Melior Discovery

Acne vulgaris (acne) effects nearly 90% of all Western teenagers, and the only pharmaceutical class of agents to treat severe forms of this skin condition are the retinoids, which are well‐described teratogens. Yet about 50% of the patients receiving this class of therapeutics are women of child‐bearing age, in their peak years of reproductive potential. On this basis, there is a significant unmet medical need for agents to treat severe forms of acne that do not carry this liability. As a means to assess potential agents of this type, here we describe methods for estimating the relative amount of sebum that a mouse produces based on the water retention on fur following a thorough wetting procedure. We have shown that a compound that is clinically effective in reducing sebum production demonstrates activity in this model. The method is therefore useful for evaluating therapeutic candidates for reducing sebum production, which would in turn be useful for treating acne. We have broken the entire procedure down into two phases/two protocols, as listed below. © 2024 Wiley Periodicals LLC. Basic Protocol 1 : Pre‐wash wet weight measurement Basic Protocol 2 : Post‐wash wet‐weight measurement

A target-based drug discovery strategy has led to a bias away from low molecular weight (MWT) drug discovery. Analysis of the ACS chemistry registration system shows that most low MWT drugs were first made in the time era before target-based drug discovery. Therapeutic activity among most low MWT drugs was identified in the era of phenotypic drug discovery when drugs were selected based on their phenotypic effects and before in vitro screening, mechanism of action considerations and experiences with fragment screening became known. The common perception that drugs cannot be found among low MWT compounds is incorrect based on both drug discovery history and our own experience with MLR-1023. The greater proportion of low MWT compounds that are commercially available compared to higher MWT compounds is a factor that should facilitate biology study. We posit that low MWT compounds are more suited to identification of new therapeutic activity using phenotypic screens provided that the phenotypic screening method has enough screening capacity. On-target and off-target therapeutic activities are discussed from both a chemistry and biology perspective because of a concern that either phenotypic or low MWT drug discovery might bias towards promiscuous compounds that combine on-target and off-target effects. Among ideal drug repositioning candidates (late-stage pre-clinical or clinically-experience compounds), pleiotropic activity (multiple therapeutic actions) is far more likely due to on-target effects arising where a single target mediates multiple therapeutic benefits, a desirable outcome for drug development purposes compared to the off-target alternative. Our exemplar of a low MWT compound, MLR-1023, discovered by phenotypic screening and subsequently found to have a single mechanism of action would have been overlooked based on current era medicinal chemistry precedent. The diverse therapeutic activities described for this compound by us, and others arise from the same pleiotropic lyn kinase activation molecular target. MLR-1023 serves as a proof-of-principle that potent, on target, low MWT drugs can be discovered by phenotypic screening.

Alcohol use disorder (AUD) and its sequelae impose a major burden on the public health of the United States, and adequate long-term control of this disorder has not been achieved. Molecular and behavioral basic science research findings are providing the groundwork for understanding the mechanisms underlying AUD and have identified multiple candidate targets for ongoing clinical trials. However, the translation of basic research or clinical findings into improved therapeutic approaches for AUD must become more efficient. Translational research is a multistage process of stream-lining the movement of basic biomedical research findings into clinical research and then to the clinical target populations. This process demands efficient bidirectional communication across basic, applied, and clinical science as well as with clinical practitioners. Ongoing work suggests rapid progress is being made with an evolving translational framework within the alcohol research field. This is helped by multiple interdisciplinary collaborative research structures that have been developed to advance translational work on AUD. Moreover, the integration of systems biology approaches with collaborative clinical studies may yield novel insights for future translational success. Finally, appreciation of genetic variation in pharmacological or behavioral treatment responses and optimal communication from bench to bedside and back may strengthen the success of translational research applications to AUD.

Purpose: MET, the receptor for hepatocyte growth factor (HGF), has been implicated in driving tumor proliferation and metastasis. High MET expression is correlated with poor prognosis in multiple cancers. Activation of MET can be induced either by HGF-independent mechanisms such as gene amplification, specific genetic mutations, and transcriptional upregulation or by HGF-dependent autocrine or paracrine mechanisms. Experimental design/results: Here, we report on LY2875358, a novel humanized bivalent anti-MET antibody that has high neutralization and internalization activities, resulting in inhibition of both HGF-dependent and HGF-independent MET pathway activation and tumor growth. In contrast to other bivalent MET antibodies, LY2875358 exhibits no functional agonist activity and does not stimulate biologic activities such as cell proliferation, scattering, invasion, tubulogenesis, or apoptosis protection in various HGF-responsive cells and no evidence of inducing proliferation in vivo in a monkey toxicity study. LY2875358 blocks HGF binding to MET and HGF-induced MET phosphorylation and cell proliferation. In contrast to the humanized one-armed 5D5 anti-MET antibody, LY2875358 induces internalization and degradation of MET that inhibits cell proliferation and tumor growth in models where MET is constitutively activated. Moreover, LY2875358 has potent antitumor activity in both HGF-dependent and HGF-independent (MET-amplified) xenograft tumor models. Together, these findings indicate that the mechanism of action of LY2875358 is different from that of the one-armed MET antibody. Conclusions: LY2875358 may provide a promising therapeutic strategy for patients whose tumors are driven by both HGF-dependent and HGF-independent MET activation. LY2875358 is currently being investigated in multiple clinical studies.

The serine hydrolase α/β-hydrolase domain 6 (ABHD6) hydrolyzes the most abundant endocannabinoid (eCB) in the brain, 2-arachidonoylglycerol (2-AG), and controls its availability at cannabinoid receptors. We show that ABHD6 inhibition decreases pentylenetetrazole (PTZ)-induced generalized tonic-clonic and myoclonic seizure incidence and severity. This effect is retained in Cnr1(-/-) or Cnr2(-/-) mice, but blocked by addition of a subconvulsive dose of picrotoxin, suggesting the involvement of GABAA receptors. ABHD6 inhibition also blocked spontaneous seizures in R6/2 mice, a genetic model of juvenile Huntington's disease known to exhibit dysregulated eCB signaling. ABHD6 blockade retained its antiepileptic activity over chronic dosing and was not associated with psychomotor or cognitive effects. While the etiology of seizures in R6/2 mice remains unsolved, involvement of the hippocampus is suggested by interictal epileptic discharges, increased expression of vGLUT1 but not vGAT, and reduced Neuropeptide Y (NPY) expression. We conclude that ABHD6 inhibition may represent a novel antiepileptic strategy.

Pregabalin has been shown to have anticonvulsant, analgesic, and anxiolytic activity in animal models. Pregabalin binds with high affinity to the α2δ1 and α2δ2 subunits of voltage-gated calcium channels. In order to better understand the relative contribution that binding to either the α2δ1 or α2δ2 subunits confers on the anticonvulsant activity of pregabalin, we characterized the anticonvulsant activity of pregabalin in different wild-type (WT) and mutant mouse strains. Two targeted mouse mutants have been made in which either the α2δ1 subunit was mutated (arginine-to-alanine mutation at amino acid 217; R217A) or the α2δ2 subunit was mutated (arginine-to-alanine mutation at amino acid 279; R279A). These mutations in α2δ1 or α2δ2 render the subunits relatively insensitive to pregabalin binding. The anticonvulsant activity of pregabalin was assessed in these different mouse lines using the maximal electroshock-induced seizure (MES) model. Pregabalin reduced the percentage of seizures and increased the latency to seizure in the MES model in two parental mouse strains used to construct the mutants. Pregabalin also reduced the percentage of seizures and increased latency to seizure similarly in the α2δ2 (R279A) and WT littermate control mice. In contrast, pregabalin's anticonvulsant efficacy was significantly reduced in α2δ1 (R217A) mutants compared with WT littermate control mice. Phenytoin showed anticonvulsant activity across all WT and mutant mice. These data show that the anticonvulsant activity of pregabalin in the MES model requires binding to the α2δ1 subunit.

Views about collaborative drug discovery changed from caution to advocacy in a few years. Milestones were the 2004 NIH Molecular Libraries Screening Center Network and library with screening data deposited to Pubchem; the 2008 Wellcome Trust transfer of proprietary chemogenomic data to the public; and in 2008, the formalization of collaborative efforts in the Clinical and Translational Science Pharmaceutical Assets Award portal. The biology/medicinal chemistry interface is difficult in academic drug discovery, with its wide range in academic drug discovery skills sets. Academics talk about innovation, thinking out of the box, maximum chemical diversity, and not being limited by preconceived rules and filters. Industry people talk about pragmatism, lessons learned, and about worthless screening compounds. Chemistry space errors impede academic drug discovery and collaborations. Screening diverse libraries is the worst way to discover a drug. Biologically active compounds occur in small tight clusters infrequently through chemistry space. Currently, well-known problematic functionality is replaced with more subtle problem compounds. Medicinal chemistry quality suffers when the academic choice is publish or perish. Hypothesis-driven research is a concern for future collaborative drug discovery because biology research driven by hypothesis can often be wrong. Complex natural products cannot be analyzed because the shapes are uncertain. This hinders exploitation of natural products in drug discovery and chemical biology.

Background Trilostane was identified in an in vivo screen of compounds in a lipopolysaccharide model of inflammation to support a repurposing effort. There is no previous documentation of any anti-inflammatory effects of trilostane. Objective The aim of this study was to elucidate the novel pharmacologic activity of trilostane in a series of inflammation and nociception signal-finding models. Methods Anti-inflammatory effects of trilostane were evaluated in lipopolysaccharide-induced systemic and lung inflammation models and in a 2,4-dinitrofluorobenzene–induced delayed-type hypersensitivity (DTH) model in the mouse ear. The analgesic activities of trilostane were evaluated in a hot plate nociception model as a function of paw-withdrawal latency and in the formalin-induced nociception model with a behavioral end point. In all studies, trilostane was administered 15 minutes before challenge. In the DTH model, the animals were given a second dose 24 hours after the first dose. Results Trilostane inhibited tumor necrosis factor-α and monocyte chemoattractant protein-1 production in the lipopolysaccharide-induced systemic and pulmonary inflammation models. It also significantly reduced ear swelling in the 2,4-dinitrofluorobenzene–induced DTH model. In the hot plate nociception model, trilostane increased the latency of paw-licking behavior. Trilostane also significantly reduced the duration of pain behaviors in the late phase of the formalin-induced inflammatory pain model. Conclusions These signal-finding studies suggest that trilostane has novel anti-inflammatory and analgesic properties.

The GABAB receptor agonist baclofen reduces urethral resistance and detrusor overactivity in patients with spasticity. However, baclofen's side effects limit its use for the treatment of overactive bladder (OAB). Here we tested a novel GABAB positive allosteric modulator (PAM) ADX71441 in models of OAB in mice and guinea pigs. Mice were left untreated or given (p.o.) vehicle (1% CMC), ADX71441 (1, 3, 10 mg kg(-1) ) or oxybutynin (100 mg kg(-1) ; Experiment 1) or vehicle (1% CMC), baclofen (1, 3, 6 mg kg(-1) ) or oxybutynin (Experiment 2). Treated mice were then overhydrated with water, challenged with furesomide, before being placed into micturition chambers and monitored for urinary parameters. In anesthetized guinea pigs, intravesical infusion of acetic acid was used to induce OAB. Then, ADX71441 (1, 3 mg kg(-1) ) or baclofen (1 mg kg(-1) ) were administered i.v. and their effects on cystometric parameters were monitored. In mice, 10 mg kg(-1) ADX71441 increased urinary latencies, while reducing the number of urinary events and the total and average urinary volumes. In guinea pigs, ADX71441 (1 and 3 mg kg(-1) ) increased intercontraction interval (ICI) and bladder capacity (BC), while reducing micturition frequency (MF) compared to vehicle. At 3 mg kg(-1) ADX71441 completely inhibited the micturition reflex and induced overflow incontinence in 5 out of 10 animals. Baclofen slightly increased ICI and BC while reducing MF. This is the first evidence that GABAB PAM has a potential as a novel approach for the treatment of OAB.

A series of novel, single chain insulin analogs containing polyethylene glycol based connecting segments were synthesized by native chemical ligation and tested for biological activity. While the full length single chain insulin analogs exhibited low potency, deletion of amino acids B26–B30 unexpectedly generated markedly higher activity. This observation is unprecedented in all previous studies of single chain insulin analogs and is consistent with the presumption that in the native hormone this sequence must translocate to achieve high potency insulin receptor interaction. Optimization of the sequence yielded an insulin analog with potency and selectivity comparable to that of native insulin. These results establish a basis for discovery of novel higher potency, single chain insulin analogs of shortened length.

2(1H)-pyrimidinone,5-(3-methylphenoxy) (MLR-1023) is a candidate for the treatment of type 2 diabetes. The current studies were aimed at determining the mechanism by which MLR-1023 mediates glycemic control. In these studies, we showed that MLR-1023 reduced blood glucose levels without increasing insulin secretion in vivo. We have further determined that MLR-1023 did not activate peroxisome proliferator-activated α, δ, and γ receptors or glucagon-like peptide-1 receptors or inhibit dipeptidyl peptidase-4 or α-glucosidase enzyme activity. However, in an in vitro broad kinase screen MLR-1023 activated the nonreceptor-linked Src-related tyrosine kinase Lyn. MLR-1023 increased the V(max) of Lyn with an EC(50) of 63 nM. This Lyn kinase activation was ATP binding site independent, indicating that MLR-1023 regulated the kinase through an allosteric mechanism. We have established a link between Lyn activation and blood glucose lowering with studies showing that the glucose-lowering effects of MLR-1023 were abolished in Lyn knockout mice, consistent with existing literature linking Lyn kinase and the insulin-signaling pathway. In summary, these studies describe MLR-1023 as a unique blood glucose-lowering agent and show that MLR-1023-mediated blood glucose lowering depends on Lyn kinase activity. These results, coupled with other results (J Pharmacol Exp Ther 342:23-32, 2012), suggest that MLR-1023 and Lyn kinase activation may be a new treatment modality for type 2 diabetes.

MLR-1023 [Tolimidone; CP-26154; 2(1H)-pyrimidinone, 5-(3-methylphenoxy)] is an allosteric Lyn kinase activator that reduces blood glucose levels in mice subjected to an oral glucose tolerance test (J Pharmacol Exp Ther 342:15-22, 2012). The current studies were designed to define the role of insulin in MLR-1023-mediated blood glucose lowering, to evaluate it in animal models of type 2 diabetes, and to compare it to the activities of selected existing diabetes therapeutics. Results from these studies show that in an acute oral glucose tolerance test MLR-1023 evoked a dose-dependent blood glucose-lowering response that was equivalent in magnitude to that of metformin without eliciting a hypoglycemic response. In streptozotocin-treated, insulin-depleted mice, MLR-1023 administration did not affect blood glucose levels. However, MLR-1023 potentiated the glucose-lowering activity of exogenously administered insulin, showing that MLR-1023-mediated blood glucose lowering was insulin-dependent. In a hyperinsulinemic/euglycemic clamp study, orally administered MLR-1023 increased the glucose infusion rate required to sustain blood glucose levels, demonstrating that MLR-1023 increased insulin receptor sensitivity. In chronically treated db/db mice, MLR-1023 elicited a dose-dependent and durable glucose-lowering effect, reduction in HbA1c levels and preservation of pancreatic β-cells. The magnitude of effect was equivalent to that seen with rosiglitazone but with a faster onset of action and without causing weight gain. These studies show that MLR-1023 is an insulin receptor-potentiating agent that produces a rapid-onset and durable blood glucose-lowering activity in diabetic animals.

Drug repositioning is an emerging strategy for filling the innovation gap in the pharmaceutical industry. Successful drug repositioning examples are nearly always explained by observations made in clinical settings or in in vivo therapeutic models. We have established an in vivo phenotypic platform, termed theraTRACE® that systematically evaluates and repositions drugs to new indications. This review article describes examples of drugs found using phenotypic models and the concepts behind the theraTRACE® approach.

Repaglinide is an FDA-approved treatment for type 2 diabetes mellitus. The anti-inflammatory effect of repaglinide in the absence of diabetes has not been reported previously. It is the objective of this set of studies to investigate the potential anti-inflammatory effects of repaglinide. The in vivo anti-inflammatory effects of repaglinide were studied in two different models of delay type hyperreactivity (DTH) response induced by sheep red blood cells (sRBC) and 2,5'-dinitrofluorobenzene (DNFB), and in two different rodent models of lipopolysaccharide (LPS) challenge. In mice systemically sensitized with sRBC, which subsequently received a local injection of sRBC in the footpad, local swelling occurred within 24 h after challenge. Repaglinide was efficacious in attenuating this response. In an orthogonal DTH model using DNFB as the antigen, the animals received topical sensitization with DNFB on their shaved backs, followed by topical challenge on the left ears. Repaglinide efficaciously downregulated the resulting ear swelling response. In mice challenged systemically or intratracheally with LPS, repaglinide significantly decreased serum tumor necrosis factor α level and bronchial alveolar lavage fluid MCP-1 levels, respectively. This set of data suggests novel anti-inflammatory effects of repaglinide in nondiabetic animals. However, the high dose required for an efficacious effect would make this application impractical in the clinic.

To investigate the effects of the second generation antipsychotic (R/S)-amisulpride, and the chirally purified enantiomers, on glucose homeostasis in diet-induced obese (DIO) mice. Normal and DIO mice were treated with pharmacologically relevant doses of amisulpride prior to oral glucose tolerance tests (OGTTs). Blood glucose, insulin, glucagon-like peptide-1, prolactin and amisulpride drug levels were determined. Racemic amisulpride significantly reduced glucose excursions during OGTT in both normal and DIO mice. This potent effect was preserved with the 'off-isomer', R-amisulpride (ED(50) 1 mg/kg). Insulin secretion was significantly increased with R-amisulpride with only a minor increase in prolactin levels. Amisulpride has antidiabetic actions in DIO mice resulting from increased insulin secretion. This provides some explanation for why amisulpride, unlike other atypical antipsychotics, is not diabetogenic in man. Furthermore, the observation that R-amisulpride is also antidiabetic and has minimal impact on prolactin levels presents the opportunity for development of this isomer as an antidiabetic agent.

Bortezomib (Velcade®) is a proteasome inhibitor that has been approved for the treatment of multiple myeloma and mantle cell lymphoma. It has been shown to inhibit the expression of cell adhesion molecules, co-stimulatory molecules, and NFκB activation, to deplete alloreactive T lymphocytes, and to decrease Th1 cytokine production. The anti-inflammatory effects of bortezomib were further investigated in this current set of studies. Systemic treatment with bortezomib was efficacious in the thioglycolate-induced MCP-1 production model, and the dinitrofluorobenzene-induced delayed-type hypersensitivity model. Psoriasis is an autoimmune disease that affects about 2% of the world population. Many treatments have been reported with varying degrees of efficacy. A topical bortezomib formulation was developed to minimize systemic exposure. Its tolerability was investigated in a topical imiquimod (IMQ)-induced psoriasis model. Daily application of IMQ on mouse skin induced inflamed scaly skin lesions resembling plaque-type psoriasis. Fatality was observed in the 1-mg/ml dose group. At 0.1 and 0.01 mg/ml, bortezomib potentiated IMQ-induced erythema, scaling, skin thickening, and caused necrotic lesions. Lower doses had no effect on the clinical observations. Histologically, bortezomib dose-dependently increased parakeratosis, hyperkeratosis, acanthosis, and inflammatory cell infiltration. This study demonstrated that topical bortezomib is not suitable for the treatment of psoriasis.

Hedgehog (Hh) signaling is critical to the patterning and development of a variety of organ systems, and both ligand-dependent and ligand-independent Hh pathway activation are known to promote tumorigenesis. Recent studies have shown that in tumors promoted by Hh ligands, activation occurs within the stromal microenvironment. Testing whether ligand-driven Hh signaling promotes tumor angiogenesis, we found that Hh antagonism reduced the vascular density of Hh-producing LS180 and SW480 xenografts. In addition, ectopic expression of sonic hedgehog in low-Hh-expressing DLD-1 xenografts increased tumor vascular density, augmented angiogenesis, and was associated with canonical Hh signaling within perivascular tumor stromal cells. To better understand the molecular mechanisms underlying Hh-mediated tumor angiogenesis, we established an Hh-sensitive angiogenesis coculture assay and found that fibroblast cell lines derived from a variety of human tissues were Hh responsive and promoted angiogenesis in vitro through a secreted paracrine signal(s). Affymetrix array analyses of cultured fibroblasts identified VEGF-A, hepatocyte growth factor, and PDGF-C as candidate secreted proangiogenic factors induced by Hh stimulation. Expression studies of xenografts and angiogenesis assays using combinations of Hh and VEGF-A inhibitors showed that it is primarily Hh-induced VEGF-A that promotes angiogenesis in vitro and augments tumor-derived VEGF to promote angiogenesis in vivo.