the pathogenesis of cancer, HIV, autoimmune and inflammatory diseases [3,4]. One such chemokine receptor is CXCR4. ...
A series of pyridine derivatives were synthesized as potential inhibitors of chemokine receptor type 4. This chemokine receptor has been linked to various disease pathways including HIV-1 proliferation, autoimmune disorders, inflammatory diseases, and cancer metastasis. The compounds were tested for activity using an affinity binding assay and an assay that tests the ability to inhibit cell invasion. Two hit compounds (2b and 2j) have been identified for further evaluation that inhibit cell invasion by at least 50% and have an effective concentration of less than 100 nM in the binding affinity assay. The structures of the synthesized compounds were confirmed by spectral data.
... CK have long been considered as potential therapeutic targets, while CKR represent some of the most "druggable" molecules in the human body [350][351][352][353]. ...
... Chemokines: [8][9][10] proteins that are mediators of hematopoiesis and the immune response. They regulate lymphocyte development, homing and trafficking to and from the bone marrow and lymphoid tissues. ...
Chemokines and their receptors are integral components of the immune response, regulating lymphocyte development, homing and trafficking, and playing a key role in the pathophysiology of many diseases. Chemokine receptors have, therefore, become the target for both small-molecule, peptide and antibody therapeutics. Chemokine receptors belong to the family of seven transmembrane receptor class A G protein-coupled receptors. The publication of the crystal structure of the archetypal class A seven transmembrane receptor protein rhodopsin, and other G protein-coupled receptors, including C-X-C chemokine receptor 4 and C-C chemokine receptor 5, provided the opportunity to create homology models of chemokine receptors. In this review, we describe an interdisciplinary approach to chemokine receptor modeling and the utility of this approach for structure-based drug design of chemokine receptor inhibitors.
... Chemokines or chemotactic cytokines are a large group of small (~ 8-15 kDa) proteins that relate to each other structurally and functionally and insert a significant function in leukocyte migration and activation (1)(2)(3)(4)(5). Chemokines mediate their influences through activation of particular proteins in surface of the cells belonging to well-known seven transmembrane spanning G-protein coupled receptors family (GPCR). ...
Quantitative relationships between structures of twenty six of 2-mercaptoimidazoles as C-C chemokine receptor type 2 (CCR2) inhibitors were assessed. Modeling of the biological activities of compounds of interest as a function of molecular structures was established by means of genetic algorithm multivariate linear regression (GA-MLR) and genetic algorithm (GA-ANN). The results showed that, the pIC50 values calculated by GA-ANN are in good agreement with the experimental data, and the performance of the artificial neural networks regression model is superior to the multivariate linear regression-based (MLR) model. With respect to the obtained results, it can be deduced that there is a non-linear relationship between the pIC50 s and the calculated structural descriptors of the 2-mercaptoimidazoles. The obtained models were able to describe about 78% and 93% of the variance in the experimental activity of molecules in training set, respectively. The study provided a novel and effective approach for predicting biological activities of 2-mercaptoimidazole derivatives as CCR2 inhibitors and disclosed that combined genetic algorithm and GA-ANN can be used as a powerful chemometric tools for quantitative structure activity relationship (QSAR) studies.
... In addition, with the recent understanding in the chemokine biology, the novel approach of targeting chemokines and chemokine receptors was boosted in 1998 when potent small-molecule antagonists for CXCR2 (by SmithKline Beecham, now GlaxoSmithKline) and CCR1 (by Berlex Biosciences) were developed [27,28]. This spurred a rigorous search for potent small-molecule antagonists which may act specifically on a wide variety of therapeutically relevant chemokine receptors (CCR1, 2, 3, 5 and 10, and CXCR2, 3, and 4) [29]. ...
The use of complimentary and alternative medicine (CAM) approaches is becoming increasingly popular among patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Arthritis is a leading cause of work-related disabilities affecting approximately 1.0% of the United States population. The lack of adequate response combined with increased risk of adverse events to conventional therapy in RA or OA patients has prompted interest in evaluating CAM options for arthritis. In this regard, the last decade has shown a growing popularity of green tea (GT) in the management of arthritis. Researchers are studying GT and its constituents to provide scientific rationale for its benefit in arthritis. This review summarizes the disease pathogenesis and novel therapeutic targets for the treatment of arthritis. I also tried to address the current treatment options that are available as well as their limitations. Finally, this article reviews the emerging role of GT and its polyphenol, epigallocatechin-3-gallate (EGCG), in arthritis. Although the recent findings provide scientific evidence of the efficacy of GT or EGCG in several in vitro and in vivo models of arthritis, further preclinical studies to validate its safety profile and additional phase-clinical trials in RA patients are warranted to authenticate its beneficial effect in arthritis and possibly other rheumatic conditions.
Chemokines are a family of small, chemoattractant cytokines that play a central role in regulating the migration of cells into inflamed tissue. The CXC-chemokine sub-family is characterized by four cysteine amino acids that form two pairs of disulfide bridges, with the two cysteines proximal to the N-terminus separated by an additional amino acid (CXC). Members of the CXC-chemokine family can be further characterized based on the presence or absence of a Glu-Leu-Arg motif, known as the ‘ELR’ motif. ELR + CXC-chemokines are angiogenic and preferentially promote the chemoattraction of neutrophils, while ELR- CXC-chemokines are angiostatic and preferentially promote lymphocyte migration. However, all CXC-chemokines stimulate the migration of several leukocytes through the activation of G-protein-coupled receptors (GPCRs, known as CXCRs). CXC-chemokines also promote cell survival, activate various leukocyte effector functions and induce gene expression. CXC-chemokines have therefore been associated with the pathogenesis of several respiratory diseases including asthma, COPD, IPF, ARDS, cancer and infectious diseases.
Targeting chemokine signaling is an attractive avenue for the treatment of inflammatory disorders. Tyrosine sulfation is an important post-translational modification (PTM) that enhances chemokine-receptor binding and is also utilized by a number of pathogenic organisms to improve binding affinity of immune-suppressive chemokine binding proteins (CKBPs). Here we report the display selection of tyrosine-sulfated cyclic peptides using a reprogrammed genetic code to discover high-affinity ligands for the chemokine CCL11 (eotaxin-1). The selected cyclic sulfopeptides possess high affinity for the target chemokine (as well as one or more of the related family members CCL2, CCL7 and CCL24) and inhibit CCL11 activation of CC chemokine receptor 3 (CCR3). This work demonstrates the utility in exploiting native PTMs as binding motifs for the generation of new leads for medicinal chemistry.
The interaction between G-Protein coupled receptor CXCR4 and its natural ligand CXCL12 has been linked to inflammation experienced by patients with Irritable Bowel Disease (IBD). Blocking this interaction could potentially reduce inflammatory symptoms in IBD patients. In this work, several thiophene-based and furan-based compounds modeled after AMD3100 and WZ811-two known antagonists that interrupt the CXCR4-CXCL12 interaction-were synthesized and analyzed. Fifteen hit compounds were identified; these compounds exhibited effective concentrations (EC) lower than 1000 nM (AMD3100) and inhibited invasion of metastatic cells by at least 45%. Selected compounds (2d, 2j, 8a) that inhibited metastatic invasion at a higher rate than WZ811 (62%) were submitted for a carrageenan inflammation test, where both 8a and 2j reduced inflammation in the same range as WZ811 (40%) but did not reduce inflammation more than 40%. Select compounds were also modeled in silico to show key residue interactions. These preliminary results with furan-based and thiophene-based analogues contribute to the new class on heterocyclic aromatic-based CXCR4 antagonists.
Herein, we describe the synthesis and structure-activity relationship of 3-aminocyclohex-2-en-1-one derivatives as novel CXCR2 antagonists. Thirteen out of 44 derivatives inhibit CXCR2 down-stream signaling in a Tango assay specific for CXCR2 with IC50 values less than 10 µM. In silico ADMET prediction suggests that all active compounds possess drug-like properties. None of these compounds show cytotoxicity, suggesting their potential application in inflammatory mediated diseases. A structure-activity relationship (SAR) map has been generated to gain better understanding of their binding mechanism to guide further optimization of these new CXCR2 antagonists.
A creation of nanotraps that could selectively recognize the chemotactic mediators of leukocyte adhesion and eliminate them from the bloodstream and tissue intercellular matrix is a promising approach for the treatment of various inflammatory and autoimmune diseases. We designed nanotraps as artificial decoy receptors based on poly(lactic acid) (PLA) nanoparticles covered by heparin and bearing on the surface two fragments of CCR5 receptor (N-terminal domain, Nt, and second extracellular loop, ECL2), responsible for chemokine binding. In order to attach Nt and ECL2 to the heparin shell, the corresponding peptides were modified with N- and/or C-terminal oligolysines. The presence of the nanotraps in the cell medium completely eliminated the activating effect of a CCR5 ligand, chemokine Rantes, with strongly decreasing the adhesion of monocytes to the human endothelial cells. We found that the modified ECL2 alone was also able to prevent monocyte adhesion, thus acting as a decoy receptor itself.
Targeting the interaction between G-Protein Coupled Receptor, CXCR4, and its natural ligand CXCL12 is a leading strategy to mitigate cancer metastasis and reduce inflammation. Several pyridine-based compounds modeled after known small molecule CXCR4 antagonists, AMD3100 and WZ811, were synthesized. Nine hit compounds were identified. These compounds showed lower binding concentrations than AMD3100 (1000nM) and six of the nine compounds had an effective concentration (EC) less than or equal to WZ811 (10nM). Two of the hit compounds (2g and 2w) inhibited invasion of metastatic cells at a higher rate than AMD3100 (62%). Compounds 2g and 2w also inhibit inflammation in the same range as WZ811 in the paw edema test at 40% reduction in inflammation. These preliminary results are the promising foundation of a new class of pyridine-based CXCR4 antagonists.
Die geregelte Kommunikation von Zellen unter physiologischen und pathologischen Bedingungen wird durch ein Netzwerk verschiedener zellständiger und sezernierter Mediatoren vermittelt. Untersuchungen der letzten zwei Jahrzehnte haben in diesem Zusammenhang eine neuartige Gruppe von Peptiden charakterisiert, die aufgrund ihrer biochemischen und funktionellen Verwandtschaft inzwischen als Chemokine fest etabliert sind [2,4,6,16,36]. Der Name leitet sich aus der ursprünglich im Zentrum der Untersuchungen stehenden biologischen Aktivität zahlreicher Mitglieder dieser Familie ab. Als Chemotaxis wird dabei das Phänomen der gerichteten Wanderung von Zellen auf einen löslichen Mediatorgradienten hin definiert, im Gegensatz zu Haptotaxis, bei der sich ein festphasengebundener Gradient findet. Zahlreiche chemisch sehr unterschiedliche Proteine und Lipide zeigen chemotaktische Aktivität und sind sowohl endogenen als auch exogenen Ursprungs. Exemplarisch seien das Komplementspaltprodukt C5a und Leukotrien B4 (LTB4) als endogene und Lipopolysaccharid (LPS) und verschiedene andere bakterielle Proteine als exogene Mediatoren genannt [25]
In 1990, Levin et al. [1] reported that high circulating levels of tumor necrosis factor (TNF-a) were found in patients with severe congestive heart failure (CHF), and suggested that this cytokine is involved in the pathogenesis of cardiac failure. Since then, increasing amounts of clinical and experimental data have accumulated to support a role of inflammatory mediators in the pathogenesis of myocardial dysfunction in CHF. The present chapter will review the current evidence on the involvement of chemokines, a subgroup of cytokines with chemo-attractant properties, in the mechanisms that lead to myocardial dysfunction and the development of CHF.
Structure−activity relationship exploration of the historical biarylurea series led to the identification of novel CNS penetrant CXCR2 antagonists with nanomolar potency, favorable PK profile and good developability potentials. More importantly, the key compound 22 showed efficacy in a cuprizone-induced demyelination model with twice daily oral administration, thereby supporting CXCR2 to be a potential therapeutic target for the treatment of demyelinating diseases such as multiple sclerosis.
Rheumatoid arthritis (RA) is a chronic inflammatory disease of multiple joints. Large numbers of inflammatory cells, including granulocytes, monocytes/macrophages, T lymphocytes and B lymphocytes accumulate in the inflamed RA synovium. The recruitment and positioning of these cells is largely controlled by chemokines, a family of chemotactic cytokines that mediate their effects through seven-transmembrane G protein-couple receptors. Depending on their function, they are grouped into inflammatory and homing subfamilies. Several members of both subfamilies and the corresponding receptors are upregulated in the RA synovial tissue suggesting a role in the pathophysiology. Therefore, the chemokines and their receptors represent attractive novel drug discovery targets.
G protein-coupled receptors (GPCRs) make up the largest group of transmembrane proteins implicated in signal transduction. These receptors are activated by a large variety of ligands, including cytokines and chemokines, peptide and non-peptide neurotransmitters, hormones, growth factors, odorant molecules and light. This chapter attempts to give a sense of the multiplicity of classical and novel signaling systems that transmit GPCR-mediated changes in cell behavior. The essential molecular events that GPCRs use to govern such a wide range of biological events seem elusive because of their apparent complexity. It emphasizes that the complexity of known GPCR-initiated signals is likely not to exist in one single cell type; that the signaling pathways available to GPCRs in a particular tissue are likely to be much more limited. However, because of the vast signaling potential of GPCRs, these critical cell-specific transduction pathways are in danger of being overridden by deregulated mechanisms that are sensitive to GPCRs. Thus, the study also focuses on mechanisms that enforce GPCR signaling fidelity to physiological pathways as a barrier against GPCR-induced pathological outcomes such as cell transformation. Recently gained understanding of GPCR-driven intracellular signaling networks and how they are organized by scaffolding proteins will provide a more global view of the general systems by which these receptors exert their numerous physiological roles, and elucidate their role in many pathological conditions.
This chapter provides a brief introduction to chemokine structure and activities. It explores the many functions of immune modulators, the family of chemokines. Chemokines are encoded by a large gene family with at least 45 members. The receptors for chemokines also belong to a gene family with at least 18 members, and all are G-protein-coupled receptors. The sequence similarities found in the chemokine gene family are reflected in their similar three-dimensional structures; however, chemokines display a diverse range of activities. Originally identified as potent leukocyte attractants in inflammatory diseases, chemokines have now been found to play critical roles in the natural development and regulation of the immune system. In addition, chemokines and their receptors have been utilized by pathogens to subvert the host immune system. Furthermore, this chapter provides a tabular description of the comprehensive chemokine nomenclature along with the receptors and cell types with which they interact. It also discusses the classification of chemokines. Chemokines can be divided into two general classes based on whether they are induced by pro-inflammatory cytokines or are constitutively expressed. The induced chemokines are upregulated very quickly at sites of infection or trauma and control the recruitment of leukocytes to the affected area. The constitutive chemokines are generally more involved in controlling migration of leukocytes through various tissues. Finally, it deals with chemokine receptors.
Das komplexe Immunsystem der Säugetiere schützt den Organismus effizient vor Infektionen durch die in der Umwelt ubiquitär vorhandenen Erreger. Mikroorganismen, Parasiten und Viren werden vom Immunsystem als fremd erkannt. Im Laufe der Evolution haben sich eine Vielzahl von spezifischen Erkennungs-, Signal- und Effektorstrukturen herausgebildet, die auf der Basis einer lokalen unspezifischen Entzündungsreaktion zu einer auf den jeweiligen „Fremdorganismus“ angepassten Immunabwehr führen. Bereits geringste Fehlfunktionen im Netzwerk dieser Immunmechanismen können zu einer defekten Abwehr von Infektionserregern oder aber einer übermäßigen Entzündungsreaktion mit jeweils schädlichen Folgen für den Organismus führen.
The human chemokine system comprises19 seven-transmembrane helix (7TM) receptors and 45 endogenous chemokines that often interact with each other in a promiscuous manner. Due to the chemokine system's primary function in leukocyte migration, it has a central role in immune homeostasis and surveillance. Chemokines are a group of 8-12 kDa large peptides with asecondary structure consisting of a flexible N-terminus and a core-domainusually stabilized by two conserved disulfide bridges.Theymainly interact with the extracellular domains of their cognate 7TM receptors. Affinity- and activity-contributing interactions areattributed to different domains and known to occur in two steps. Here, knowledge on chemokine and receptor domains involved in the first binding-step and the second activation-stepis reviewed. A mechanism comprising at least two steps seems consistent; however, several intermediate interactions possibly occur, resulting in a multi-step process, as recently proposed for other 7TM receptors.Overall, the N-terminus of chemokine receptors is pivotal for binding of all chemokines. During receptor activation, differences between the two majorchemokine subgroups occur, as CC-chemokines mainly interact with or rely on transmembrane receptor residues, while CXC-chemokines use residues located further exterior.Moreover, different chemokinesfor the same receptor often bind at different sites, uncovering the existence of several orthosteric sites thereby adding another level of complexity. This gives rise to a probe-dependency of small molecule "drug-like" ligands, which, depending on the chemokine interaction, may bind allosteric for some, and orthosteric for other chemokines targeting the same receptor, thereby resulting in probe-dependent pharmacodynamics.
Polymorphonuclear neutrophils (PMN) play a key role in host innate immune responses by migrating to the sites of inflammation. Furthermore, PMN recruitment also plays a significant role in the pathophysiology of a plethora of inflammatory disorders such as chronic obstructive pulmonary disease (COPD), gram negative sepsis, inflammatory bowel disease (IBD), lung injury, and arthritis. Of note, chemokine-dependent signalling is implicated in the amplification of immune responses by virtue of its role in PMN chemotaxis in most of the inflammatory diseases. It has been clinically established that impediment of PMN recruitment ameliorates disease severity and provides relief in majority of other immune-associated disorders. This review focuses on different novel approaches clinically proven to be effective in blocking chemokine signalling associated with PMN recruitment that includes CXCR2 antagonists, chemokine analogs, anti-CXCR2 monoclonal antibodies, and CXCR2 knock-out models. It also highlights the significance of the utility of nanoparticles in drugs used for blocking migration of PMN to the sites of inflammation.
CC Chemokine Receptor 2 (CCR2) is a member of the G protein-coupled receptor (GPCR) superfamily that serves as the receptor for monocyte chemoattractant proteins 1–4 (MCP-1 to -4), a group of pro-inflammatory chemotactic cytokines (chemokines). CCR2 is the primary chemokine receptor on inflammatory monocytes, and is also expressed on T-cells, dendritic cells, and endothelial cells. A number of studies have demonstrated that antagonism of CCR2 and/or MCP-1 reduces disease scores in pre-clinical models of arthritis. Recently, the first reports of the actions of small molecule antagonists in both collagen-induced arthritis (CIA) and adjuvant arthritis models have appeared, and they confirm the earlier findings: blockade of CCR2 reduced disease score. The effects of genetic deletion of CCR2 in mouse CIA showed that CCR2–/– mice exhibited exacerbated disease. CCR2–/–mice developed chronic polyarthritis after infection with Mycobacterium avium, whereas wild-type (WT) littermates did not. Recent data have highlighted that CCR2 plays a more central role in immunology than had been previously anticipated, in that it governs the emigration of activated monocytes from the bone marrow in addition to directing their migration toward certain points of inflammation. New pre-clinical validation for CCR2 antagonism in rodent disease models has also been obtained, using both genetic and chemical approaches.
Over the last decade extensive preclinical studies, including various animal disease models, have uncovered a role of CCR2 in the inflammation-related diseases such as rheuma-toid arthritis, multiple sclerosis, atherosclerosis, chronic obstruc-tive pulmonary disease (COPD) and type 2 diabetes mellitus.
An improved and efficient synthetic route towards piperidino-piperazine based CCR5 antagonists was developed. The new approach was flexible for introducing various substituents in the pharmacophore sites via Grignard reagent addition and reductive amination. l-Amino acids were used as a chiral pool to introduce and then induce the desired stereochemistries, meanwhile rendering the variable substitution. The efficient construction of the piperazino-piperidine nucleus was achieved in a highly convergent manner with a key building block of N1-Boc-4-substituent-4-aminopiperidine, exhibiting significant advantages in terms of concise synthetic route and environmental-friendly reagents over the previously described stepwise synthesis, in which a modified Strecker reaction was involved with highly toxic reagents such as diethylaluminum cyanide.
The long-term goal of this research project is to develop CCR5 antagonists with the structure feature of anibamine as novel anti prostate cancer agents. The tasks of this project include: 1. Chemical synthesis of the ligand designed as anibamine derivatives; 2. Radio-ligand competition binding assay to evaluate the binding affinity of the ligands synthesized; 3. Characterization of CCL5 and CCR5 expression in SV40T-immortalized human prostate epithelial cell lines of the same cellular lineage with varying tumorigenicity and metastatic capacity in vivo (P69, M2182, M2205 and M12); 4. Investigation of the impact of chemokine receptor CCR5 antagonist on prostate cancer cell growth and progression using M12, PC-3, DU-145 and LNCaP cell lines; 5. Molecular modeling study. In order to achieve these aims, we have first designed and synthesized a series of anibamine analogs based on ?Deconstruction-Reconstruction- Elaboration? method. So far thirty-two novel ligands have been prepared as derivatives of anibamine for biological screening assays. Second, several progressive biological assays to evaluate the anti prostate cancer activity of the ligands have been set up and pursued. Primarily, a RANTES-binding inhibition assay protocol has been set up and ready to test our drug candidates. Additionally, biological screening includes the test of capacity of these novel compounds to inhibit proliferation and/or apoptosis by the human prostate cancer cell lines M12, PC-3, DU-145 and LNCaP has been conducted to evaluate their efficacy. Also we have assessed changes in the expression of CCL5 and CCR5 in human prostate epithelial cell lines of the same cellular lineage but with differing in vivo phenotypes (P69SV40TAg, M2182, M2205, and M12).
Chemokine receptor 5 (CCR5) is an important receptor used by human immunodeficiency virus type 1 (HIV-1) to gain viral entry into host cell. In this study, we used a combined approach of comparative modeling, molecular docking, and three dimensional quantitative structure activity relationship (3D-QSAR) analyses to elucidate detailed interaction of CCR5 with their inhibitors. Docking study of the most potent inhibitor from a series of compounds was done to derive the bioactive conformation. Parameters such as random selection, rational selection, different charges and grid spacing were utilized in the model development to check their performance on the model predictivity. Final comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) models were chosen based on the rational selection method, Gasteiger-Hückel charges and a grid spacing of 0.5 Å. Rational model for CoMFA (q
2 = 0.722, r
2 = 0.884, Q
2 = 0.669) and CoMSIA (q
2 = 0.712, r
2 = 0.825, Q
2 = 0.522) was obtained with good statistics. Mapping of contour maps onto CCR5 interface led us to better understand of the ligand–protein interaction. Docking analysis revealed that the Glu283 is crucial for interaction. Two new amino acid residues, Tyr89 and Thr167 were identified as important in ligand–protein interaction. No site directed mutagenesis studies on these residues have been reported.
Generally referred to as Europe's largest Biotech company, Serono, with global headquarters in Geneva, has long been known for its portfolio of therapeutic proteins, which has, so far, resulted in market approvals of several recombinant products in the areas of reproductive health (Gonal-F,
Luveris, Ovidrel), metabolism-endocrinology (Saizen, Serostim), neurology (Rebif), and dermatology (Raptiva). In the late 90s, Serono's management made the strategic decision to add small molecule drug discovery to their research portfolio, with the aim of mimicking and further extending the
spectrum of action of the existing protein therapeutics with orally bioavailable next-generation products. As a hallmark of this new research paradigm, in late 1997, Serono acquired the GBRI (Glaxo Biomedical Research Institute), now SPRI (Serono Pharmaceutical Research Institute), located
just outside Geneva, and during the following year therein established a new, state-of-the-art Chemistry Department, with the necessary manpower, expertise, as well as the medicinal, analytical, and combinatorial chemistry equipment, including extensive structure- and ligand-based design capabilities.
In conjunction with a somewhat smaller Chemistry Department previously established at Serono's Boston-based research site SRBI (Serono Reproductive Biology Institute), Serono's chemists have now been working for around five years on a variety of small molecule drug discovery projects. As the
first small molecules emerging from these efforts have started entering human clinical trials, the present article will give an account on some of the work performed to date.
Recent studies have indicated that the CCR5 chemokine receptor may be a potential target for treating prostate cancer. Thus, development of CCR5 antagonists may provide novel prostate cancer therapy. Anibamine, a novel pyridine quaternary alkaloid isolated from Aniba sp., was found to effectively compete with (125)I-gp120 in binding to the chemokine receptor CCR5, with an IC(50) = 1 μM. Anibamine is the first natural product reported as a CCR5 antagonist, and thus provides a novel structural skeleton unique from other lead compounds that have generally been identified from high-throughput screening efforts. In order to refine the lead compound's structure and improve the therapeutic index of anibamine derivatives as potential anti prostate cancer agents, the approach of "deconstruction-reconstruction-elaboration" was applied in the structure-activity relationship studies of this work. Here, we report the design, syntheses and anti prostate cancer activities of anibamine and 17 analogues. The results from the in vitro and in vivo studies described here show that this class of compounds has potential to provide novel leads as anti prostate cancer agents.
Peptide auf das Wesentliche beschränkt: Das Pentapeptid cyclo(-L-Nal 1-Gly 2-D-Tyr 3-L-Arg 4-L-Arg 5-) wurde durch Kombination einer konformationsbasierten mit einer sequenzbasierten Peptid-Bibliothek als möglicher CXCR4-Antagonist ermittelt (Nal=L-3-(2-Naphthyl)alanin; Bild: Projektion der fünf energetisch günstigsten Strukturen). Mit einem IC50-Wert von 4 nM ist es vergleichbar effektiv wie die 14-Aminosäuren-Stammverbindung T140.
Introduction Chemokine Receptors in HIV-1 Entry Structure-Function Relationship of Chemokine Receptors Structure-Function Relationship of Chemokine Ligands Importance of Chemokines and Chemokine Receptors in Other Pathological Processes SMM-Chemokines:A New Class of Unnatural Synthetic Molecules as Chemical Probes of Chemokine Receptor Biology and Leads for Therapeutic Development Conclusions and Future Prospects Acknowledgments References
The question of how the migration of cells can be specifically controlled was first addressed at least 15 years ago with the
identification of the neutrophil chemattractant, IL-8, and the monocyte attractants, MCP-1 and MIP-1α. The rapid expansion
of this family of proteins, known as chemoattractant cytokines and re-named chemokines, showed that the idea of one chemokine being responsible for the recruitment of a single cell type
was over simplistic in most cases.
The Duffy blood group antigen has been postulated to be a receptor on red blood cells (RBCs) for the malarial parasite Plasmodium vivax and a promiscuous receptor for the chemokine superfamily of inflammatory proteins. Recently, the Duffy antigen glycoprotein D cDNA has been cloned (Chaudhuri et al: Proc Natl Acad Sci USA 90:10793, 1993). We have analyzed the binding properties of the cloned Duffy antigen. Duffy- antigen cDNAs expressed in human embryonic kidney cells produced cell- surface proteins that reacted with two known anti-Duffy monoclonal antibodies. Direct ligand binding and displacement experiments using recombinant chemokine proteins also show that the cloned Duffy protein is the RBC chemokine receptor. Radiolabeled chemokines of both the C-C (RANTES and MCP-1) and C-X-C (IL-8 and MGSA/gro) subclasses bound reversibly to transfected cells with dissociation constants in the nanomolar range. Chemokines of either class displaced heterologous chemokines, indicating that they were competing for a single site on the transfected cells. Although the chemokines bound to the transfected cells with high affinity, there was no evidence for signal transduction, as measured by transient increases in intracellular calcium ion concentration, through the Duffy antigen/RBC chemokine receptor in transfected cells. Lastly, we have performed a computer analysis on the amino acid structure of the Duffy antigen/RBC chemokine receptor. Although the cloned Duffy antigen has been postulated to be a nine-transmembrane-spanning receptor, our analysis suggests that the molecule most likely belongs to the seven-transmembrane-spanning receptor superfamily and is therefore similar to other chemokine receptors previously identified.
Antagonists of multiple chemokines could be more effective than inhibitors of specific chemokines for controlling cell migration
and inflammation. To attempt to identify such antagonists we characterized a number of truncated analogs of regulated on activation
normal T cell expressed protein (RANTES), monocyte chemoattractant protein (MCP)-3, and MCP-1. On the basis of their ability
to compete for binding of their parent chemokines, three analogs were selected for cross-reactivity studies: RANTES (9-68),
MCP-3 (10-76), and MCP-1 (9-76). These analogs bound to THP-1 monocytic cells with dissociation constants that were within
4-6-fold of their native counterparts, but they did not promote detectable chemotaxis of THP-1 cells or enzyme release from
purified human monocytes. The RANTES (9-68) analog competed for the binding and inhibited the activities of all three chemokines.
In contrast, native RANTES was specific for RANTES binding sites. However, truncation of either MCP-1 or MCP-3 did not change
their respective binding specificity. MCP-3 and MCP-3 (10-76) competed for binding of all three labeled chemokines. MCP-1
(9-76) competed strongly for binding of labeled MCP-1, but only weakly for the other two labeled ligands and inhibited the
activities induced by MCP-1 and MCP-3 but not RANTES. Although RANTES (9-68) and MCP-3 (10-76) inhibited all three chemokines,
the RANTES analog was significantly more potent for RANTES-induced activity. The results indicate that NH-terminal residues partly determine the receptor specificity of RANTES, and deletions within this region permit binding to
multiple chemokine receptors. The findings suggest the feasibility of design of high affinity multi-specific CC chemokine
antagonists.
Abstract
Interleukin-8 (IL-8) and closely related Glu-Leu-Arg (ELR) containing CXC chemokines, including growth-related oncogene (GRO)alpha, GRObeta, GROgamma, and epithelial cell-derived neutrophil-activating peptide-78 (ENA-78), are potent neutrophil chemotactic and activating peptides, which are proposed to be major mediators of inflammation. IL-8 activates neutrophils by binding to two distinct seven-transmembrane (7-TMR) G-protein coupled receptors CXCR1 (IL-8RA) and CXCR2 (IL-8RB), while GROalpha, GRObeta, GROgamma, and ENA-78 bind to and activate only CXCR2. A chemical lead, which selectively inhibited CXCR2 was discovered by high throughput screening and chemically optimized. SB 225002 (N-(2-hydroxy-4-nitrophenyl)-N'-(2-bromophenyl)urea) is the first reported potent and selective non-peptide inhibitor of a chemokine receptor. It is an antagonist of 125I-IL-8 binding to CXCR2 with an IC50 = 22 nM. SB 225002 showed >150-fold selectivity over CXCR1 and four other 7-TMRs tested. In vitro, SB 225002 potently inhibited human and rabbit neutrophil chemotaxis induced by both IL-8 and GROalpha. In vivo, SB 225002 selectively blocked IL-8-induced neutrophil margination in rabbits. The present findings suggest that CXCR2 is responsible for neutrophil chemotaxis and margination induced by IL-8. This selective antagonist will be a useful tool compound to define the role of CXCR2 in inflammatory diseases where neutrophils play a major role.
The β-chemokine receptor CCR5 is considered to be an attractive target for inhibition of macrophage-tropic (CCR5-using or
R5) HIV-1 replication because individuals having a nonfunctional receptor (a homozygous 32-bp deletion in the CCR5 coding
region) are apparently normal but resistant to infection with R5 HIV-1. In this study, we found that TAK-779, a nonpeptide
compound with a small molecular weight (Mr 531.13), antagonized the binding of RANTES (regulated on activation, normal T cell expressed and secreted) to CCR5-expressing
Chinese hamster ovary cells and blocked CCR5-mediated Ca2+ signaling at nanomolar concentrations. The inhibition of β-chemokine receptors by TAK-779 appeared to be specific to CCR5
because the compound antagonized CCR2b to a lesser extent but did not affect CCR1, CCR3, or CCR4. Consequently, TAK-779 displayed
highly potent and selective inhibition of R5 HIV-1 replication without showing any cytotoxicity to the host cells. The compound
inhibited the replication of R5 HIV-1 clinical isolates as well as a laboratory strain at a concentration of 1.6–3.7 nM in
peripheral blood mononuclear cells, though it was totally inactive against T-cell line-tropic (CXCR4-using or X4) HIV-1.
The CC chemokine receptor-1 (CCR1) is a prime therapeutic target for treating autoimmune diseases. Through high capacity screening
followed by chemical optimization, we identified a novel non-peptide CCR1 antagonist, R-N-[5-chloro-2-[2-[4-[(4-fluorophenyl)methyl]-2-methyl-1-piperazinyl]-2-oxoethoxy]phenyl]urea hydrochloric acid salt (BX 471).
Competition binding studies revealed that BX 471 was able to displace the CCR1 ligands macrophage inflammatory protein-1α
(MIP-1α), RANTES, and monocyte chemotactic protein-3 (MCP-3) with high affinity (K
i ranged from 1 nm to 5.5 nm). BX 471 was a potent functional antagonist based on its ability to inhibit a number of CCR1-mediated effects including Ca2+ mobilization, increase in extracellular acidification rate, CD11b expression, and leukocyte migration. BX 471 demonstrated
a greater than 10,000-fold selectivity for CCR1 compared with 28 G-protein-coupled receptors. Pharmacokinetic studies demonstrated
that BX 471 was orally active with a bioavailability of 60% in dogs. Furthermore, BX 471 effectively reduces disease in a
rat experimental allergic encephalomyelitis model of multiple sclerosis. This study is the first to demonstrate that a non-peptide
chemokine receptor antagonist is efficacious in an animal model of an autoimmune disease. In summary, we have identified a
potent, selective, and orally available CCR1 antagonist that may be useful in the treatment of chronic inflammatory diseases.
Interleukin-4 (IL-4) represents a prototypic lymphokine (for a recent review see Paul, 1991). It promotes differentiation of B-cells and the proliferation of T- and B-cell, and other cell types of the lymphoid system. An antagonist of human IL-4 was discovered during the studies presented here after Tyr124 of the recombinant protein had been substituted by an aspartic acid residue. This IL-4 variant, Y124D, bound with high affinity to the IL-4 receptor (KD = 310 pM), but retained no detectable proliferative activity for T-cells and inhibited IL-4-dependent T-cell proliferation competitively (K(i) = 620 pM). The loss of efficacy in variant Y124D was estimated to be greater than 100-fold on the basis of a weak partial agonist activity for the very sensitive induction of CD23 positive B-cells. The substitution of Tyr124 by either phenylalanine, histidine, asparagine, lysine or glycine resulted in partial agonist variants with unaltered receptor binding affinity and relatively small deficiencies in efficacy. These results demonstrate that high affinity binding and signal generation can be uncoupled efficiently in a ligand of a receptor belonging to the recently identified hematopoietin receptor family. In addition we show for the first time, that a powerful antagonist acting on the IL-4 receptor system can be derived from the IL-4 protein.
Monocyte chemoattractant protein (MCP)-1 analogues were designed to determine the role of the NH2-terminal region in structure and function. The NH2-terminal residue was important for function and receptor binding, as it could not be deleted or extended. However the NH2-terminal pyroglutamate residue of the wild type was not essential as it could be replaced by several other noncyclic amino acids without loss of activity. Residues 7-10 were essential for receptor desensitization, but were not sufficient for function, and the integrity of residues 1-6 were required for functional activity. A peptide corresponding to MCP-1, 1-10 lacked detectable receptor-binding activities, indicating that residues 1-10 are essential for MCP-1 function, but that other residues are also involved. Several truncated analogues, including 8-76, 9-76, and 10-76, desensitized MCP-1-induced Ca2+ induction, but were not significantly active. These analogues were antagonists of MCP-1 activity with the most potent being the 9-76 analogue (IC50 = 20 nM) The 9-76 specifically bound to MCP-1 receptors with a Kd of 8.3 nM, which was three-fold higher than MCP-1 (Kd 2.8 nM). The 9-76 analogue desensitized the Ca2+ response to MCP-1 and MCP-3, but not to other CC chemokines, suggesting that it is MCP receptor specific. The availability of these compounds will be helpful in evaluating MCP receptor antagonists as anti-inflammatory therapeutics.
Monocyte chemoattractant protein-1 (MCP-1) is a monocyte-specific chemoattractant and activator and is a member of the chemokine-beta family of cytokines. To identify regions of MCP-1 which are required for its biological activity, we constructed human MCP-1 mutants that were expressed in eukaryotic cells and tested for their ability to attract monocytes in vitro. Deletion of amino acids 2-8 destroyed activity, suggesting that the amino-terminal region is necessary for activity. Within the deleted region, mutation of aspartate 3 to alanine produced a protein with 9% of wild-type activity, whereas mutation of asparagine 6 to alanine produced a protein with 52.9% of wild-type activity. Mutation of amino acids within the first intercysteine loop yielded variable results. Changing tyrosine 28 to aspartate or arginine 30 to leucine each produced proteins with essentially no monocyte chemoattractant activity. The side chains of these amino acids are predicted to point into a putative receptor binding cleft, and these loss-of-function mutations are consistent with this model. Also consistent is the retention of 60% of wild-type activity after mutation of serine 27 to glutamine, since the side chain of serine 27 is predicted to point away from the binding cleft. However, mutation of arginine 24, which lies outside of this area, to phenylalanine produced a protein with only 5% of wild-type activity, suggesting more complex interactions. Truncations of the carboxyl terminus, as well as mutation of aspartate 68 to leucine, generated proteins with 10-20% of wild-type activity. (Another carboxyl-terminal insertional mutation demonstrated that O-linked carbohydrate in MCP-1 alpha may be added to a threonine in the carboxyl-terminal region.) These findings are consistent with a structural model of dimeric MCP-1 which is similar to interleukin-8, in which amino acids that point into a cleft between the two carboxyl-terminal alpha-helices of the subunits are important for receptor binding. In addition, however, amino acids at the amino terminus and others outside of the interhelical cleft are also essential for activity. The carboxyl-terminal alpha-helix is not required for signaling per se but is required for maximal specific activity. Finally, four mutant proteins partially inhibited the ability of wild-type MCP-1 to attract monocytes in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)
The stem cell inhibitor, macrophage inflammatory protein-1 alpha (MIP-1 alpha) or LD78, protects multipotent hematopoietic progenitors in murine models from the cytotoxic effects of chemotherapy. Clinical use of human MIP-1 alpha during chemotherapy could therefore lead to faster hematologic recovery and may allow dose intensification. We have also shown that human MIP-1 alpha causes the rapid mobilization of hematopoietic cells, suggesting an additional clinical use in peripheral blood stem cell transplantation. However, the clinical evaluation of human MIP-1 alpha is complicated by its tendency to associate and form high molecular weight polymers. We have produced a variant of rhMIP-1 alpha, BB-10010, carrying a single amino acid substitution of Asp26 > Ala, with a reduced tendency to form large polymers at physiologic pH and ionic strength. This greatly increases its solubility, facilitating its production and clinical formulation. We confirmed the potency of BB-10010 as a human MIP-1 alpha-like agonist in receptor binding, calcium mobilization, inhibition of colony formation, and thymidine suicide assays. The myeloprotective activity of BB-10010 was shown in a murine model of repeated chemotherapy using hydroxyurea. BB-10010 is therefore an ideal variant with which to evaluate the therapeutic potential of recombinant human MIP-1 alpha.
Extension of recombinant human RANTES by a single residue at the amino terminus is sufficient to produce a potent and selective
antagonist. RANTES is a proinflammatory cytokine that promotes cell accumulation and activation in chronic inflammatory diseases.
When mature RANTES was expressed heterologously in Escherichia coli, the amino-terminal initiating methionine was not removed by the endogenous amino peptidases. This methionylated protein
was fully folded but completely inactive in RANTES bioassays of calcium mobilization and chemotaxis of the promonocytic cell
line THP-1. However, when assayed as an antagonist of both RANTES and macrophage inflammatory polypeptide-1α (MIP-1α) in these
assays, the methionylated RANTES (Met-RANTES) inhibited the actions of both chemokines. T cell chemotaxis was similarly inhibited.
The antagonistic effect was selective since Met-RANTES had no effect on interleukin-8- or monocyte chemotractant protein-1-induced
responses in these cells. Met-RANTES can compete with both [I]RANTES and [I]MIP-1α binding to THP-1 cells or to stably transfected HEK cells recombinantly expressing their common receptor, CC-CKR-1.
These data show that the integrity of the amino terminus of RANTES is crucial to receptor binding and cellular activation.
An antagonist of human monocyte chemoattractant protein (MCP)-1, which consists of MCP-1(9-76), had previously been characterized and shown to inhibit MCP-1 activity in vitro. To test the hypothesis that, by inhibiting endogenous MCP-1, the antagonist has antiinflammatory activity in vivo, we examined its effect in the MRL-lpr mouse model of arthritis. This strain spontaneously develops a chronic inflammatory arthritis that is similar to human rheumatoid arthritis. Daily injection of the antagonist, MCP-1(9-76), prevented the onset of arthritis as monitored by measuring joint swelling and by histopathological evaluation of the joints. In contrast, controls treated with native MCP-1 had enhanced arthritis symptoms, indicating that the inhibitory effect is specific to the antagonist. In experiments where the antagonist was given only after the disease had already developed, there was a marked reduction in symptoms and histopathology, although individuals varied in the magnitude of the response. The mechanism of inhibition of disease is not known, although the results suggest that it could be more complex than the competitive inhibition of ligand binding that is observed in vitro. The demonstration of the beneficial effects of an MCP-1 antagonist in arthritis suggests that chemokine receptor antagonists could have therapeutic application in inflammatory diseases.
Interleukin 5 (IL-5) is the key cytokine involved in regulating the production and many of the specialized functions of mature eosinophils including priming, adhesion, and survival. We have generated a point mutant of human IL-5, IL-5 (E12K), which is devoid of agonist activity in both a TF-1 cell proliferation assay and a human eosinophil adhesion assay. However, IL-5 (E12K) is a potent and specific antagonist of both these IL-5-dependent functional responses. In both receptor binding and cross-linking studies the wild-type and IL-5 (E12K) mutant exhibit virtually identical properties. This mutant protein was unable to stimulate tyrosine phosphorylation in human eosinophils, and blocked the phosphorylation stimulated by IL-5. In contrast, IL-5 (E12K) is a full agonist in a human eosinophil survival assay, although with reduced potency compared to the wild-type protein. This IL-5 mutant enables us to clearly distinguish between two IL-5-dependent functional responses and reveals distinct mechanisms of receptor/cellular activation.
Bicyclams are a novel class of antiviral compounds that are highly potent and selective inhibitors of the replication of HIV-1 and HIV-2. Surprisingly, however, when the prototype compound AMD3100 was tested against M-tropic virus strains such as BaL, ADA, JR-CSF, and SF-162 in human peripheral blood mononuclear cells, the compound was completely inactive. Because of the specific and potent inhibitory effect of AMD3100 on T-tropic viruses, but not M-tropic viruses, it was verified that AMD3100 interacts with the CXC-chemokine receptor CXCR4, the main coreceptor used by T-tropic viruses. AMD3100 dose dependently inhibited the binding of a specific CXCR4 monoclonal antibody to SUP-T1 cells as measured by flow cytometry. It did not inhibit the binding of the biotinylated CC-chemokine macrophage inflammatory protein (MIP) 1alpha or MIP-1beta, ligands for the chemokine receptor CCR5 (the main coreceptor for M-tropic viruses). In addition, AMD3100 completely blocked (a) the Ca2+ flux at 100 ng/ml in lymphocytic SUP-T1 and monocytic THP-1 cells, and (b) the chemotactic responses of THP-1 cells induced by stromal cell-derived factor 1alpha, the natural ligand for CXCR4. Finally, AMD3100 had no effect on the Ca2+ flux induced by the CC-chemokines MIP-1alpha, regulated on activation normal T cell expressed and secreted (RANTES; also a ligand for CCR5), or monocyte chemoattractant protein 3 (a ligand for CCR1 and CCR2b), nor was it able to induce Ca2+ fluxes by itself. The bicyclams are, to our knowledge, the first low molecular weight anti-HIV agents shown to act as potent and selective CXCR4 antagonists.
CCR5, a chemokine receptor expressed on T cells and macrophages, is the principal coreceptor for M-tropic HIV-1 strains. Recently, we described an NH2-terminal modification of the CCR5 ligand regulated on activation, normal T cell expressed and secreted (RANTES), aminooxypentane-RANTES (AOP-RANTES), that showed potent inhibition of macrophage infection by HIV-1 under conditions where RANTES was barely effective. To investigate the mechanism of AOP-RANTES inhibition of HIV infectivity we examined the surface expression of CCR5 using a monoclonal anti-CCR5 antibody, MC-1. We demonstrate that AOP-RANTES rapidly caused >90% decrease in cell surface expression of CCR5 on lymphocytes, monocytes/ macrophages, and CCR5 transfected Chinese hamster ovary (CHO) cells. RANTES also caused a loss of cell surface CCR5, although its effect was less than with AOP-RANTES. Significantly, AOP-RANTES inhibited recycling of internalized CCR5 to the cell surface, whereas RANTES did not. When peripheral blood mononuclear cells are cultured for prolonged periods of time in the presence of RANTES, CCR5 expression is comparable to that seen on cells treated with control medium, whereas there is no CCR5 surface expression on cells cultured in the presence of AOP-RANTES. Immunofluorescence indicated that both AOP-RANTES and RANTES induced downmodulation of cell surface CCR5, and that the receptor was redistributed into endocytic organelles containing the transferrin receptor. When RANTES was removed, the internalized receptor was recycled to the cell surface; however, the receptor internalized in the presence of AOP-RANTES was retained in endosomes. Using human osteosarcoma (GHOST) 34/CCR5 cells, the potency of AOP-RANTES and RANTES to inhibit infection by the M-tropic HIV-1 strain, SF 162, correlated with the degree of downregulation of CCR5 induced by the two chemokines. These differences between AOP-RANTES and RANTES in their effect on receptor downregulation and recycling suggest a mechanism for the potent inhibition of HIV infection by AOP-RANTES. Moreover, these results support the notion that receptor internalization and inhibition of receptor recycling present new targets for therapeutic agents to prevent HIV infection.
The CC chemokines macrophage inflammatory protein-1alpha (MIP-1alpha) and RANTES (regulated on activation normal T cell expressed) have been implicated in rheumatoid arthritis and multiple sclerosis. Since their effects are mediated through the CCR1 chemokine receptor, we set up a small molecule CCR1 antagonist program to search for inhibitors. Through high capacity screening we discovered a number of 4-hydroxypiperidine compounds with CCR1 antagonist activity and report their synthesis and in vitro pharmacology here. Scatchard analysis of the competition binding data revealed that the compounds had Ki values ranging from 40 to 4000 nM. The pharmacological profile of the most potent member of this series, compound 1 (2-2-diphenyl-5-(4-chlorophenyl)piperidin-lyl)valeronitri te), was further evaluated. Compound 1 showed concentration-dependent inhibition of MIP-1alpha-induced extracellular acidification and Ca2+ mobilization demonstrating functional antagonism. When given alone, the compound did not elicit any responses, indicating the absence of intrinsic agonist activity. Compound 1 inhibited MIP-1alpha- and RANTES-induced migration in peripheral blood mononuclear cells in a dose-responsive manner. Selectivity testing against a panel of seven transmembrane domain receptors indicated that compound 1 is inactive on a number of receptors at concentrations up to 10 microM. This is the first description of CCR1 receptor antagonists that may be useful in the treatment of chronic inflammatory diseases involving MIP-1alpha, RANTES, and CCR1.
IP10 and MIG are two members of the CXC branch of the chemokine superfamily whose expression is dramatically up-regulated
by interferon (IFN)-γ. The proteins act largely on natural killer (NK)-cells and activated T-cells and have been implicated
in mediating some of the effects of IFN-γ and lipopolysaccharides (LPSs), as well as T-cell-dependent anti-tumor responses.
Recently both chemokines have been shown to be functional agonists of the same G-protein-coupled receptor, CXCR3. We now report
the pharmacological characterization of CXCR3 and find that, when heterologously expressed, CXCR3 binds IP10 and MIG with
Ki
values of 0.14 and 4.9 nm, respectively. The receptor has very modest affinity for SDF-1α and little or no affinity for other CXC-chemokines. The properties
of the endogenous receptor expressed on activated T-cells are similar. Surprisingly, several CC-chemokines, particularly eotaxin
and MCP-4, also compete with moderate affinity for the binding of IP10 to CXCR3. Eotaxin does not activate CXCR3 but, in CXCR3-transfected
cells, can block IP10-mediated receptor activation. Eotaxin, therefore, may be a natural CXCR3 antagonist.
The natural ligands for the CCR5 chemokine receptor, macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, and RANTES (regulated on T-cell activation, normal T-cell expressed and secreted), are known to inhibit human immunodeficiency virus (HIV) entry, and N-terminally modified RANTES analogues are more potent than native RANTES in blocking infection. However, potent CCR5 blocking agents may select for HIV-1 variants that use alternative coreceptors at less than fully inhibitory concentrations. In this study, two N-terminal chemical modifications of RANTES produced by total synthesis, aminooxypentane (AOP)-RANTES[2-68] and N-nonanoyl (NNY)-RANTES[2-68], were tested for their ability to prevent HIV-1 infection and to select for coreceptor switch variants in the human peripheral blood lymphocyte-SCID mouse model. Mice were infected with a CCR5-using HIV-1 isolate that requires only one or two amino acid substitutions to use CXCR4 as a coreceptor. Even though it achieved lower circulating concentrations than AOP-RANTES (75 to 96 pM as opposed to 460 pM under our experimental conditions), NNY-RANTES was more effective in preventing HIV-1 infection. However, in a subset of treated mice, these levels of NNY-RANTES rapidly selected viruses with mutations in the V3 loop of envelope that altered coreceptor usage. These results reinforce the case for using agents that block all significant HIV-1 coreceptors for effective therapy.
The beta-chemokine receptor CCR5 is considered to be an attractive target for inhibition of macrophage-tropic (CCR5-using or R5) HIV-1 replication because individuals having a nonfunctional receptor (a homozygous 32-bp deletion in the CCR5 coding region) are apparently normal but resistant to infection with R5 HIV-1. In this study, we found that TAK-779, a nonpeptide compound with a small molecular weight (Mr 531.13), antagonized the binding of RANTES (regulated on activation, normal T cell expressed and secreted) to CCR5-expressing Chinese hamster ovary cells and blocked CCR5-mediated Ca2+ signaling at nanomolar concentrations. The inhibition of beta-chemokine receptors by TAK-779 appeared to be specific to CCR5 because the compound antagonized CCR2b to a lesser extent but did not affect CCR1, CCR3, or CCR4. Consequently, TAK-779 displayed highly potent and selective inhibition of R5 HIV-1 replication without showing any cytotoxicity to the host cells. The compound inhibited the replication of R5 HIV-1 clinical isolates as well as a laboratory strain at a concentration of 1.6-3.7 nM in peripheral blood mononuclear cells, though it was totally inactive against T-cell line-tropic (CXCR4-using or X4) HIV-1.
BB-10010 is a variant of the human form of macrophage inflammatory protein-1 (MIP-1), which has been shown in mice to block the entry of hematopoietic stem cells into S-phase and to increase their self-renewal capacity during recovery from cytotoxic damage. Its use may constitute a novel approach for protecting the quality of the stem cell population and its capacity to regenerate after periods of cytotoxic treatment. Thirty patients with locally advanced or metastatic breast cancer were entered into the first randomized, parallel group controlled phase II study. This was designed to evaluate the potential myeloprotective effects of a 7-day regimen of BB-10010 administered to patients receiving six cycles of 5-fluorouracil (5-FU), adriamycin, and cyclophosphamide (FAC) chemotherapy. Patients were randomized, 10 receiving 100 μg/kg BB-10010, 11 receiving 30 μg/kg BB-10010, and nine control patients receiving no BB-10010. BB-10010 was well-tolerated in all patients with no severe adverse events related to the drug. Episodes of febrile neutropenia complicated only 4% of the treatment cycles and there was no difference in incidence between the treated and nontreated groups. Studies to assess the generation of progenitor cells in long-term bone marrow cultures were performed immediately preceding chemotherapy and at the end of six dosing cycles in 18 patients. Circulating neutrophils, platelets, CD 34+ cells, and granulocyte/macrophage colony-forming cell (GM-CFC) levels were determined at serial time points in cycles 1, 3, and 6. The results showed similar hemoglobin and platelet kinetics in all three groups. On completion of the six treatment cycles, the average pretreatment neutrophil levels were reduced from 5.3 to 1.7 × 109/L in the control patients and from 4.3 to 1.9 and 4.5 to 2.5 × 109/L in the 30/100 μg/kg BB-10010 groups, respectively. Relative to their pretreatment values, 50% of the patients receiving BB-10010 completed the treatment with neutrophil values significantly higher than any of the controls (P = .02). Mobilization of GM-CFC was enhanced by BB-10010 with an additional fivefold increase over that generated by chemotherapy alone, giving a maximal 25-fold increase over pretreatment values. Bone marrow progenitor assays before and after this standard regimen of chemotherapy indicated little long-term cumulative impairment to recovery from chemotherapy. Despite the limited cumulative damage to the bone marrow, which may have minimized the protective value of BB-10010 during this regimen of chemotherapy, better recovery of neutrophils in the later treatment cycles with BB-10010 was indicated in a number of patients.
© 1998 by The American Society of Hematology.
The concept of leukocyte-specific chemoattractants was enunciated almost as soon as microscopy revealed the existence of leukocyte-specific inflammatory infiltrates (1). With the advent of reproducible in vitro chemotaxis assays, evidence for the existence of chemoattractants was bolstered by activities found in crude biological preparations. For example, the monocytic infiltrates associated with a wide variety of tumor types were suggested to be elicited by a tumor-derived chemotactic factor (TDCF) that could attract monocytes to the exclusion of neutrophils (2). In the late 1970s and early 1980s, a monocyte-specific chemoattractant activity could be demonstrated in medium conditioned by malignant cell lines whose cognate tumors were associated with monocytic infiltration in vivo (2–4).
ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
Truncation of the original piperidino-2(S)-methyl piperazine lead structure 2, from a family of muscarinic antagonists, gave compound 8 which has improved selectivity for the HIV-1 co-receptor CCR5 over muscarinic receptors. Further optimization for pharmacokinetic properties afforded Sch-350634 (1), a prototypical piperazine-based CCR5 antagonist, which is a potent inhibitor of HIV-1 entry and replication in PBMCs. The title compound (1) has excellent oral bioavailability in rat, dog, and monkey.
Chemokines are a family of small cytokines involved in the recruitment and activation of a large variety of cell types. They were originally identified because of their role in recruiting cells during inflammation, but have since been shown to be involved in such diverse activities as the orchestration of the immune response in routine immuno-surveillance, embryonic development and angiogenesis. Early studies showed that different leukocyte populations expressed different chemokine receptors, implying that chemokine receptor antagonists could potentially be highly selective anti-inflammatory molecules. The pharmaceutical interest in an antichemokine receptor strategy expanded with the discovery that two chemokine receptors are also critical for the pathway by which HIV invades host cells. In this review, we discuss the recent progress made in identifying potential therapeutics that can modulate chemokine networks, starting with antibodies, modified chemokines and chemokine binding proteins. Over the last two years the patent literature on non-peptidic small molecule antagonists has started to mature. We review the state of the art, and speculate on the future directions of this exciting field.
Ligands for the CCR1 receptor (MIP-1α and RANTES) have been implicated in a number of chronic inflammatory diseases, most notably multiple sclerosis and rheumatoid arthritis. Because these ligands share a common receptor, CCR1, we sought to discover antagonists for this receptor as an approach to treating these disorders. A novel series of 4-hydroxypiperidines has been discovered by high throughput screening (HTS) which potently inhibits the binding of MIP-1α and RANTES to the recombinant human CCR1 chemokine receptor. The structure−activity relationships of various segments of this template are described as the initial HTS lead 1 was optimized synthetically to the highly potent receptor antagonist 6s. This compound has been shown to have at least 200-fold selectivity for inhibition of CCR1 over other human 7-TM receptors, including other chemokine receptors. In addition, data obtained from in vitro functional assays demonstrate the functional antagonism of compound 6s and structurally related analogues against the CCR1 receptor in a concentration dependent manner. The discovery and optimization of potent and selective CCR1 receptor antagonists represented by compound 6s potentially represent a novel approach to the treatment of chronic inflammatory diseases.
In an effort to determine the role of the acidic group in the receptor binding ofN-(2-hydroxy-4-nitrophenyl)-N′-(phenyl) urea, an interleukin-8B receptor antagonist, its binding and that of several analogs was measured as a function
of pH. These titrations indicate that these ureas bind most strongly in their anionic form. Studies of antagonists, with different
acidities, demonstrated that the greatest change in binding of each urea occurred around the pK
a
of the compound being examined. The studies suggest that the increase in binding of the antagonists at higher pH is a result
of the increased negative charge on the compounds rather than the effects of pH on the receptor or radioligand.
The classic signs of acute cellular rejection during organ transplantation include the infiltration of mononuclear cells into the interstitium. This recruitment of leukocytes into the transplanted tissue is promoted by chemokines like RANTES. Since RANTES is a potent agonist for the CC chemokine receptor CCR1, we examined whether the CCR1 antagonist BX 471 was efficacious in a rabbit kidney transplant rejection model. BX 471 was able to compete with high affinity with the CCR1 ligands MIP-1α and RANTES for binding to HEK 293 cells expressing rabbit CCR1. BX 471 was a competitive antagonist of rabbit CCR1 in Ca2+ flux studies. Two separate studies in which animals were subcutaneously implanted with slow release pellets of BX 471 demonstrated that animals implanted with BX 471 had increased survival compared with untreated controls or animals implanted with placebo. The mean survival time for the placebo group was 12.33±1.7 days. The animals in the BX 471 treated group had mean survival times of 16.9±2.1 and 16.0±1.7 days, respectively, for the two studies. Analysis of the combined data by Student t-test gave a P value of 0.03 that is significant at the 0.05 level. In addition, there was a marked reduction in the urea and creatinine levels in the BX 471 treated animals compared with the control and placebo groups in both studies. Finally, pathologic analysis of the kidneys in the rabbit renal transplantation model from animals in the different groups showed that BX 471 was similar to cyclosporin in its ability to prevent extensive infarction of transplanted kidneys. Based on the data from these studies, BX 471 shows clear efficacy at the single dose tested compared with animals treated with placebo.
T-cell priming occurs in the lymphoid tissue via T cell–dendritic cell interaction. Conversely, delayed-type hypersensitivity or allergic reactions can occur in any tissue, following interaction of T helper 1 (Th1) or Th2 cells with effector leukocytes. Here, Federica Sallusto and colleagues review the role played by chemokines and chemokine receptors in positioning T cells for the immune response.
Interleukin-8 (IL-8) is a member of a family of pro-inflammatory cytokines. Although the best characterized activities of IL-8 include the chemoattraction and activation of neutrophils, other members of this family have a wide range of specific actions including the chemotaxis and activation of monocytes, the selective chemotaxis of memory T cells, the inhibition of hematopoietic stem cell proliferation, and the induction of neutrophil infiltration in vivo. A complementary DNA encoding the IL-8 receptor from human neutrophils has now been isolated. The amino acid sequence shows that the receptor is a member of the superfamily of receptors that couple to guanine nucleotide binding proteins (G proteins). The sequence is 29% identical to that of receptors for the other neutrophil chemoattractants, fMet-Leu-Phe and C5a. Mammalian cells transfected with the IL-8 receptor cDNA clone bind IL-8 with high affinity and respond specifically to IL-8 by transiently mobilizing calcium. The IL-8 receptor may be part of a subfamily of related G protein-coupled receptors that transduce signals for the IL-8 family of pro-inflammatory cytokines.
Interleukin-8 (IL-8) is an inflammatory cytokine that activates neutrophil chemotaxis, degranulation, and the respiratory
burst. Neutrophils express receptors for IL-8 that are coupled to guanine nucleotide-binding proteins (G proteins); binding
of IL-8 to its receptor induces the mobilization of intracellular calcium stores. A cDNA clone from HL-60 neutrophils, designated
p2, has now been isolated that encodes a human IL-8 receptor. When p2 is expressed in oocytes from Xenopus laevis, the oocytes
bind 125I-labeled IL-8 specifically and respond to IL-8 by mobilizing calcium stores with an EC50 of 20 nM. This IL-8 receptor
has 77% amino acid identity with a second human neutrophil receptor isotype that binds IL-8 with higher affinity. It also
exhibits 69% amino acid identity with a protein reported to be an N-formyl peptide receptor from rabbit neutrophils, but less
than 30% identity with all other known G protein-coupled receptors, including the human N-formyl peptide receptor.
The Duffy blood group antigen has been postulated to be a receptor on red blood cells (RBCs) for the malarial parasite Plasmodium vivax and a promiscuous receptor for the chemokine superfamily of inflammatory proteins. Recently, the Duffy antigen glycoprotein D cDNA has been cloned (Chaudhuri et al: Proc Natl Acad Sci USA 90:10793, 1993). We have analyzed the binding properties of the cloned Duffy antigen. Duffy-antigen cDNAs expressed in human embryonic kidney cells produced cell-surface proteins that reacted with two known anti-Duffy monoclonal antibodies. Direct ligand binding and displacement experiments using recombinant chemokine proteins also show that the cloned Duffy protein is the RBC chemokine receptor. Radiolabeled chemokines of both the C-C (RANTES and MCP-1) and C-X-C (IL-8 and MGSA/gro) subclasses bound reversibly to transfected cells with dissociation constants in the nanomolar range. Chemokines of either class displaced heterologous chemokines, indicating that they were competing for a single site on the transfected cells. Although the chemokines bound to the transfected cells with high affinity, there was no evidence for signal transduction, as measured by transient increases in intracellular calcium ion concentration, through the Duffy antigen/RBC chemokine receptor in transfected cells. Lastly, we have performed a computer analysis on the amino acid structure of the Duffy antigen/RBC chemokine receptor. Although the cloned Duffy antigen has been postulated to be a nine-transmembrane-spanning receptor, our analysis suggests that the molecule most likely belongs to the seven-transmembrane-spanning receptor superfamily and is therefore similar to other chemokine receptors previously identified.
The chemokine family of chemotactic cytokines plays a key role in orchestrating the immune response. The family has been divided into 2 subfamilies, alpha and beta, based on the spacing of the first 2 cysteine residues, function, and chromosomal location. Members within each subfamily have 25-70% sequence identity, whereas the amino acid identity between members of the 2 subfamilies ranges from 20 to 40%. A quantitative analysis of the hydrophobic properties of 11 alpha and 9 beta chemokine sequences, based on the coordinates of the prototypic alpha and beta chemokines, interleukin-8 (IL-8), and human macrophage inflammatory protein-1 beta (hMIP-1 beta), respectively, is presented. The monomers of the alpha and beta chemokines have their strongest core hydrophobic cluster at equivalent positions, consistent with their similar tertiary structures. In contrast, the pattern of monomer surface hydrophobicity between the alpha and beta chemokines differs in a manner that is fully consistent with the observed differences in quaternary structure. The most hydrophobic surface clusters on the monomer subunits are located in very different regions of the alpha and beta chemokines and comprise in each case the amino acids that are buried at the interface of their respective dimers. The theoretical analysis of hydrophobicity strongly supports the hypothesis that the distinct dimers observed for IL-8 and hMIP-1 beta are preserved for all the alpha and beta chemokines, respectively. This provides a rational explanation for the lack of receptor crossbinding and reactivity between the alpha and beta chemokine subfamilies.
The three-dimensional structure of a member of the beta subfamily of chemokines, human macrophage inflammatory protein-1 beta (hMIP-1 beta), has been determined with the use of solution multidimensional heteronuclear magnetic resonance spectroscopy. Human MIP-1 beta is a symmetric homodimer with a relative molecular mass of approximately 16 kilodaltons. The structure of the hMIP-1 beta monomer is similar to that of the related alpha chemokine interleukin-8 (IL-8). However, the quaternary structures of the two proteins are entirely distinct, and the dimer interface is formed by a completely different set of residues. Whereas the IL-8 dimer is globular, the hMIP-1 beta dimer is elongated and cylindrical. This provides a rational explanation for the absence of cross-binding and reactivity between the alpha and beta chemokine subfamilies. Calculation of the solvation free energies of dimerization suggests that the formation and stabilization of the two different types of dimers arise from the burial of hydrophobic residues.
Antagonists of multiple chemokines could be more effective than inhibitors of specific chemokines for controlling cell migration and inflammation. To attempt to identify such antagonists we characterized a number of truncated analogs of regulated on activation normal T cell expressed protein (RANTES), monocyte chemoattractant protein (MCP)-3, and MCP-1. On the basis of their ability to compete for binding of their parent chemokines, three analogs were selected for cross-reactivity studies: RANTES (9-68), MCP-3 (10-76), and MCP-1 (9-76). These analogs bound to THP-1 monocytic cells with dissociation constants that were within 4-6-fold of their native counterparts, but they did not promote detectable chemotaxis of THP-1 cells or enzyme release from purified human monocytes. The RANTES (9-68) analog competed for the binding and inhibited the activities of all three chemokines. In contrast, native RANTES was specific for RANTES binding sites. However, truncation of either MCP-1 or MCP-3 did not change their respective binding specificity. MCP-3 and MCP-3 (10-76) competed for binding of all three labeled chemokines. MCP-1 (9-76) competed strongly for binding of labeled MCP-1, but only weakly for the other two labeled ligands and inhibited the activities induced by MCP-1 and MCP-3 but not RANTES. Although RANTES (9-68) and MCP-3 (10-76) inhibited all three chemokines, the RANTES analog was significantly more potent for RANTES-induced activity. The results indicate that NH2-terminal residues partly determine the receptor specificity of RANTES, and deletions within this region permit binding to multiple chemokine receptors. The findings suggest the feasibility of design of high affinity multi-specific CC chemokine antagonists.
The chemokine receptors CXCR4 and CCR5 have recently been shown to act as coreceptors, in concert with CD4, for human immunodeficiency
virus–type 1 (HIV-1) infection. RANTES and other chemokines that interact with CCR5 and block infection of peripheral blood
mononuclear cell cultures inhibit infection of primary macrophages inefficiently at best. If used to treat HIV-1–infected
individuals, these chemokines could fail to influence HIV replication in nonlymphocyte compartments while promoting unwanted
inflammatory side effects. A derivative of RANTES that was created by chemical modification of the amino terminus, aminooxypentane
(AOP)–RANTES, did not induce chemotaxis and was a subnanomolar antagonist of CCR5 function in monocytes. It potently inhibited
infection of diverse cell types (including macrophages and lymphocytes) by nonsyncytium-inducing, macrophage-tropic HIV-1
strains. Thus, activation of cells by chemokines is not a prerequisite for the inhibition of viral uptake and replication.
Chemokine receptor antagonists like AOP-RANTES that achieve full receptor occupancy at nanomolar concentrations are strong
candidates for the therapy of HIV-1–infected individuals.
Kaposi's sarcoma–associated herpesvirus encodes a chemokine called vMIP-II. This protein displayed a broader spectrum of receptor
activities than any mammalian chemokine as it bound with high affinity to a number of both CC and CXC chemokine receptors.
Binding of vMIP-II, however, was not associated with the normal, rapid mobilization of calcium from intracellular stores;
instead, it blocked calcium mobilization induced by endogenous chemokines. In freshly isolated human monocytes the virally
encoded vMIP-II acted as a potent and efficient antagonist of chemotaxis induced by chemokines. Because vMIP-II could inhibit
cell entry of human immunodeficiency virus (HIV) mediated through CCR3 and CCR5 as well as CXCR4, this protein may serve as
a lead for development of broad-spectrum anti-HIV agents.
The bicyclam AMD3100 (formula weight 830) blocks HIV-1 entry and membrane fusion via the CXCR4 co-receptor, but not via CCR5. AMD3100 prevents monoclonal antibody 12G5 from binding to CXCR4, but has no effect on binding of monoclonal antibody 2D7 to CCR5. It also inhibits binding of the CXC-chemokine, SDF-1alpha, to CXCR4 and subsequent signal transduction, but does not itself cause signaling and has no effect on RANTES signaling via CCR5. Thus, AMD3100 prevents CXCR4 functioning as both a HIV-1 co-receptor and a CXC-chemokine receptor. Development of small molecule inhibitors of HIV-1 entry is feasible.
BB-10010 is a variant of the human form of macrophage inflammatory protein-1alpha (MIP-1alpha), which has been shown in mice to block the entry of hematopoietic stem cells into S-phase and to increase their self-renewal capacity during recovery from cytotoxic damage. Its use may constitute a novel approach for protecting the quality of the stem cell population and its capacity to regenerate after periods of cytotoxic treatment. Thirty patients with locally advanced or metastatic breast cancer were entered into the first randomized, parallel group controlled phase II study. This was designed to evaluate the potential myeloprotective effects of a 7-day regimen of BB-10010 administered to patients receiving six cycles of 5-fluorouracil (5-FU), adriamycin, and cyclophosphamide (FAC) chemotherapy. Patients were randomized, 10 receiving 100 microgram/kg BB-10010, 11 receiving 30 microgram/kg BB-10010, and nine control patients receiving no BB-10010. BB-10010 was well-tolerated in all patients with no severe adverse events related to the drug. Episodes of febrile neutropenia complicated only 4% of the treatment cycles and there was no difference in incidence between the treated and nontreated groups. Studies to assess the generation of progenitor cells in long-term bone marrow cultures were performed immediately preceding chemotherapy and at the end of six dosing cycles in 18 patients. Circulating neutrophils, platelets, CD 34(+) cells, and granulocyte/macrophage colony-forming cell (GM-CFC) levels were determined at serial time points in cycles 1, 3, and 6. The results showed similar hemoglobin and platelet kinetics in all three groups. On completion of the six treatment cycles, the average pretreatment neutrophil levels were reduced from 5.3 to 1.7 x 10(9)/L in the control patients and from 4.3 to 1.9 and 4.5 to 2.5 x 10(9)/L in the 30/100 microgram/kg BB-10010 groups, respectively. Relative to their pretreatment values, 50% of the patients receiving BB-10010 completed the treatment with neutrophil values significantly higher than any of the controls (P = .02). Mobilization of GM-CFC was enhanced by BB-10010 with an additional fivefold increase over that generated by chemotherapy alone, giving a maximal 25-fold increase over pretreatment values. Bone marrow progenitor assays before and after this standard regimen of chemotherapy indicated little long-term cumulative impairment to recovery from chemotherapy. Despite the limited cumulative damage to the bone marrow, which may have minimized the protective value of BB-10010 during this regimen of chemotherapy, better recovery of neutrophils in the later treatment cycles with BB-10010 was indicated in a number of patients.
This study demonstrates that several CC-chemokines, including those that inhibit entry and replication of macrophage-tropic strains of HIV, increase the replication of T cell (T)-tropic strains in CD4(+) T cells. Enhancement of T-tropic HIV replication is observed at early stages of replication, requires signaling through inhibitory guanine nucleotide-binding regulatory (Gi) proteins, and is associated with increased cell surface colocalization of CD4 and the T-tropic HIV coreceptor CXCR4. These findings may further our understanding of the factors that influence the replication and spread of T-tropic strains of HIV in vivo and suggest that the use of cell signaling CC-chemokines as therapeutic agents for the purpose of limiting HIV replication in vivo should be approached with caution.
HIV-1 entry into cells is an attractive target for new antiviral agents. But will inhibitors aimed at the CCR5 co-receptor force HIV-1 to evolve more virulent forms?