Allosteric coupling asymmetry mediates paradoxical activation of BRAF by type II inhibitors.

The type II class of RAF inhibitors currently in clinical trials paradoxically activate BRAF at subsaturating concentrations. Activation is mediated by induction of BRAF dimers, but why activation rather than inhibition occurs remains unclear. Using biophysical methods tracking BRAF dimerization and conformation, we built an allosteric model of inhibitor-induced dimerization that resolves the allosteric contributions of inhibitor binding to the two active sites of the dimer, revealing key differences between type I and type II RAF inhibitors. For type II inhibitors the allosteric coupling between inhibitor binding and BRAF dimerization is distributed asymmetrically across the two dimer binding sites, with binding to the first site dominating the allostery. This asymmetry results in efficient and selective induction of dimers with one inhibited and one catalytically active subunit. Our allosteric models quantitatively account for paradoxical activation data measured for 11 RAF inhibitors. Unlike type II inhibitors, type I inhibitors lack allosteric asymmetry and do not activate BRAF homodimers. Finally, NMR data reveal that BRAF homodimers are dynamically asymmetric with only one of the subunits locked in the active αC-in state. This provides a structural mechanism for how binding of only a single αC-in inhibitor molecule can induce potent BRAF dimerization and activation.

Discovery of WRN inhibitor HRO761 with synthetic lethality in MSI cancers.

The Werner syndrome RecQ helicase WRN was identified as a synthetic lethal target in cancer cells with microsatellite instability (MSI) by several genetic screens1-6. Despite advances in treatment with immune checkpoint inhibitors7-10, there is an unmet need in the treatment of MSI cancers11-14. Here we report the structural, biochemical, cellular and pharmacological characterization of the clinical-stage WRN helicase inhibitor HRO761, which was identified through an innovative hit-finding and lead-optimization strategy. HRO761 is a potent, selective, allosteric WRN inhibitor that binds at the interface of the D1 and D2 helicase domains, locking WRN in an inactive conformation. Pharmacological inhibition by HRO761 recapitulated the phenotype observed by WRN genetic suppression, leading to DNA damage and inhibition of tumour cell growth selectively in MSI cells in a p53-independent manner. Moreover, HRO761 led to WRN degradation in MSI cells but not in microsatellite-stable cells. Oral treatment with HRO761 resulted in dose-dependent in vivo DNA damage induction and tumour growth inhibition in MSI cell- and patient-derived xenograft models. These findings represent preclinical pharmacological validation of WRN as a therapeutic target in MSI cancers. A clinical trial with HRO761 (NCT05838768) is ongoing to assess the safety, tolerability and preliminary anti-tumour activity in patients with MSI colorectal cancer and other MSI solid tumours.

Allosteric Modulation of the YAP/TAZ-TEAD Interaction by Palmitoylation and Small-Molecule Inhibitors.

The Hippo signaling pathway is a highly conserved signaling network that plays a central role in regulating cellular growth, proliferation, and organ size. This pathway consists of a kinase cascade that integrates various upstream signals to control the activation or inactivation of YAP/TAZ proteins. Phosphorylated YAP/TAZ is sequestered in the cytoplasm; however, when the Hippo pathway is deactivated, it translocates into the nucleus, where it associates with TEAD transcription factors. This partnership is instrumental in regulating the transcription of progrowth and antiapoptotic genes. Thus, in many cancers, aberrantly hyperactivated YAP/TAZ promotes oncogenesis by contributing to cancer cell proliferation, metastasis, and therapy resistance. Because YAP and TAZ exert their oncogenic effects by binding with TEAD, it is critical to understand this key interaction to develop cancer therapeutics. Previous research has indicated that TEAD undergoes autopalmitoylation at a conserved cysteine, and small molecules that inhibit TEAD palmitoylation disrupt effective YAP/TAZ binding. However, how exactly palmitoylation contributes to YAP/TAZ-TEAD interactions and how the TEAD palmitoylation inhibitors disrupt this interaction remains unknown. Utilizing molecular dynamics simulations, our investigation not only provides detailed atomistic insight into the YAP/TAZ-TEAD dynamics but also unveils that the inhibitor studied influences the binding of YAP and TAZ to TEAD in distinct manners. This discovery has significant implications for the design and deployment of future molecular interventions targeting this interaction.

Akt Inhibitor Advancements: From Capivasertib Approval to Covalent-Allosteric Promises.

Akt kinase is vital in cell growth, survival, metabolism, and migration. Dysregulation of Akt signaling is implicated in cancer and metabolic disorders. In the context of cancer, overactive Akt promotes cell survival and proliferation. This has spurred extensive research into developing Akt inhibitors as potential therapeutic agents to disrupt aberrant Akt signaling. Akt inhibitors are classified into three main types: ATP-competitive, allosteric, and covalent-allosteric inhibitors (CAAIs). ATP-competitive inhibitors compete with ATP for binding to Akt, allosteric inhibitors interact with the Pleckstrin homology (PH) domain, and covalent-allosteric inhibitors form covalent bonds, making them more potent and selective. Notably, capivasertib (AZD5363), a potent ATP-competitive Akt inhibitor, received FDA approval in November 2023 for use in combination with the estrogen receptor degrader fulvestrant to treat breast cancer. Challenges remain, including improving selectivity, identifying biomarkers to tailor treatments, and enhancing therapeutic efficacy while minimizing adverse effects. Particularly covalent-allosteric inhibitors hold promise for future more effective and personalized treatments.

Bitter taste receptor activation by cholesterol and an intracellular tastant.

Bitter taste sensing is mediated by type 2 taste receptors (TAS2Rs (also known as T2Rs)), which represent a distinct class of G-protein-coupled receptors1. Among the 26 members of the TAS2Rs, TAS2R14 is highly expressed in extraoral tissues and mediates the responses to more than 100 structurally diverse tastants2-6, although the molecular mechanisms for recognizing diverse chemicals and initiating cellular signalling are still poorly understood. Here we report two cryo-electron microscopy structures for TAS2R14 complexed with Ggust (also known as gustducin) and Gi1. Both structures have an orthosteric binding pocket occupied by endogenous cholesterol as well as an intracellular allosteric site bound by the bitter tastant cmpd28.1, including a direct interaction with the α5 helix of Ggust and Gi1. Computational and biochemical studies validate both ligand interactions. Our functional analysis identified cholesterol as an orthosteric agonist and the bitter tastant cmpd28.1 as a positive allosteric modulator with direct agonist activity at TAS2R14. Moreover, the orthosteric pocket is connected to the allosteric site via an elongated cavity, which has a hydrophobic core rich in aromatic residues. Our findings provide insights into the ligand recognition of bitter taste receptors and suggest activities of TAS2R14 beyond bitter taste perception via intracellular allosteric tastants.

Chemoproteomic discovery of a covalent allosteric inhibitor of WRN helicase.

WRN helicase is a promising target for treatment of cancers with microsatellite instability (MSI) due to its essential role in resolving deleterious non-canonical DNA structures that accumulate in cells with faulty mismatch repair mechanisms1-5. Currently there are no approved drugs directly targeting human DNA or RNA helicases, in part owing to the challenging nature of developing potent and selective compounds to this class of proteins. Here we describe the chemoproteomics-enabled discovery of a clinical-stage, covalent allosteric inhibitor of WRN, VVD-133214. This compound selectively engages a cysteine (C727) located in a region of the helicase domain subject to interdomain movement during DNA unwinding. VVD-133214 binds WRN protein cooperatively with nucleotide and stabilizes compact conformations lacking the dynamic flexibility necessary for proper helicase function, resulting in widespread double-stranded DNA breaks, nuclear swelling and cell death in MSI-high (MSI-H), but not in microsatellite-stable, cells. The compound was well tolerated in mice and led to robust tumour regression in multiple MSI-H colorectal cancer cell lines and patient-derived xenograft models. Our work shows an allosteric approach for inhibition of WRN function that circumvents competition from an endogenous ATP cofactor in cancer cells, and designates VVD-133214 as a promising drug candidate for patients with MSI-H cancers.

Wighteone, a prenylated flavonoid from licorice, inhibits growth of SW480 colorectal cancer cells by allosteric inhibition of Akt.

ETHNOPHARMACOLOGICAL RELEVANCE: Licorice is a frequently used herbal medicine worldwide, and is used to treat cough, hepatitis, cancer and influenza in clinical practice of traditional Chinese medicine. Modern pharmacological studies indicate that prenylated flavonoids play an important role in the anti-tumor activity of licorice, especially the tumors in stomach, lung, colon and liver. Wighteone is one of the main prenylated flavonoids in licorice, and its possible effect and target against colorectal cancer have not been investigated. AIM OF THE STUDY: This study aimed to investigate the anti-colorectal cancer effect and underlying mechanism of wighteone. MATERIALS AND METHODS: SW480 human colorectal cancer cells were used to evaluate the in vitro anti-colorectal cancer activity and Akt regulation effect of wighteone by flow cytometry, phosphoproteomic and Western blot analysis. Surface plasmon resonance (SPR) assay, molecular docking and dynamics simulation, and kinase activity assay were used to investigate the direct interaction between wighteone and Akt. A nude mouse xenograft model with SW480 cells was used to verify the in vivo anti-colorectal cancer activity of wighteone. RESULTS: Wighteone inhibited phosphorylation of Akt and its downstream kinases in SW480 cells, which led to a reduction in cell viability. Wighteone had direct interaction with both PH and kinase domains of Akt, which locked Akt in a "closed" conformation with allosteric inhibition, and Gln79, Tyr272, Arg273 and Lys297 played the most critical role due to their hydrogen bond and hydrophobic interactions with wighteone. Based on Akt overexpression or activation in SW480 cells, further mechanistic studies suggested that wighteone-induced Akt inhibition led to cycle arrest, apoptosis and autophagic death of SW480 cells. Moreover, wighteone exerted in vivo anti-colorectal cancer effect and Akt inhibition activity in the nude mouse xenograft model. CONCLUSION: Wighteone could inhibit growth of SW480 cells through allosteric inhibition of Akt, which led to cell cycle arrest, apoptosis and autophagic death. The results contributed to understanding of the anti-tumor mechanism of licorice, and also provided a rationale to design novel Akt allosteric inhibitors for the treatment of colorectal cancer.

Channeling Nicotinamide Phosphoribosyltransferase (NAMPT) to Address Life and Death.

Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the rate-limiting step in NAD+ biosynthesis via salvage of NAM formed from catabolism of NAD+ by proteins with NADase activity (e.g., PARPs, SIRTs, CD38). Depletion of NAD+ in aging, neurodegeneration, and metabolic disorders is addressed by NAD+ supplementation. Conversely, NAMPT inhibitors have been developed for cancer therapy: many discovered by phenotypic screening for cancer cell death have low nanomolar potency in cellular models. No NAMPT inhibitor is yet FDA-approved. The ability of inhibitors to act as NAMPT substrates may be associated with efficacy and toxicity. Some 3-pyridyl inhibitors become 4-pyridyl activators or "NAD+ boosters". NAMPT positive allosteric modulators (N-PAMs) and boosters may increase enzyme activity by relieving substrate/product inhibition. Binding to a "rear channel" extending from the NAMPT active site is key for inhibitors, boosters, and N-PAMs. A deeper understanding may fulfill the potential of NAMPT ligands to regulate cellular life and death.

Phosphorylation-dependent allosteric regulation of Cx43 gap junction inhibitor potency.

Physiological and pathological processes such as homeostasis, embryogenesis, development, tumorigenesis, and cell movement depend on the intercellular communication through gap junctions (GJIC). Connexin (Cx)-based GJ channels are formed of two apposing hemichannels in the contiguous cells and provide a direct pathway for electrical and metabolic intercellular communication. The main modulators of GJ conductance are transjunctional voltage, intracellular pH, Ca2+, Mg2+, and phosphorylation. Chemical modulators of GJIC are being used in cases of various intercellular communication-dependent diseases. In this study, we used molecular docking, dual whole-cell patch-clamp, and Western blotting to investigate the impact of connexin phosphorylation on GJ chemical gating by α-pinene and other GJ inhibitors (octanol, carbenoxolone, mefloquine, intracellular pH, glycyrrhetinic acid, and sevoflurane) in HeLa cells expressing exogenous Cx43 (full length and truncated at amino acid 258) and other connexins typical of heart and/or nervous system (Cx36, Cx40, Cx45, and Cx47), and in cells expressing endogenous Cx43 (Novikoff and U-87). We found that Ca2+-regulated kinases, such as Ca2+/calmodulin-dependent kinase II, atypical protein kinase C, cyclin-dependent kinase, and Pyk2 kinase may allosterically modulate the potency of α-pinene through phosphorylation of Cx43 C-terminus. The identified new phenomenon was Cx isoform-, inhibitor-, and cell type-dependent. Overall, these results suggest that compounds, the potency of which depends on receptor phosphorylation, might be of particular interest in developing targeted therapies for diseases accompanied by high kinase activity, such as cardiac arrhythmias, epilepsy, stroke, essential tremor, inflammation, and cancer.

The allostery and modification of hGHRH molecules and specific dimer produced significant fertility effect by proliferating and activating in-situ ovarian mesenchymal stem cells.

The negative coordination of growth hormone secretagogue receptor (GHS-R) and growth hormone-releasing hormone receptor (GHRH-R) involves in the repair processes of cellular injury. The allosteric U- or H-like modified GHRH dimer Grinodin and 2Y were comparatively evaluated in normal Kunming mice and hamster infertility models induced by CPA treatment. 1-3-9 µg of Grinodin or 2Y per hamster stem-cell-exhaustion model was subcutaneously administered once a week, respectively inducing 75-69-46 or 45-13-50 % of birth rates. In comparison, the similar mole of human menopausal gonadotropin (hMG) or human growth hormone (hGH) was administered once a day but caused just 25 or 20 % of birth rates. Grinodin induced more big ovarian follicles and corpora lutea than 2Y, hMG, hGH. The hMG-treated group was observed many distorted interstitial cells and more connective tissues and the hGH-treated group had few ovarian follicles. 2Y had a plasma lifetime of 21 days and higher GH release in mice, inducing lower birth rate and stronger individual specificity in reproduction as well as only promoting the proliferation of mesenchymal-stem-cells (MSCs) in the models. In comparison, Grinodin had a plasma lifetime of 30 days and much lower GH release in mice. It significantly promoted the proliferation and activation of ovarian MSCs together with the development of follicles in the models by increasing Ki67 and GHS-R expressions, and decreasing GHRH-R expression in a dose-dependent manner. However, the high GH and excessive estrogen levels in the models showed a dose-dependent reduction in fertility. Therefore, unlike 2Y, the low dose of Grinodin specifically shows low GHS-R and high GHRH-R expressions thus evades GH and estrogen release and improves functions of organs, resulting in an increase of fertility.

Modulation of mGlu5 reduces rewarding associative properties of nicotine via changes in mesolimbic plasticity: Relevance to comorbid cigarette smoking in psychosis.

RATIONALE: Antipsychotic medications that are used to treat psychosis are often limited in their efficacy by high rates of severe side effects. Treatment success in schizophrenia is further complicated by high rates of comorbid nicotine use. Dopamine D2 heteroreceptor complexes have recently emerged as targets for the development of more efficacious pharmaceutical treatments for schizophrenia. OBJECTIVE: The current study sought to explore the use of the positive allosteric modulator of the mGlu5 receptor 3-Cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB) as a treatment to reduce symptoms related to psychosis and comorbid nicotine use. METHODS: Neonatal treatment of animals with the dopamine D2-like receptor agonist quinpirole (NQ) from postnatal day (P)1-21 produces a lifelong increase in D2 receptor sensitivity, showing relevance to psychosis and comorbid tobacco use disorder. Following an 8-day conditioning paradigm, brain tissue in the mesolimbic pathway was analyzed for several plasticity markers, including brain derived neurotrophic factor (BDNF), phosphorylated p70 ribosomal S6 kinase (phospho-p70S6K), and cadherin-13 (Cdh13). RESULTS: Pretreatment with CDPPB was effective to block enhanced nicotine conditioned place preference observed in NQ-treated animals. Pretreatment was additionally effective to block the nicotine-induced increase in BDNF and sex-dependent increases in cadherin-13 in the ventral tegmental area (VTA), as well as increased phospho-p70S6K in the nucleus accumbens (NAcc) shell found in NQ-treated animals. CONCLUSION: In conjunction with prior work, the current study suggests positive allosteric modulation of the mGlu5 receptor, an emerging target for schizophrenia therapeutics, may be effective for the treatment of comorbid nicotine abuse in psychosis.

Butyrate acts as a positive allosteric modulator of the 5-HT transporter to decrease availability of 5-HT in the ileum.

BACKGROUND AND PURPOSE: Radiation therapy-induced gastrointestinal distress is partly associated with the elimination of gut microbiota. The effectiveness of 5-HT receptor antagonists to treat radiation therapy-induced emesis implies a pathophysiological role of 5-HT. Peripheral 5-HT is derived from intestinal epithelium. We have investigated the role of gut microbiota in regulating intestinal 5-HT availability. EXPERIMENTAL APPROACH: A radiation therapy murine model accompanied by faecal microbiota transplantation from donors fed different diets was investigated, and mouse ileal organoids were used for mechanistic studies. The clinical relevance was validated by a small-scale human study. KEY RESULTS: Short-term high-fat diet (HFD) induced gut bacteria to produce butyrate. Irradiated mice receiving HFD-induced microbiome had the lowest ileal levels of 5-HT, compared with other recipients. Treatment with butyrate increased 5-HT uptake in mouse ileal organoids, assayed by the real-time tracking of a fluorescent substrate for monoamine transporters. Silencing the 5-HT transporter (SERT) in the organoids abolished butyrate-stimulated 5-HT uptake. The competitive tests using different types of selective 5-HT reuptake inhibitors suggested that butyrate acted as a positive allosteric modulator of SERT. In human gut microbiota, butyrate production was associated with the interconversion between acetate and butyrate. Faecal contents of both acetate and butyrate were negatively associated with serum 5-HT, but only butyrate was positively correlated with body mass index in humans. CONCLUSION AND IMPLICATIONS: Short-term HFD may be beneficial for alleviating gastrointestinal reactions by increasing butyrate to suppress local 5-HT levels and providing energy to cancer patients given radiation therapy.

The energetic and allosteric landscape for KRAS inhibition.

Thousands of proteins have been validated genetically as therapeutic targets for human diseases1. However, very few have been successfully targeted, and many are considered 'undruggable'. This is particularly true for proteins that function via protein-protein interactions-direct inhibition of binding interfaces is difficult and requires the identification of allosteric sites. However, most proteins have no known allosteric sites, and a comprehensive allosteric map does not exist for any protein. Here we address this shortcoming by charting multiple global atlases of inhibitory allosteric communication in KRAS. We quantified the effects of more than 26,000 mutations on the folding of KRAS and its binding to six interaction partners. Genetic interactions in double mutants enabled us to perform biophysical measurements at scale, inferring more than 22,000 causal free energy changes. These energy landscapes quantify how mutations tune the binding specificity of a signalling protein and map the inhibitory allosteric sites for an important therapeutic target. Allosteric propagation is particularly effective across the central ß-sheet of KRAS, and multiple surface pockets are genetically validated as allosterically active, including a distal pocket in the C-terminal lobe of the protein. Allosteric mutations typically inhibit binding to all tested effectors, but they can also change the binding specificity, revealing the regulatory, evolutionary and therapeutic potential to tune pathway activation. Using the approach described here, it should be possible to rapidly and comprehensively identify allosteric target sites in many proteins.

Allosteric inhibition of the epidermal growth factor receptor through disruption of transmembrane interactions.

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase (RTK) commonly targeted for inhibition by anticancer therapeutics. Current therapeutics target EGFR's kinase domain or extracellular region. However, these types of inhibitors are not specific for tumors over healthy tissue and therefore cause undesirable side effects. Our lab has recently developed a new strategy to regulate RTK activity by designing a peptide that specifically binds to the transmembrane (TM) region of the RTK to allosterically modify kinase activity. These peptides are acidity-responsive, allowing them to preferentially target acidic environments like tumors. We have applied this strategy to EGFR and created the PET1 peptide. We observed that PET1 behaves as a pH-responsive peptide that modulates the configuration of the EGFR TM through a direct interaction. Our data indicated that PET1 inhibits EGFR-mediated cell migration. Finally, we investigated the mechanism of inhibition through molecular dynamics simulations, which showed that PET1 sits between the two EGFR TM helices; this molecular mechanism was additionally supported by AlphaFold-Multimer predictions. We propose that the PET1-induced disruption of native TM interactions disturbs the conformation of the kinase domain in such a way that it inhibits EGFR's ability to send migratory cell signals. This study is a proof-of-concept that acidity-responsive membrane peptide ligands can be generally applied to RTKs. In addition, PET1 constitutes a viable approach to therapeutically target the TM of EGFR.

Monobody Inhibitor Selective to the Phosphatase Domain of SHP2 and its Use as a Probe for Quantifying SHP2 Allosteric Regulation.

SHP2 is a phosphatase/adaptor protein that plays an important role in various signaling pathways. Its mutations are associated with cancers and developmental diseases. SHP2 contains a protein tyrosine phosphatase (PTP) and two SH2 domains. Selective inhibition of these domains has been challenging due to the multitude of homologous proteins in the proteome. Here, we developed a monobody, synthetic binding protein, that bound to and inhibited the SHP2 PTP domain. It was selective to SHP2 PTP over close homologs. A crystal structure of the monobody-PTP complex revealed that the monobody bound both highly conserved residues in the active site and less conserved residues in the periphery, rationalizing its high selectivity. Its epitope overlapped with the interface between the PTP and N-terminal SH2 domains that is formed in auto-inhibited SHP2. By using the monobody as a probe for the accessibility of the PTP active site, we developed a simple, nonenzymatic assay for the allosteric regulation of SHP2. The assay showed that, in the absence of an activating phospho-Tyr ligand, wild-type SHP2 and the "PTP-dead" C459E mutant were predominantly in the closed state in which the PTP active site is inaccessible, whereas the E76K and C459S mutants were in the open, active state. It also revealed that previously developed monobodies to the SH2 domains, ligands lacking a phospho-Tyr, weakly favored the open state. These results provide corroboration for a conformational equilibrium underlying allosteric regulation of SHP2, provide powerful tools for characterizing and controlling SHP2 functions, and inform drug discovery against SHP2.

Mechanistic insights on novel small molecule allosteric activators of cGMP-dependent protein kinase PKG1α.

cGMP-dependent protein kinase (PKG) represents a compelling drug target for treatment of cardiovascular diseases. PKG1 is the major effector of beneficial cGMP signaling which is involved in smooth muscle relaxation and vascular tone, inhibition of platelet aggregation and signaling that leads to cardioprotection. In this study, a novel piperidine series of activators previously identified from an ultrahigh-throughput screen were validated to directly bind partially activated PKG1α and subsequently enhance its kinase activity in a concentration-dependent manner. Compounds from initial optimization efforts showed an ability to activate PKG1α independent of the endogenous activator, cGMP. We demonstrate these small molecule activators mimic the effect of cGMP on the kinetic parameters of PKG1α by positively modulating the KM of the peptide substrate and negatively modulating the apparent KM for ATP with increase in catalytic efficiency, kcat. In addition, these compounds also allosterically modulate the binding affinity of cGMP for PKG1α by increasing the affinity of cGMP for the high-affinity binding site (CNB-A) and decreasing the affinity of cGMP for the low-affinity binding site (CNB-B). We show the mode of action of these activators involves binding to an allosteric site within the regulatory domain, near the CNB-B binding site. To the best of our knowledge, these are the first reported non-cGMP mimetic small molecules shown to directly activate PKG1α. Insights into the mechanism of action of these compounds will enable future development of cardioprotective compounds that function through novel modes of action for the treatment of cardiovascular diseases.

Allosteric Regulation of the Soluble Epoxide Hydrolase by Nitro Fatty Acids: a Combined Experimental and Computational Approach.

The human soluble epoxide hydrolase (hsEH) is a key regulator of epoxy fatty acid (EpFA) metabolism. Inhibition of sEH can maintain endogenous levels of beneficial EpFAs and reduce the levels of their corresponding diol products, thus ameliorating a variety of pathological conditions including cardiovascular, central nervous system and metabolic diseases. The quest for orthosteric drugs that bind directly to the catalytic crevice of hsEH has been prolonged and sustained over the past decades, but the disappointing outcome of clinical trials to date warrants alternative pharmacological approaches. Previously, we have shown that hsEH can be allosterically inhibited by the endogenous electrophilic lipid 15-deoxy-Δ12,14-Prostaglandin-J2, via covalent adduction to two cysteines, C423 and C522. In this study, we explore the properties and behaviour of three electrophilic lipids belonging to the class of the nitro fatty acids, namely 9- and 10-nitrooleate and 10-nitrolinoleate. Biochemical and biophysical investigations revealed that, in addition to C423 and C522, nitro fatty acids can covalently bind to additional nucleophilic residues in hsEH C-terminal domain (CTD), two of which predicted in this study to be latent allosteric sites. Systematic mapping of the protein mutational space and evaluation of possible propagation pathways delineated selected residues, both in the allosteric patches and in other regions of the enzyme, envisaged to play a role in allosteric signalling. The responses elicited by the ligands on the covalent adduction sites supports future fragment-based design studies of new allosteric effectors for hsEH with increased efficacy and selectivity.

Development of Selective Phosphatidylinositol 5-Phosphate 4-Kinase γ Inhibitors with a Non-ATP-competitive, Allosteric Binding Mode.

Phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are emerging as attractive therapeutic targets in diseases, such as cancer, immunological disorders, and neurodegeneration, owing to their central role in regulating cell signaling pathways that are either dysfunctional or can be modulated to promote cell survival. Different modes of binding may enhance inhibitor selectivity and reduce off-target effects in cells. Here, we describe efforts to improve the physicochemical properties of the selective PI5P4Kγ inhibitor, NIH-12848 (1). These improvements enabled the demonstration that this chemotype engages PI5P4Kγ in intact cells and that compounds from this series do not inhibit PI5P4Kα or PI5P4Kß. Furthermore, the first X-ray structure of PI5P4Kγ bound to an inhibitor has been determined with this chemotype, confirming an allosteric binding mode. An exemplar from this chemical series adopted two distinct modes of inhibition, including through binding to a putative lipid interaction site which is 18 Å from the ATP pocket.

A PDK-1 allosteric agonist neutralizes insulin signaling derangements and beta-amyloid toxicity in neuronal cells and in vitro.

The Alzheimer's brain is affected by multiple pathophysiological processes, which include a unique, organ-specific form of insulin resistance that begins early in its course. An additional complexity arises from the four-fold risk of Alzheimer's Disease (AD) in type 2 diabetics, however there is no definitive proof of causation. Several strategies to improve brain insulin signaling have been proposed and some have been clinically tested. We report findings on a small allosteric molecule that reverses several indices of insulin insensitivity in both cell culture and in vitro models of AD that emphasize the intracellular accumulation of ß-amyloid (Aßi). PS48, a chlorophenyl pentenoic acid, is an allosteric activator of PDK-1, which is an Akt-kinase in the insulin/PI3K pathway. PS48 was active at 10 nM to 1 µM in restoring normal insulin-dependent Akt activation and in mitigating Aßi peptide toxicity. Synaptic plasticity (LTP) in prefrontal cortical slices from normal rat exposed to Aß oligomers also benefited from PS48. During these experiments, neither overstimulation of PI3K/Akt signaling nor toxic effects on cells was observed. Another neurotoxicity model producing insulin insensitivity, utilizing palmitic acid, also responded to PS48 treatment, thus validating the target and indicating that its therapeutic potential may extend outside of ß-amyloid reliance. The described in vitro and cell based-in vitro coupled enzymatic assay systems proved suitable platforms to screen a preliminary library of new analogs.

Targeting the Non-Catalytic Functions: a New Paradigm for Kinase Drug Discovery?

Protein kinases have been highly fruitful targets for cancer drug discovery in the past two decades, while most of these drugs bind to the "adenosine triphosphate (ATP)-site" and inhibit kinase catalytic activity. Recently, accumulated evidence suggests that kinases possess functions beyond catalysis through their scaffolds, and the scaffolding functions could play critical roles in multiple cellular signaling and cell fate controls. Small molecules modulating the noncatalytic functions of kinases are rarely reported but emerge as new promising therapeutic strategies for various diseases. Herein, we summarize the characterized noncatalytic functions of kinases, and highlight the recent progress on developing small-molecule modulators of the noncatalytic functions of kinases. Mechanisms and characteristics of different kinds of modulators are also discussed. It is also speculated that targeting the noncatalytic functions would represent a new direction for kinase-based drug discovery.