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SSR128129E (SSR) is a unique small‐molecule inhibitor of fibroblast growth factor receptors (FGFRs). SSR is a high‐affinity allosteric binder that selectively blocks one of the two major FGFR‐mediated pathways. The mechanisms of SSR activity were studied previously in much detail, allowing the identification of its binding site, located in the hydrophobic groove of the receptor D3 domain. The binding site overlaps with the position of an N‐terminal helix, an element exclusive for the FGF8b growth factor, which could potentially convert SSR from an allosteric inhibitor into an orthosteric blocker for the particular FGFR/FGF8b system. In this regard, we report here on the structural and functional investigation of FGF8b/FGFR3c system and the effects imposed on it by SSR. We show that SSR is equally or more potent in inhibiting FGF8b‐induced FGFR signaling compared to FGF2‐induced activation. On the other hand, when studied in the context of separate extracellular domains of FGFR3c in solution with NMR spectroscopy, SSR is unable to displace the N‐terminal helix of FGF8b from its binding site on FGFR3c and behaves as a weak orthosteric inhibitor. The substantial inconsistency between the results obtained with cell culture and for the individual water‐soluble subdomains of the FGFR proteins points to the important role played by the cell membrane. [ABSTRACT FROM AUTHOR]

The heat shock protein 70 (Hsp70) family of molecular chaperones are highly expressed in tumors. Inhibitors containing a pyridinium-modified benzothiazole, such as JG-98, bind to a conserved, allosteric site in Hsp70, showing promising anti-proliferative activity in cancer cells. When bound to Hsp70, the charged pyridinium makes favorable contacts; however, this moiety also increases the inhibitor's fluorescence, giving rise to undesirable interference in biochemical and cell-based assays. Here, we explore whether the pyridinium can be replaced with a neutral pyridine. We report that pyridine-modified benzothiazoles, such as compound 17h (JG2-38), have reduced fluorescence, yet retain promising anti-proliferative activity (EC50 values ~0.1 to 0.07 µM) in breast and prostate cancer cell lines. These chemical probes are expected to be useful in exploring the roles of Hsp70s in tumorigenesis and cell survival.

The main protease enzyme (Mpro) of SARS-CoV-2 is one of the most promising targets for COVID-19 treatment. Accordingly, in this work, a structure-based virtual screening of 3.8 million ligand libraries was carried out. After rigorous filtering, docking, and post screening assessments, 78 compounds were selected for biological evaluation, 3 of which showed promising inhibition of the Mpro enzyme. The obtained hits (CB03, GR04, and GR20) had reasonable potencies with Ki values in the medium to high micromolar range. Interestingly, while our most potent hit, GR20, was suggested to act via a reversible covalent mechanism, GR04 was confirmed as a noncompetitive inhibitor that seems to be one of a kind when compared to the other allosteric inhibitors discovered so far. Moreover, all three compounds have small sizes (~300 Da) with interesting fittings in their relevant binding sites, and they possess lead-like characteristics that can introduce them as very attractive candidates for the future development of COVID-19 treatments. [ABSTRACT FROM AUTHOR]

PI3Kα is a lipid kinase that phosphorylates PIP2 and generates PIP3. The hyperactive PI3Kα mutation, H1047R, accounts for about 14% of breast cancer, making it a highly attractive target for drug discovery. Here, we report the cryo-EM structures of PI3KαH1047R bound to two different allosteric inhibitors QR-7909 and QR-8557 at a global resolution of 2.7 Å and 3.0 Å, respectively. The structures reveal two distinct binding pockets on the opposite sides of the activation loop. Structural and MD simulation analyses show that the allosteric binding of QR-7909 and QR-8557 inhibit PI3KαH1047R hyper-activity by reducing the fluctuation and mobility of the activation loop. Our work provides a strong rational basis for a further optimization and development of highly selective drug candidates to treat PI3KαH1047R-driven cancers. [Display omitted] • Cryo-EM structures of PI3KαH1047R reveal two allosteric inhibitor binding sites • Inhibitor QR-7909 makes direct contacts with the tumor-associated H1047R residue • Two distinct allosteric inhibitory modes revealed involvement of the activation loop • PI3KαH1047R-inhibitor interactions were further analyzed with MD simulations Huang et al. determined the cryo-EM structures of PI3KαH1047R with two different allosteric inhibitors. The structures reveal an H1047R-specific allosteric inhibitory mechanism for PI3Kα involving the activation loop. [ABSTRACT FROM AUTHOR]

Specifically targeting the With‐No‐Lysine (WNK1) kinase, which is implicated in hypertension, renders a significant challenge in discovering competitive inhibitors due to the highly conserved ATP‐binding pocket. However, an allosteric inhibitor may impart high specificity against the WNK kinase isoforms since it targets the less conserved site and can provide greater efficacy even under high physiological ATP concentration. In the current study, we have investigated the structural and energetic basis of the specificity of the allosteric inhibitor WNK476 against WNK kinase isoforms by combining molecular dynamics simulations and free energy calculations using molecular mechanics Poisson–Boltzmann surface area. Our study reveals that the conformational stabilization of αC‐helix near the allosteric binding site, including conformational changes in activation and glycine‐rich loop regions, favors the specificity of WNK476 toward WNK1. The MM/PBSA calculations suggest that the non‐polar contribution from hydrophobic residues and polar solvation energy influences WNK/WNK476 complexation. Despite more favorable electrostatic and van der Waals interactions in WNK2/WNK476, WNK476 is more potent against WNK1 due to the lower contribution of disfavoring components—polar solvation and entropy. Further, we have identified that the hydrophobic residues of DLG, αC‐helix, β4, and β5 regions, and H‐bond network near the β4 strand play a critical role in the specificity of WNK476 against WNK1. Finally, our study reveals that residues Leu272, Val281, Phe283, and Leu369 of WNK1 actively contribute to the overall hydrophobic interactions for WNK1/WNK476. Overall, our study might help in the rational design of novel allosteric inhibitors against hypertension. [ABSTRACT FROM AUTHOR]

Small C‐terminal domain phosphatase 1(SCP1)'s biological function is significant in many cellular activities. Still, a recent study on neuroglioma cells emphasized the requirement of negative regulation of SCP1 in cancer invasion suppression. Due to the structural conservation of C‐terminal domain (CTD) phosphatases, we aimed to determine the peripherally targeting inhibitors, which reciprocally bind to an eccentric site on SCP1 using a multidisciplinary approach. From biochemical screening, we have identified two potential inhibitors, which showed twofold to threefold selectivity toward SCP1 compared to Dullard. Dullard was utilized as a negative control as it is a small CTD phosphatase that contains structural similarities to SCP1. Besides, from in silico approaches like protein–ligand docking and molecular dynamics analyses, we have successfully discovered two allosteric inhibitors of napthoquinone family compounds explicitly binding to the unique hydrophobic pocket of SCP1 from 1000 molecular dockings and 125 ns of dynamics simulation studies, respectively. [ABSTRACT FROM AUTHOR]

• Cloning, purification and biochemical characterization of CysE from Klebsiella pneumoniae • 3-D crystal structure of Kpn CysE complexed with L-cysteine at 2.8 Å • In silico identification and in vitro validation of inhibitory potential of quercetin • Identification of an allosteric site at the trimer-trimer interface in the hexameric CysE The emergence and spread of multidrug-resistant strains of Klebsiella pneumoniae is a major concern that necessitates the development of unique therapeutics. The essential requirement of serine acetyltransferase (SAT/CysE) for survival of several human pathogens makes it a very promising target for inhibitor designing and drug discovery. In this study, as an initial step to structure-based drug discovery, CysE from K. pneumonia was structurally and biochemically characterized. Subsequently, blind docking of selected natural products into the X-ray crystallography determined 3D structure of the target was carried out. Experimental validation of the inhibitory potential of the top-scorers established quercetin as an uncompetitive inhibitor of Kpn CysE. Molecular dynamics simulations carried out to elucidate the binding mode of quercetin reveal that this small molecule binds at the trimer-trimer interface of hexameric CysE, a site physically distinct from the active site of the enzyme. Detailed analysis of conformational differences incurred in Kpn CysE structure on binding to quercetin provides mechanistic understanding of allosteric modulation. Binding of quercetin to CysE leads to conformation changes in the active site loops and proximal loops that affect its internal dynamics and consequently its affinity for substrate/co-factor binding, justifying the reduced enzyme activity. [ABSTRACT FROM AUTHOR]

Organotellurium compounds have been reported as an immune-modulator sensitizing chemotherapeutics. Herein, we report the design and synthesis of a series of novel tellurodibenzoic acids as mimics of diphenylarsenic acid (DPAA) and potential selective KGA inhibitors. Representative compound 3B exhibited potent inhibition of KGA and glutamine-dependent HCT-116 cells. Stability experiments indicated that 3B has excellent stability under acidic (HCOOH), basic (NH 3 ·H 2 O) and oxidative (H 2 O 2) conditions, but reacts with β-ME, DTT and lysine which suggested that compound 3B may interact with cysteine or lysine residues. Moreover, molecular docking disclosed that compound 3B binds to the allosteric site of the GAC tetramer containing Arg317-Lys320-Leu321-Phe322-Tyr394-Glu325, which helped to rationalize the SAR and further design and optimization. Taken together, compound 3B could be used as a starting point for the development of new KGA inhibitors. [ABSTRACT FROM AUTHOR]

Tropomyosin receptor kinase A (TrkA) is considered a therapeutic target in pain treatment, and several TrkA inhibitors have been designed and developed as potential analgesics. The subselectivity toward TrkA is closely associated with safety and efficacy and was achieved by developing an allosteric inhibitor to occupy the less conserved non-ATP binding site. Druggable modifications of potent TrkA inhibitors could be challenging due to the unique requirement of functional groups that supply potency but are accompanied by poor druggability. We developed carboxyl acid 5 as a potent (IC 50 = 22.4 nM) and subselective TrkA inhibitor (>8000-fold toward TrkB and TrkC). We also converted 5 to corresponding prodrug 39 as a useful tool compound in future analgesic development with promising antinociceptive effects in both hot plate testing and formalin-induced pain models with a suitable ED 50 value (7.8 mg/kg). [Display omitted] • We developed compound 5 as a highly potent and sub-selective TrkA inhibitor. • We converted 5 to the prodrug 39 with promising antinociceptive effects on mice. • Prodrug 39 possessed a suitable ED 50 value (7.8 mg/kg) on mice. [ABSTRACT FROM AUTHOR]

This study presents alternatives to a previously described binding model for a novel class of nitrothiazole inhibitors of c-Jun-N-terminal kinase (JNK). In these models, the conformational flexibility of the protein structure has been incorporated to accommodate the ligand in the allosteric site while it forms physically strong hydrogen bonding and hydrophobic enclosure interactions with the amino acid residues of the binding site. Moreover, unlike in the previously reported model, these interactions take place without any physically meaningless stereochemical short contacts. The present models could potentially serve as a new basis for the design of potent and selective JNK ligands. [ABSTRACT FROM AUTHOR]

[Display omitted] p38α mitogen activated protein kinase (MAPK) plays important roles in multiple cellular functions by phosphorylating a wide variety of substrates, and therefore, p38α MAPK has been considered as an important drug target. In this study, we designed peptide-based inhibitors for p38α MAPK, which can only inhibit the Smad3 phosphorylation specifically, by targeting the KIM binding site of p38α MAPK. Peptide 6 showed a significant inhibitory potency for the Smad3 phosphorylation by p38α MAPK. Peptide 6 showed no ATP dependency, and did not inhibit the phosphorylation of other substrates by p38α MAPK. The discovery of peptide 6 by targeting the KIM binding site likely provide an opportunity for the discovery of a novel class of allosteric and substrate-specific p38α MAPK inhibitors. [ABSTRACT FROM AUTHOR]

MKK7 (MEK7) is a key regulator of the JNK stress signaling pathway and targeting MKK7 has been proposed as a chemotherapeutic strategy. Detailed understanding of the MKK7 structure and factors that affect its activity is therefore of critical importance. Here, we present a comprehensive set of MKK7 crystal structures revealing insights into catalytic domain plasticity and the role of the N-terminal regulatory helix, conserved in all MAP2Ks, mediating kinase activation. Crystal structures harboring this regulatory helix revealed typical structural features of active kinase, providing exclusively a first model of the MAP2K active state. A small-molecule screening campaign yielded multiple scaffolds, including type II irreversible inhibitors a binding mode that has not been reported previously. We also observed an unprecedented allosteric pocket located in the N-terminal lobe for the approved drug ibrutinib. Collectively, our structural and functional data expand and provide alternative targeting strategies for this important MAP2K kinase. • The N-terminal regulatory helix allosterically stabilizes an active state in MKK7 • Diverse covalent type I and type II inhibitors target flexible region in MKK7 • Unprecedented discovery of an allosteric binding site for ibrutinib Schröder et al. presents a first structure of active MKK7 revealing an allosteric role of the N-terminal helix, providing a model for the MAP2K activation mechanism. Screening identified potent chemically diverse MKK7 inhibitors with diverse binding modes abrogating JNK signaling. Crystal structure revealed ibrutinib binding at an unprecedented allosteric site. [ABSTRACT FROM AUTHOR]

A series of allosteric kidney-type glutaminase (GLS) inhibitors possessing a mercaptoethyl (SCH 2 CH 2) linker were synthesized in an effort to further expand the structural diversity of chemotypes derived from bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES), a prototype allosteric inhibitor of GLS. BPTES analog 3a with a mercaptoethyl linker between the two thiadiazole rings was found to potently inhibit GLS with an IC 50 value of 50 nM. Interestingly, the corresponding derivative with an n -propyl (CH 2 CH 2 CH 2) linker showed substantially lower inhibitory potency (IC 50 = 2.3 μM) while the derivative with a dimethylsulfide (CH 2 SCH 2) linker showed no inhibitory activity at concentrations up to 100 μM, underscoring the critical role played by the mercaptoethyl linker in the high affinity binding to the allosteric site of GLS. Additional mercaptoethyl-linked compounds were synthesized and tested as GLS inhibitors to further explore SAR within this scaffold including derivatives possessing a pyridazine as a replacement for one of the two thiadiazole moiety. [ABSTRACT FROM AUTHOR]

Summary Ubiquitin-like (Ubl) post-translational modifications are potential targets for therapeutics. However, the only known mechanism for inhibiting a Ubl-activating enzyme is through targeting its ATP-binding site. Here we identify an allosteric inhibitory site in the small ubiquitin-like modifier (SUMO)-activating enzyme (E1). This site was unexpected because both it and analogous sites are deeply buried in all previously solved structures of E1s of ubiquitin-like modifiers (Ubl). The inhibitor not only suppresses SUMO E1 activity, but also enhances its degradation in vivo , presumably due to a conformational change induced by the compound. In addition, the lead compound increased the expression of miR-34b and reduced c-Myc levels in lymphoma and colorectal cancer cell lines and a colorectal cancer xenograft mouse model. Identification of this first-in-class inhibitor of SUMO E1 is a major advance in modulating Ubl modifications for therapeutic aims. Graphical Abstract Highlights • We identified an allosteric inhibitory site for an activating enzyme • The lead compound inhibits the ATP-dependent step of SUMO E1 catalysis • The compound has specificity to 1 out of 18 non-disulfide bonded Cys residues • The compound increased miR-34b and reduced c-Myc in cellular and xenograft models Ubiquitin-like (Ubl) post-translational modifications are potential targets for developing novel therapeutics for life-threatening diseases. Here, we have identified a new allosteric, covalent inhibitory site for inhibiting an activating enzyme of Ubl. This finding could spur innovative drug discovery efforts targeting Ubl modifications. [ABSTRACT FROM AUTHOR]

HIV‐1 protease (HIV‐PR) performs a vital step in the virus life cycle which makes it an excellent target for drug therapy. However, due to the error‐prone of HIV reverse transcriptase, mutations in HIV‐PR often occur, inducing drug‐resistance to inhibitors. Some HIV‐PR mutations can make the flaps of the enzyme more flexible thus increasing the flaps opening rate and inhibitor releasing. It has been shown that by targeting novel binding sites on HIV‐PR with small molecules, it is possible to alter the equilibrium of flap conformational states. A previous fragment‐based crystallographic screen have found two novel binding sites for small fragments in the inhibited, closed form of HIV‐PR, termed flap and exo sites. While these experiments were performed in wild type HIV‐PR, it still remains to be proven whether these small fragments can stabilize the closed conformation of flaps in resistant forms of the enzyme. Here we performed Molecular Dynamics simulations of wild type and mutant form of HIV‐PR bound to inhibitor TL‐3. Simulations show that on going from wild type to 6X mutant the equilibrium shifts from closed to semi‐open conformation of flaps. However, when fragment Br6 is placed at flap site of mutant form, the enzyme is restored back to closed conformation. This finding supports the hypothesis that allosteric inhibitors, together with active site inhibitors could increase the number of point mutations necessary for appreciable clinical resistance to AIDS therapy. [ABSTRACT FROM AUTHOR]