Small-molecule Allosteric Modulators Research Articles

Activation mechanism of the cardiac calcium pump by a small-molecule allosteric modulator

Activation mechanism of the cardiac calcium pump by a small-molecule allosteric modulator

Small-molecule allosteric modulators of the AML-associated Src-family kinase, Hck

Small-molecule allosteric modulators of the AML-associated Src-family kinase, Hck

Characterization of allosteric modulators that disrupt androgen receptor co-activator protein-protein interactions to alter transactivation-Drug leads for metastatic castration resistant prostate cancer.

Three series of compounds were prioritized from a high content screening campaign that identified molecules that blocked dihydrotestosterone (DHT) induced formation of Androgen Receptor (AR) protein-protein interactions (PPIs) with the Transcriptional Intermediary Factor 2 (TIF2) coactivator and also disrupted preformed AR-TIF2 PPI complexes; the hydrobenzo-oxazepins (S1), thiadiazol-5-piperidine-carboxamides (S2), and phenyl-methyl-indoles (S3). Compounds from these series inhibited AR PPIs with TIF2 and SRC-1, another p160 coactivator, in mammalian 2-hybrid assays and blocked transcriptional activation in reporter assays driven by full length AR or AR-V7 splice variants. Compounds inhibited the growth of five prostate cancer cell lines, with many exhibiting differential cytotoxicity towards AR positive cell lines. Representative compounds from the 3 series substantially reduced both endogenous and DHT-enhanced expression and secretion of the prostate specific antigen (PSA) cancer biomarker in the C4-2 castration resistant prostate cancer (CRPC) cell line. The comparatively weak activities of series compounds in the H3-DHT and/or TIF2 box 3 LXXLL-peptide binding assays to the recombinant ligand binding domain of AR suggest that direct antagonism at the orthosteric ligand binding site or AF-2 surface respectively are unlikely mechanisms of action. Cellular enhanced thermal stability assays (CETSA) indicated that compounds engaged AR and reduced the maximum efficacy and right shifted the EC50 of DHT-enhanced AR thermal stabilization consistent with the effects of negative allosteric modulators. Molecular docking of potent representative hits from each series to AR structures suggest that S1-1 and S2-6 engage a novel binding pocket (BP-1) adjacent to the orthosteric ligand binding site, while S3-11 occupies the AR binding function 3 (BF-3) allosteric pocket. Hit binding poses indicate spaces and residues adjacent to the BP-1 and BF-3 pockets that will be exploited in future medicinal chemistry optimization studies. Small molecule allosteric modulators that prevent/disrupt AR PPIs with coactivators like TIF2 to alter transcriptional activation in the presence of orthosteric agonists might evade the resistance mechanisms to existing prostate cancer drugs and provide novel starting points for medicinal chemistry lead optimization and future development into therapies for metastatic CRPC.

Fluorescence lifetime FRET assay for live-cell high-throughput screening of the cardiac SERCA pump yields multiple classes of small-molecule allosteric modulators

We have used FRET-based biosensors in live cells, in a robust high-throughput screening (HTS) platform, to identify small-molecules that alter the structure and activity of the cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA2a). Our primary aim is to discover drug-like small-molecule activators that improve SERCA’s function for the treatment of heart failure. We have previously demonstrated the use of an intramolecular FRET biosensor, based on human SERCA2a, by screening two different small validation libraries using novel microplate readers that detect the fluorescence lifetime or emission spectrum with high speed, precision, and resolution. Here we report results from FRET-HTS of 50,000 compounds using the same biosensor, with hit compounds functionally evaluated using assays for Ca2+-ATPase activity and Ca2+-transport. We focused on 18 hit compounds, from which we identified eight structurally unique scaffolds and four scaffold classes as SERCA modulators, approximately half of which are activators and half are inhibitors. Five of these compounds were identified as promising SERCA activators, one of which activates Ca2+-transport even more than Ca2+-ATPase activity thus improving SERCA efficiency. While both activators and inhibitors have therapeutic potential, the activators establish the basis for future testing in heart disease models and lead development, toward pharmaceutical therapy for heart failure.

Application of Mixed-Solvent Molecular Dynamics Simulations for Prediction of Allosteric Sites on G Protein-Coupled Receptors.

The development of small molecule allosteric modulators acting at G protein-coupled receptors (GPCRs) is becoming increasingly attractive. Such compounds have advantages over traditional drugs acting at orthosteric sites on these receptors, in particular target specificity. However, the number and locations of druggable allosteric sites within most clinically relevant GPCRs are unknown. In the present study, we describe the development and application of a mixed-solvent molecular dynamics (MixMD)-based method for the identification of allosteric sites on GPCRs. The method employs small organic probes with druglike qualities to identify druggable hotspots in multiple replicate short-timescale simulations. As proof of principle, we first applied the method retrospectively to a test set of five GPCRs (cannabinoid receptor type 1, C-C chemokine receptor type 2, M2 muscarinic receptor, P2Y purinoceptor 1, and protease-activated receptor 2) with known allosteric sites in diverse locations. This resulted in the identification of the known allosteric sites on these receptors. We then applied the method to the μ-opioid receptor. Several allosteric modulators for this receptor are known, although the binding sites for these modulators are not known. The MixMD-based method revealed several potential allosteric sites on the mu-opioid receptor. Implementation of the MixMD-based method should aid future efforts in the structure-based drug design of drugs targeting allosteric sites on GPCRs. SIGNIFICANCE STATEMENT: Allosteric modulation of G protein-coupled receptors (GPCRs) has the potential to provide more selective drugs. However, there are limited structures of GPCRs bound to allosteric modulators, and obtaining such structures is problematic. Current computational methods utilize static structures and therefore may not identify hidden or cryptic sites. Here we describe the use of small organic probes and molecular dynamics to identify druggable allosteric hotspots on GPCRs. The results reinforce the importance of protein dynamics in allosteric site identification.

Perturbation of HCN1 response to small molecule modulation.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels are proteins with essential roles in pacemaking activity, neural signaling, and the development of neuropathic pain. Several allosteric ligands have been shown to inhibit HCN1; however, the structural basis of their action has yet to be defined. We uncovered putative binding sites for the intravenous general anesthetic propofol (2,6-di-isopropylphenol), a known HCN1 small molecule allosteric modulator, by cryoEM. Location of the sites correlate well with computational models. Moreover, these sites align with our bioinformatically derived allosteric network for channel activation, suggesting a mode of inhibition. Perturbation of one of the structurally identified binding sites resulted in differential inhibitor response compared to wildtype HCN1, as measured by Xenopus laevis oocyte two electrode voltage clamp. Future studies will further characterize these inhibitor binding sites and mechanistically investigate the source of isoform specificity.

Abstract 1714: Small Molecule Allosteric Modulators of the AML-associated Src-family Kinase, Hck

Abstract 1714: Small Molecule Allosteric Modulators of the AML-associated Src-family Kinase, Hck

Structures of Adrenoceptors.

The first structure of an adrenoceptor (AR), the human β2-adrenoceptor (hβ2AR) was published in 2007 and since then a total of 78 structures (up to June 2022) have been determined by X-ray crystallography and electron cryo-microscopy (cryo-EM) of all three βARs (β1, β2 and β3) and four out of six αARs (α1B, α2A, α2B, α2C). The structures are in a number of different conformational states, including the inactive state bound to an antagonist, an intermediate state bound to agonist and active states bound to agonist and an intracellular transducer (G protein or arrestin) or transducer mimetic (nanobody). The structures identify molecular details of how ligands bind in the orthosteric binding pocket (OBP; 19 antagonists, 18 agonists) and also how three different small molecule allosteric modulators bind. The structures have been used to define the molecular details of receptor activation and also the molecular determinants for transducer coupling. This chapter will give a brief overview of the structures, receptor activation, a comparison across the different subfamilies and commonalities of ligand-receptor interactions.

Methods to assess small molecule allosteric modulators of the STRAD pseudokinase.

With the increased appreciation of the biological relevance of pseudokinase (PSK) allostery, the broadening of small molecule strategies to target PSK function is of particular importance. We and others have pursued the development of small molecule allosteric modulators of the STRAD pseudokinase by targeting its ATP binding pocket. The purpose of this effort is to modulate the function of the LKB1 tumor suppressor kinase, which exists in a trimer with the STRAD PSK and the adaptor protein MO25. Here we provide detailed guidance regarding the different methods we have used for medium throughput screening to identify STRAD ligands and measure their impact on LKB1 kinase activity. Our experience supports preferential use of direct measurements of LKB1 kinase activity, and demonstrates the limitations of indirect assessment methods in the development trans-acting allosteric modulators.

Identification of small-molecule allosteric modulators that act as enhancers/disrupters of rhodopsin oligomerization

The elongated cilia of the outer segment of rod and cone photoreceptor cells can contain concentrations of visual pigments of up to 5 mM. The rod visual pigments, G protein–coupled receptors called rhodopsins, have a propensity to self-aggregate, a property conserved among many G protein–coupled receptors. However, the effect of rhodopsin oligomerization on G protein signaling in native cells is less clear. Here, we address this gap in knowledge by studying rod phototransduction. As the rod outer segment is known to adjust its size proportionally to overexpression or reduction of rhodopsin expression, genetic perturbation of rhodopsin cannot be used to resolve this question. Therefore, we turned to high-throughput screening of a diverse library of 50,000 small molecules and used a novel assay for the detection of rhodopsin dimerization. This screen identified nine small molecules that either disrupted or enhanced rhodopsin dimer contacts in vitro. In a subsequent cell-free binding study, we found that all nine compounds decreased intrinsic fluorescence without affecting the overall UV-visible spectrum of rhodopsin, supporting their actions as allosteric modulators. Furthermore, ex vivo electrophysiological recordings revealed that a disruptive, hit compound #7 significantly slowed down the light response kinetics of intact rods, whereas compound #1, an enhancing hit candidate, did not substantially affect the photoresponse kinetics but did cause a significant reduction in light sensitivity. This study provides a monitoring tool for future investigation of the rhodopsin signaling cascade and reports the discovery of new allosteric modulators of rhodopsin dimerization that can also alter rod photoreceptor physiology.

Improved Hemolytic Anemia with GBT1118 Is Reno-Protective in Transgenic Sickle Mice

Intravascular hemolysis releases cell-free hemoglobin and heme into the circulation. When haptoglobin and hemopexin are depleted, as observed in sickle cell anemia, cell-free hemoglobin and heme can cause direct oxidative injury, upregulation of inflammatory and immune response pathways, and depletion of nitric oxide. The kidneys are the primary route for clearance of non-scavenged cell-free hemoglobin and heme and are particularly susceptible to these deleterious pathways (PMID 31455889).In patients with sickle cell anemia, increased markers of hemolysis and hemoglobinuria are risk factors for kidney disease and kidney disease progression (PMID 24329963). Voxelotor is a small molecule allosteric modulator that binds and maintains sickle hemoglobin in the oxygenated state, thereby preventing hemoglobin S polymerization and red blood cell sickling. In a phase 3 study, voxelotor improved the degree of hemolysis, as reflected by a rise in hemoglobin concentration and a reduction in indirect bilirubin and reticulocyte percentage (PMID 31199090). The benefits of reducing hemolytic anemia with voxelotor on kidney function are unknown.We investigated whether GBT1118, an analogue of voxelotor with improved pharmacokinetic properties in transgenic sickle mice, would improve biomarkers of kidney damage and kidney function in sickle cell anemia. Transgenic sickle mice (Hb SS; Townes model, Jackson Laboratory) were treated with either GBT1118 (5 male, 5 female) or control (5 male, 5 female) chow from 12 to 24 weeks of age. Urine was collected using metabolic cages for 24 hours at 3 week intervals and blood was collected at baseline, 6 weeks, and 12 weeks of therapy. We compared markers of hemolysis (hemoglobin, reticulocyte percentage), reactive oxygen species (thibarbituric acid reactive substances [TBARS], kidney injury (kidney injury molecule-1 [KIM-1], nephrin), and kidney function (urine albumin and protein, serum blood urea nitrogen [BUN] and creatinine) at the respective time points using ANOVA and adjusting for gender. Mean and standard error values are provided.Consistent with the clinical results observed in the voxelotor phase 3 study, we observed significant improvements in the hemoglobin concentrations and reticulocyte percentages of Hb SS mice treated with GBT1118 versus control (Figure 1A and 1B). The hemoglobin occupancy of GBT1118 after 6 weeks (33 ± 2%) and 12 weeks (28 ± 2%) of therapy were also similar to what was observed in the phase 3 clinical study. An improvement in hemolysis led to reduced hemoglobinuria (6 weeks: treated = 0.07 ± 0.03 vs. control = 0.72 ± 0.2 ng/day, P = 0.004; 12 weeks: treated = 0.04 ± 0.03 vs. control = 1.06 ± 0.32 ng/day, P = 0.006) and less oxidant damage in the kidneys, assessed by urine TBARS concentration, in the treated versus control mice (6 weeks: treated = 31 ± 6 vs. control = 60 ± 10 nmol/day, P = 0.001; 12 weeks: treated = 29 ± 4 vs. control = 71 ± 9 nmol/day, P < 0.001).At 24 weeks of age, markers of glomerular (nephrin: treated = 3.5 ± 0.7 vs. control = 8.5 ± 1.6 ng/day) and tubular injury (KIM-1: treated = 169.6 ± 19.4 vs. control = 348.9 ± 48.2 pg/day) were significantly improved in the treated versus control Hb SS mice (P< 0.001) (Figure 1C). Albuminuria remained stable in the treated Hb SS mice and was relatively close in value to the degree of albuminuria observed in Hb AA mice (Figure 1D). In contrast, albuminuria progressively increased with older age in the control Hb SS mice. A similar pattern was observed for proteinuria (24 week: Hb SS treated = 2.02 ± 0.17 mg/day, Hb SS control = 3.65 ± 0.44 mg/day, Hb AA = 1.65 ± 0.27 mg/day). At 24 weeks of age, the treated Hb SS mice had serum BUN and creatinine values that were similar to what was observed at 12 weeks of age in the Hb SS mice and to the 24-week old Hb AA mice (Figure 1E, 1F). Furthermore, the values for serum BUN and creatinine were significantly lower in the treated versus control Hb SS mice at 24 weeks of age (P ≤ 0.04).In conclusion, we demonstrate that an improvement in hemolysis results in preserved kidney function in transgenic sickle mice. Our findings highlight the clinical importance of hemolytic anemia in the pathophysiology of sickle cell nephropathy. Our results also provide support for developing strategies to mitigate hemolysis in sickle cell anemia in order to provide a targeted approach to improve kidney disease, a devastating complication associated with high morbidity and mortality, in sickle cell anemia. [Display omitted] DisclosuresGordeuk: Modus Therapeutics: Consultancy; Novartis: Research Funding; Incyte: Research Funding; Emmaus: Consultancy, Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; CSL Behring: Consultancy. Saraf: Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding.

Structural Insights and Characterization of a Novel Transmembrane Binding Site for Potential Allosteric Modulation of the Mu‐opioid Receptor

Objective Opioids such as morphine, codeine, and oxycodone represent the gold standard for the treatment of pain. These drugs elicit their analgesic action by activating the mu-opioid receptor (mu-OR), a class A G protein-coupled receptor (GPCR). These agonists suffer from serious side effects, including respiratory depression, constipation, tolerance, and drug abuse. Recent advances in the structural biology of GPCRs revealed novel transmembrane lipid-facing binding pockets for allosteric modulators of the beta-2-adrenergic and cannabinoid (CB1) receptors. Allosteric ligands offer distinct advantages such as greater receptor subtype selectivity and the potential to “finetune” endogenous agonism while preserving the temporal and spatial characteristics of the signaling. Most importantly, allosteric modulators can offer unique safety by providing a “ceiling level” for their effects. Our central hypothesis is that a druggable transmembrane allosteric site exists for mu-OR, and high-affinity small-molecule ligands that stabilize the receptor in an active conformation can be identified. In this study, we identified such a novel transmembrane allosteric binding site on mu-OR and several high-affinity positive allosteric modulators through a comprehensive multidisciplinary (computational and experimental) approach. Our results provide the structural and mechanistic basis for the allosteric modulation of the receptor through this site and offer insights into the key binding site residues and membrane interactions that are critical for ligand affinity and receptor stabilization in the active state. Methods We employed a comprehensive computational approach that includes a sequence- and structure-based alignment of several experimental structures of GPCRs bound to their allosteric modulators to identify and characterize lipid-facing transmembrane binding sites. Structure-based pharmacophore models were developed, refined, and used for virtual screening of commercially available compound libraries to identify potential ligands using MOE software. Molecular docking, molecular dynamics simulations, and funnel metadynamics were used to assess the relative binding energies, using GROMACS and PLUMED software. Top ligands were evaluated by multiple in vitro assays for the positive allosteric modulation of mu-OR. Results A lipid-facing transmembrane binding site distinct from the orthosteric site was identified, and several high-affinity small-molecule allosteric modulators with drug-like properties were obtained by a combined computational and experimental approach. A variety of activation signature distances monitored were able to distinguish an active receptor from an inactive. The molecules will be further validated by in vivo methods for potential analgesic actions. Conclusion Our results illustrate a potential allosteric binding site on the transmembrane helices of the µ-opioid receptor that requires the active participation of membrane lipids for stable binding of ligands. The obtained ligands may translate into a safe and potential therapeutic alternative for opioids in the treatment of pain.

Identification of small molecule allosteric modulators of 5,10-methylenetetrahydrofolate reductase (MTHFR) by targeting its unique regulatory domain

The folate and methionine cycles, constituting one-carbon metabolism, are critical pathways for cell survival. Intersecting these two cycles, 5,10-methylenetetrahydrofolate reductase (MTHFR) directs one-carbon units from the folate to methionine cycle, to be exclusively used for methionine and S-adenosylmethionine (AdoMet) synthesis. MTHFR deficiency and upregulation result in diverse disease states, rendering it an attractive drug target. The activity of MTHFR is inhibited by the binding of AdoMet to an allosteric regulatory domain distal to the enzyme’s active site, which we have previously identified to constitute a novel fold with a druggable pocket. Here, we screened 162 AdoMet mimetics using differential scanning fluorimetry, and identified 4 compounds that stabilized this regulatory domain. Three compounds were sinefungin analogues, closely related to AdoMet and S-adenosylhomocysteine (AdoHcy). The strongest thermal stabilisation was provided by (S)-SKI-72, a potent inhibitor originally developed for protein arginine methyltransferase 4 (PRMT4). Using surface plasmon resonance, we confirmed that (S)-SKI-72 binds MTHFR via its allosteric domain with nanomolar affinity. Assay of MTHFR activity in the presence of (S)-SKI-72 demonstrates inhibition of purified enzyme with sub-micromolar potency and endogenous MTHFR from HEK293 cell lysate in the low micromolar range, both of which are lower than AdoMet. Nevertheless, unlike AdoMet, (S)-SKI-72 is unable to completely abolish MTHFR activity, even at very high concentrations. Combining binding assays, kinetic characterization and compound docking, this work indicates the regulatory domain of MTHFR can be targeted by small molecules and presents (S)-SKI-72 as an excellent candidate for development of MTHFR inhibitors.

Binding Characterization of GPCRs-Modulator by Molecular Complex Characterizing System (MCCS).

Increasing attention has been devoted to allosteric modulators as the preferred therapeutic agents for their colossal advantages such as higher selectivity, fewer side effects, and lower toxicity since they bind at allosteric sites that are topographically distinct from the classic orthosteric sites. However, the allosteric binding pockets are not conserved and there are no cogent methods to comprehensively characterize the features of allosteric sites with the binding of modulators. To overcome this limitation, our lab has developed a novel algorithm that can quantitatively characterize the receptor-ligand binding feature named Molecular Complex Characterizing System (MCCS). To illustrate the methodology and application of MCCS, we take G protein coupled receptors (GPCRs) as an example. First, we summarized and analyzed the reported allosteric binding pockets of class A GPCRs using MCCS. Sequentially, a systematic study was conducted between cannabinoid receptor type 1 (CB1) and its allosteric modulators, where we used MCCS to analyze the residue energy contribution and the interaction pattern. Finally, we validated the predicted allosteric binding site in CB2 via MCCS in combination with molecular dynamics (MD) simulation. Our results demonstrate that the MCCS program is advantageous in recapitulating the allosteric regulation pattern of class A GPCRs of the reported pockets as well as in predicting potential allosteric binding pockets. This MCCS program can serve as a valuable tool for the discovery of small-molecule allosteric modulators for class A GPCRs.

Focused structure-activity relationship profiling around the 2-phenylindole scaffold of a cannabinoid type-1 receptor agonist-positive allosteric modulator: site-III aromatic-ring congeners with enhanced activity and solubility

Specific tuning of cannabinoid 1 receptor (CB1R) activity by small-molecule allosteric modulators is a therapeutic modality with multiple properties inherently advantageous to therapeutic applications. We previously generated a library of unique CB1R positive allosteric modulators (PAMs) derived from GAT211, which has three pharmacophoric sites critical to its ago-PAM activity. To elaborate our CB1R PAM library, we report the rational design and molecular-pharmacology profiling of several 2-phenylindole analogs modified at the “site-III” aromatic ring. The comprehensive structure–activity relationship (SAR) investigation demonstrates that attaching small lipophilic functional groups on the ortho-position of the GAT211 site-III phenyl ring could markedly enhance CB1R ago-PAM activity. Select site-III modifications also improved GAT211’s water solubility. The SAR reported both extends the structural diversity of this compound class and demonstrates the utility of GAT211’s site-III for improving the parent compound’s drug-like properties of potency and/or aqueous solubility.

Abstract PR4: Modulation of integrin CD11b as a novel therapeutic strategy against lung cancer

Abstract Lung cancer is the leading cause of cancer-related deaths in the United States. With a 5-year survival rate at 19%, lung cancer patients are urgently in need of new therapeutic options. A major problem facing cancer therapy is the immune suppression by the tumor and its subsequent escape from the immune response, which is mediated by the tumor microenvironment. Tumors have large numbers of myeloid-derived suppressor cells (MDSCs), which suppress the adaptive immune response, increase neovascularization of the tumor, and promote tumor survival. Integrin heterodimer CD11b/CD18 (aka Mac-1 or αMβ2) is highly expressed on MDSCs and plays an important role in innate cell recruitment and other biologic functions. GB1275 (previously known as ADH503) is a novel small-molecule CD11b allosteric modulator. In experimental models of pancreatic cancer, GB1275 reduces MDSC infiltration and M2 macrophage polarization leading to tumor growth inhibition. GB1275 is currently in phase 1/2 clinical development in patients with pancreatic, esophageal, gastric/GEJ, triple-negative breast, castration-resistant prostate, or microsatellite stable colorectal cancer. To investigate the impact of CD11b modulation in the context of lung cancer, GB1275 treatment effects on tumor growth and immune infiltrates were assessed in the Lewis Lung Carcinoma (LLC) syngeneic tumor model, propagated in C57BL/6 wild-type (WT) mice. Additionally, we developed a transgenic CD11b knockin (KI) mouse that renders CD11b in a partially active state. CD11b KI mice were engineered to express partially active CD11b by introducing a point mutation at position 332 causing an isoleucine to glycine switch (I332G). We studied LLC tumor growth in CD11b KI mice as an orthogonal approach to test the mechanism of action. We will present data showing that pharmacologic modulation of CD11b with GB1275 significantly reduces LLC tumor growth. Concomitantly, we will present our studies with the CD11b KI mice that show a significant reduction in both the size and the rate of LLC tumor growth, as compared to the WT mice, mimicking treatment effects with GB1275. Our comprehensive pharmacologic and genetic studies show that CD11b activation reprograms MDSCs to enhance the adaptive immune response and is a novel strategy against lung cancer. This abstract is also being presented as Poster B71. Citation Format: Terese D. Geraghty, Anugraha Rajagopalan, Rabail Aslam, Vineet Gupta. Modulation of integrin CD11b as a novel therapeutic strategy against lung cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr PR4.

Finding the gas pedal on a slow sirtuin.

The class III histone deacetylase sirtuin 6 (SIRT6) modulates numerous functions in the cell by deacetylating histone lysine residues. Interestingly, SIRT6's efficiency in in vitro experiments is far greater against substrates carrying long-chain fatty acyl modifications such as myristoylated lysine compared with acetylated counterparts, but the deacetylase activity can be stimulated by fatty acids and small-molecule allosteric modulators. A new study helps to explain this puzzling activation using a novel activator, thorough kinetic investigation, and mutagenesis studies. These data help elucidate the molecular requirements for activation of SIRT6 and provide a foundation for development of activators for therapeutic purposes.

The different aspects of the GABAB receptor allosteric modulation.

The GABAB receptor is activated by the main inhibitory neurotransmitter of the central nervous system, the γ-aminobutyric acid (GABA). The receptor is expressed in almost all neuronal and glial cells and plays a central role in the modulation of many physiological and pathological processes. The GABAB receptor has been considered for years as an interesting target for the treatment of spasticity, pain, addiction, anxiety or depression. This has prompted many studies aiming at understanding the activation of the receptor and its modulation. While it belongs to the super-family of G protein-coupled receptors (GPCRs), it was rapidly evident that the GABAB receptor is peculiar in the variety of allosteric modulations governing its activation. Here, I wish to gather the different aspects of the GABAB receptor allosteric modulation. After presenting the main small molecule allosteric modulators known to date, the intramolecular transitions controlling the receptor activation will be summarized. In addition, recent findings obtained in the last decade on the existence of GABAB receptor complexes and their influence on the receptor function will be introduced, including the GABAB receptor oligomers and the auxiliary proteins associated with the receptor. These new concepts will certainly be of major interest in the future analysis of GABAB receptor allosteric modulation.

Discovery of Non-peptide Small Molecule Allosteric Modulators of the Src-family Kinase, Hck.

The eight mammalian Src-family tyrosine kinases are dynamic, multi-domain structures, which adopt distinct “open” and “closed” conformations. In the closed conformation, the regulatory SH3 and SH2 domains pack against the back of the kinase domain, providing allosteric control of kinase activity. Small molecule ligands that engage the regulatory SH3-SH2 region have the potential to modulate Src-family kinase activity for therapeutic advantage. Here we describe an HTS-compatible fluorescence polarization assay to identify small molecules that interact with the unique-SH3-SH2-linker (U32L) region of Hck, a Src-family member expressed exclusively in cells of myeloid lineage. Hck has significant potential as a drug target in acute myeloid leukemia, an aggressive form of cancer with substantial unmet clinical need. The assay combines recombinant Hck U32L protein with a fluorescent probe peptide that binds to the SH3 domain in U32L, resulting in an increased FP signal. Library compounds that interact with the U32L protein and interfere with probe binding reduce the FP signal, scoring as hits. Automated 384-well high-throughput screening of 60,000 compounds yielded Z'-factor coefficients > 0.7 across nearly 200 assay plates, and identified a series of hit compounds with a shared pyrimidine diamine substructure. Surface plasmon resonance assays confirmed direct binding of hit compounds to the Hck U32L target protein as well as near-full-length Hck. Binding was not observed with the individual SH3 and SH2 domains, demonstrating that these compounds recognize a specific three-dimensional conformation of the regulatory regions. This conclusion is supported by computational docking studies, which predict ligand contacts with a pocket formed by the juxtaposition of the SH3 domain, the SH3-SH2 domain connector, and the SH2-kinase linker. Each of the four validated hits stimulated recombinant, near-full-length Hck activity in vitro, providing evidence for allosteric effects on the kinase domain. These results provide a path to discovery and development of chemical scaffolds to target the regulatory regions of Hck and other Src family kinases as a new approach to pharmacological kinase control.

The Muscarinic Acetylcholine Receptor M5: Therapeutic Implications and Allosteric Modulation.

The muscarinic acetylcholine receptor (mAChR) subtype 5 (M5) was the most recent mAChR to be cloned and has since emerged as a potential therapeutic target for a number of indications. Early studies with knockout animals have provided clues to the receptor's role in physiological processes related to Alzheimer's disease, schizophrenia, and addiction, and until recently, useful subtype-selective tools to further probe the pharmacology of M5 have remained elusive. Small-molecule allosteric modulators have since gained traction as a means by which to selectively examine muscarinic pharmacology. This review highlights the discovery and optimization of M5 positive allosteric modulators (PAMs) and negative allosteric modulators (NAMs).