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Although many cellular processes depend upon enzymatic reactions, protein-protein interactions (PPIs) mediate a large number of important regulatory pathways and thus play a central role in disease development. In order to understand and selectively inhibit cellular signalling pathways, there is a pressing need for small molecules that target PPIs,...
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Citations
... This work was motivated by the benefits that peptides provide, such as modularity, scalability, favorable pharmacokinetics, and low immunogenicity [26]. Small peptide conjugates can be designed to give effective nuclear or optical readouts of cell surface or intracellular proteins; these probes can also potentiate imaging of dynamic cellular processes [27]. Specifically, we focused on identifying, designing, synthesizing, and evaluating peptide conjugates specific for CD38, which is a clinically viable target for MM and other pathologies. ...
Purpose
Multiple myeloma (MM) affects over 35,000 patients each year in the US. There remains a need for versatile Positron Emission Tomography (PET) tracers for the detection, accurate staging, and monitoring of treatment response of MM that have optimal specificity and translational attributes. CD38 is uniformly overexpressed in MM and thus represents an ideal target to develop CD38-targeted small molecule PET radiopharmaceuticals to address these challenges.
Procedures
Using phage display peptide libraries and pioneering algorithms, we identified novel CD38 specific peptides. Imaging bioconjugates were synthesized using solid phase peptide chemistry, and systematically analyzed in vitro and in vivo in relevant MM systems.
Results
The CD38-targeted bioconjugates were radiolabeled with copper-64 ( ⁶⁴ Cu) with100% radiochemical purity and an average specific activity of 3.3 – 6.6 MBq/nmol. The analog NODAGA-PEG4-SL022-GGS (SL022: Thr-His-Tyr-Pro-Ile-Val-Ile) had a K d of 7.55 ± 0.291 nM and was chosen as the lead candidate. ⁶⁴ Cu-NODAGA-PEG4-SL022-GGS demonstrated high binding affinity to CD38 expressing human myeloma MM.1S-CBR-GFP-WT cells, which was blocked by the non-radiolabeled version of the peptide analog and anti-CD38 clinical antibodies, daratumumab and isatuximab, by 58%, 73%, and 78%, respectively. The CD38 positive MM.1S-CBR-GFP-WT cells had > 68% enhanced cellular binding when compared to MM.1S-CBR-GFP-KO cells devoid of CD38. Furthermore, our new CD38-targeted radiopharmaceutical allowed visualization of tumors located in marrow rich bones, remaining there for up to 4 h. Clearance from non-target organs occurred within 60 min. Quantitative PET data from a murine disseminated tumor model showed significantly higher accumulation in the bones of tumor-bearing animals compared to tumor-naïve animals (SUV max 2.06 ± 0.4 versus 1.24 ± 0.4, P = 0.02). Independently, tumor uptake of the target compound was significantly higher (P = 0.003) compared to the scrambled peptide, ⁶⁴ Cu-NODAGA-PEG4-SL041-GGS (SL041: Thr-Tyr-His-Ile-Pro-Ile-Val). The subcutaneous MM model demonstrated significantly higher accumulation in tumors compared to muscle at 1 and 4 h after tracer administration (SUV max 0.8 ± 0.2 and 0.14 ± 0.04, P = 0.04 at 1 h; SUV max 0.89 ± 0.01 and 0.09 ± 0.01, P = 0.0002 at 4 h).
Conclusions
The novel CD38-targeted, radiolabeled bioconjugates were specific and allowed visualization of MM, providing a starting point for the clinical translation of such tracers for the detection of MM.
... For instance, Maraviroc is an FDA approved medicine against HIV infection [19]. The pharmacological effect of such medicine is mediated by blocking PPIs between HIV protein and cell membrane receptor CCR5 [19,20]. Therefore, identifying novel PPIs can help finding novel targets to interfere and block the pathological processes. ...
Protein–protein interactions (PPIs) involve the physical or functional contact between two or more proteins. Generally, proteins that can interact with each other always have special relationships. Some previous studies have reported that gene ontology (GO) terms are related to the determination of PPIs, suggesting the special patterns on the GO terms of proteins in PPIs. In this study, we explored the special GO term patterns on human PPIs, trying to uncover the underlying functional mechanism of PPIs. The experimental validated human PPIs were retrieved from STRING database, which were termed as positive samples. Additionally, we randomly paired proteins occurring in positive samples, yielding lots of negative samples. A simple calculation was conducted to count the number of positive samples for each GO term pair, where proteins in samples were annotated by GO terms in the pair individually. The similar number for negative samples was also counted and further adjusted due to the great gap between the numbers of positive and negative samples. The difference of the above two numbers and the relative ratio compared with the number on positive samples were calculated. This ratio provided a precise evaluation of the occurrence of GO term pairs for positive samples and negative samples, indicating the latent GO term patterns for PPIs. Our analysis unveiled several nuclear biological processes, including gene transcription, cell proliferation, and nutrient metabolism, as key biological functions. Interactions between major proliferative or metabolic GO terms consistently correspond with significantly reported PPIs in recent literature.
... A drug-like subset is rule-of-five 46,47 compliant and offers the opportunity to identify hits with high affinities while likely Nature reviews | Chemistry also having good drug properties. The third subset goes beyond the rule-of-five and is typically used to investigate challenging but promising targets like protein-protein interactions [48][49][50][51] . Being based on naturalproduct-inspired frameworks, the library features organic compounds with a high content of sp 3 -hybridized C atoms, an aspect that typically translates to improved medicinal chemistry properties 52 . ...
... The physico-chemical properties of this collection include a distribution of molar masses M in the range 275-650. This library can be stratified and screened separately as three subsets: lead-like 42,43,45 , drug-like 44 and a higher M subset used for disrupting proteinprotein interactions (PPIs) [48][49][50][51] . c | The fraction of C atoms that are sp 3 -hybridized (Fsp3) is centred at 0.45 and is considerably higher than traditional high-throughput screening collections, which should lead to hits with improved medicinal properties 52 . ...
Affinity selection mass spectrometry (AS-MS) has gained momentum in drug discovery. This review summarizes how this technology has slowly risen as a new paradigm in hit identification and its potential synergy with DNA encoded library technology. It presents an overview of the recent results on challenging targets and perspectives on new areas of research, such as RNA targeting with small molecules. The versatility of the approach is illustrated and strategic drivers discussed in terms of the experience of a small-medium CRO and a big pharma organization. Teaser: This review highlights the interest aroused by AS-MS as a screening technology, assessing the shared experience of a small-medium contract research organization (CRO) and a big pharma organization.
... [5] Stapled peptides have the ability to mimic one of the protein epitopes, bind to a topologically shallow surface of the protein partner, and thus selectively disrupt these molecular recognition events. [6][7][8][9][10] In addition, stapled peptides overcome some of the physicochem-ical inadequacies of native peptides. Constraining a peptide into a helical conformation decreases the polarity through the adoption of an extended intramolecular hydrogen bonding network between the NH and carbonyl of the backbone amide functionality. ...
Stapled peptides are a unique class of cyclic α‐helical peptides that are conformationally constrained via their amino acid side‐chains. They have been transformative to the field of chemical biology and peptide drug discovery through addressing many of the physicochemical limitations of linear peptides. However, there are several issues with current chemical strategies to produce stapled peptides. For example, two distinct unnatural amino acids are required to synthesize i, i+7 alkene stapled peptides, leading to high production costs. Furthermore, low purified yields are obtained due to cis/trans isomers produced during ring‐closing metathesis macrocyclisation. Here we report the development of a new i, i+7 diyne‐girder stapling strategy that addresses these issues. The asymmetric synthesis of nine unnatural Fmoc‐protected alkyne‐amino acids facilitated a systematic study to determine the optimal (S,S)‐stereochemistry and 14‐carbon diyne‐girder bridge length. Diyne‐girder stapled T‐STAR peptide 29 was demonstrated to have excellent helicity, cell permeability and stability to protease degradation. Finally, we demonstrate that the diyne‐girder constraint is a Raman chromophore with potential use in Raman cell microscopy. Development of this highly effective, bifunctional diyne‐girder stapling strategy leads us to believe that it can be used to produce other stapled peptide probes and therapeutics.
... In the third case, structural information is present for both ligands and the receptor protein. Usually a pharmacophore model represents the key features of a small molecule that allow it to bind to some receptor molecule, but this idea can be reversed and pharmacophore queries built from features of a protein active site 21 . These features describe the principle interactions between the protein and its ligands, and can be mapped onto the bioactive conformation of the ligand. ...
The pharmacophore concept was first put forward as a useful picture of drug interactions almost a century ago, and with the rise in computational power over the last few decades, has become a well-established CADD method with numerous different applications in drug discovery. Depending on the prior knowledge of the system, pharmacophores can be used to identify derivatives of compounds, change the scaffold to new compounds with a similar target, virtual screen for novel inhibitors, profile compounds for ADME-tox, investigate possible off-targets, or just complement other molecular methods “chemical groups” or functions in a molecule were responsible for a biological effect, and molecules with similar effect had similar functions in common. The word pharmacophore was coined much later, by Schueler in his 1960 book Chemobiodynamics and Drug Design, and was defined as “a molecular framework that carries (phoros) the essential features responsible for a drug’s (Pharmacon) biological activity.
... 5 Stapled peptides have the ability to mimic one of the protein epitopes, bind to a topologically shallow surface of the protein partner, and thus selectively disrupt these molecular recognition events. [6][7][8][9][10] In addition, stapled peptides overcome some of the physicochemical inadequacies of native peptides. Constraining a peptide into a helical conformation decreases the polarity through the adoption of an extended intramolecular hydrogen bonding network between the NH and carbonyl of the backbone amide functionality. ...
Stapled peptides are a unique class of cyclic alpha-helical peptides that are conformationally constrained via their amino acid side-chains. They have been transformative to the field of chemical biology and peptide drug discovery through addressing many of the physicochemical limitations of linear peptides. However, there are several issues with current chemical strategies to produce stapled peptides. For example, two distinct unnatural amino acids (R8 and S5) are required for synthesis of i, i + 7 alkene stapled peptides, leading to high cost of production. Furthermore, low purified yields are obtained due to cis/trans isomers being produced during the key ring-closing metathesis macrocyclisation step. Here we report the development of a new i, i + 7 diyne-girder stapling strategy that addresses these issues. The asymmetric synthesis of nine unnatural Fmoc-protected alkyne-amino acids facilitated a systematic study to determine the optimal (S,S)-stereochemistry and 14-carbon diyne-girder bridge length. Diyne-girder stapled T-STAR peptide 29 was demonstrated to have excellent helicity, was cell permeable and stable to protease degradation. Finally, we demonstrate that the diyne-girder constraint is a bifunctional Raman chromophore with potential use in Raman cell microscopy. Development of this highly effective, bifunctional diyne-girder stapling strategy leads us to believe that it can be used to produce other stapled peptide probes and therapeutics.
... Structural characterization of protein-protein interactions (PPIs) is of great importance for the mechanistic investigation of cellular processes and therapeutic design [1][2][3]. However, currently, we still lack structural information for most of known PPIs, as determining complex structures of PPIs using techniques like Xray crystallography and cryogenic electron microscopy is time consuming and expensive [4][5][6]. ...
The knowledge of contacting residue pairs between interacting proteins is very useful for the structural characterization of protein-protein interactions (PPIs). However, accurately identifying the tens of contacting ones from hundreds of thousands of inter-protein residue pairs is extremely challenging, and performances of the state-of-the-art inter-protein contact prediction methods are still quite limited. In this study, we developed a deep learning method for inter-protein contact prediction, which is referred to as DRN-1D2D_Inter. Specifically, we employed pretrained protein language models to generate structural information-enriched input features to residual networks formed by dimensional hybrid residual blocks to perform inter-protein contact prediction. Extensively bechmarking DRN-1D2D_Inter on multiple datasets, including both heteromeric PPIs and homomeric PPIs, we show DRN-1D2D_Inter consistently and significantly outperformed two state-of-the-art inter-protein contact prediction methods, including GLINTER and DeepHomo, although both the latter two methods leveraged the native structures of interacting proteins in the prediction, and DRN-1D2D_Inter made the prediction purely from sequences. We further show that applying the predicted contacts as constraints for protein-protein docking can significantly improve its performance for protein complex structure prediction.
... Stable isolated peptides with a defined short α-helical segment would be ideal inhibitors of macromolecular interactions. However, those with less than ~15 amino acid residues rarely adopt a defined conformation in isolation [3][4][5][6], and often lack the ability to fold into their bioactive conformation due to an entropic penalty for folding. New techniques for stabilizing short peptide helices may aid the design of inhibitors or mimics of protein function [7][8][9][10][11][12]. ...
A novel hydrogen bond surrogate-based (HBS) α-helix mimetic was designed by the combination of covalent H-bond replacement and the use of an ether linkage to substitute an amide bond within a short peptide sequence. The new helix template could be placed in position other than the N-terminus of a short peptide, and the CD studies demonstrate that the template adopts stable conformations in aqueous buffer at exceptionally high temperatures.
... Over the past 30 years, attempts to target and inhibit therapeutically relevant PPIs has been met with varying degrees of success. On the one hand, the ability to modulate large-scale PPIs with small molecules has been especially challenging, as small molecules often cannot inhibit biomolecular interactions that occur over large surface areas (Wilson, 2009). To circumvent this problem, researchers have gravitated towards using protein-based molecules, such as miniature proteins or antibody fragments, to target and inhibit larger-scale PPIs (Chrunyk et al., 2000;Zabrady et al., 2014). ...
This article outlines the design and development of scyllatoxin (ScTx)–based BH3 domain mimetics with diverse patterns of native disulfide bonds. More specifically, this method summarizes the total chemical synthesis of ScTx‐based peptides that contain zero, one, two, or three disulfide linkages, including techniques to generate variants with any combination of native disulfides. Each peptide reported herein is generated on solid‐phase support using microwave‐assisted coupling procedures, and all reaction parameters related to the peptide synthesis are described in detail. The various disulfide patterns of the ScTx‐based constructs are established during peptide synthesis and are ultimately verified by mass analysis of trypsin‐digested fragments. The BH3 domain mimetics developed herein were generated by transposing residues from the helical BH3 domain of the pro‐apoptotic BCL2 protein Bax to the α‐helix of wild‐type ScTx. Interestingly, we found that the relative binding affinities of ScTx‐Bax peptides for the anti‐apoptotic BCL2 protein Bcl‐2 (proper) were heavily influenced by the number and position of disulfide linkages within the ScTx‐Bax sequence. As a consequence, we were able to utilize ScTx‐Bax BH3 domain mimetics with varied patterns of disulfide bonds to survey how structural rigidity within the helical Bax BH3 domain affects binding to promiscuous anti‐apoptotic BCL2 proteins. More broadly, the ability to generate ScTx‐based molecules that contain any combination of native disulfide bonds expands the utility of such constructs as tools to study the molecular nature of protein‐protein interactions. © 2022 Wiley Periodicals LLC.
Basic Protocol 1 : Synthesis and characterization of ScTx‐based Bax BH3 domain mimetics
Basic Protocol 2 : Oxidation of ScTx‐Bax BH3 domain mimetics containing one, two, or three disulfide linkages
Support Protocol : Mapping of disulfide linkages in oxidized ScTx‐Bax BH3 domain mimetics
... As a result, the antibody-antigen bond can resist large changes in salt, pH etc. Here, we consider only protein antigens, whose binding surface is termed an epitope. 1 Small molecules bind to proteins with much smaller buried surface areas and as a result, are expected to lack the ability to interfere in antibody-epitope binding due to a limited surface area for interaction and a relatively low affinity to an epitope in the absence of other molecules. 2 Consequently, identifying small molecules to modulate the antibody-epitope interaction has been largely overlooked and its potential applications have been rarely explored. ...
... 10 In addition, allosteric small molecules could exhibit an epitope alteration effect due to conformational changes. 2 Based on our discovery and previous reports, we speculated that the alteration of antibody-epitope recognition could be used to identify small molecule hits for a target-ofinterest. ...
... This may arise from the at and large interface of this epitope that makes it even more difficult for BMS-202 to compete with the binding. 2 In contrast, the readout of the WL12-treated group showed a signicantly decreased signal, indicating the inhibiting role of WL12 for the Phe19-Thr239 epitope. Fluorescence polarization assay showed no signicant readout change of WL12 with an AlexaFluo488 labelled primary or secondary antibody (ESI Fig. 2a and b †). ...
Small molecules and antibodies are normally considered separately in drug discovery, except in the case of covalent conjugates. We unexpectedly discovered several small molecules that could inhibit or enhance antibody-epitope interactions which opens new possibilities in drug discovery and therapeutic modulation of auto-antibodies. We first discovered a small molecule, CRANAD-17, that enhanced the binding of an antibody to amyloid beta (Aβ), one of the major hallmarks of Alzheimer's disease, by stable triplex formation. Next, we found several small molecules that altered antibody-epitope interactions of tau and PD-L1 proteins, demonstrating the generality of this phenomenon. We report a new screening technology for ligand discovery, screening platform based on epitope alteration for drug discovery (SPEED), which is label-free for both the antibody and small molecule. SPEED, applied to an Aβ antibody, led to the discovery of a small molecule, GNF5837, that inhibits Aβ aggregation and another, obatoclax, that binds Aβ plaques and can serve as a fluorescent reporter in brain slices of AD mice. We also found a small molecule that altered the binding between Aβ and auto-antibodies from AD patient serum. SPEED reveals the sensitivity of antibody-epitope interactions to perturbation by small molecules and will have multiple applications in biotechnology and drug discovery.
