Class Of Allosteric Inhibitors Research Articles

Structure-Based Discovery of Allosteric Inhibitors Targeting a New Druggable Site in the Respiratory Syncytial Virus Polymerase.

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory infections for which effective treatment options remain limited. Herein, we employed a computational structure-based design strategy aimed at identifying potential targets for a new class of allosteric inhibitors. Our investigation led to the discovery of a previously undisclosed allosteric binding site within the RSV polymerase, the large (L) protein. This discovery was achieved through a combination of virtual screening and molecular dynamics simulations. Subsequently, we identified two inhibitors, 6a and 10b, which both exhibited promising antiviral activity in the low micromolar range. Resistance profiling revealed a distinctive pattern in how RSV evaded treatment with this class of inhibitors. This pattern strongly suggested that this class of small molecules was targeting a new binding site in the RSV L protein, aligning with the computational predictions made in our study. This study paves the way for the development of more potent inhibitors for combating RSV infections by targeting a new druggable pocket within the RdRp which does not overlap with previously known resistance sites.

Structural elements of a pH-sensitive inhibitor binding site in NMDA receptors

Context-dependent inhibition of N-methyl-D-aspartate (NMDA) receptors has important therapeutic implications for the treatment of neurological diseases that are associated with altered neuronal firing and signaling. This is especially true in stroke, where the proton concentration in the afflicted area can increase by an order of magnitude. A class of allosteric inhibitors, the 93-series, shows greater potency against GluN1-GluN2B NMDA receptors in such low pH environments, allowing targeted therapy only within the ischemic region. Here we map the 93-series compound binding site in the GluN1-GluN2B NMDA receptor amino terminal domain and show that the interaction of the N-alkyl group with a hydrophobic cage of the binding site is critical for pH-dependent inhibition. Mutation of residues in the hydrophobic cage alters pH-dependent potency, and remarkably, can convert inhibitors into potentiators. Our study provides a foundation for the development of highly specific neuroprotective compounds for the treatment of neurological diseases.

Recent developments in solution nuclear magnetic resonance (NMR)-based molecular biology.

Visualizing post-translational modifications, conformations, and interaction surfaces of protein structures at atomic resolution underpins the development of novel therapeutics to combat disease. As computational resources expand, in silico calculations coupled with experimentally derived structures and functional assays have led to an explosion in structure-based drug design (SBDD) with several compounds in clinical trials. It is increasingly clear that "hidden" transition-state structures along activation trajectories can be harnessed to develop novel classes of allosteric inhibitors. The goal of this mini-review is to empower the clinical researcher with a general knowledge of the strengths and weaknesses of nuclear magnetic resonance (NMR) spectroscopy in molecular medicine. Although NMR can determine protein structures at atomic resolution, its unrivaled strength lies in sensing subtle changes in a nuclei's chemical environment as a result of intrinsic conformational dynamics, solution conditions, and binding interactions. These can be recorded at atomic resolution, without explicit structure determination, and then incorporated with static structures or molecular dynamics simulations to produce a complete biological picture.

Abstract 1155: Regulation of glutamine metabolism: Allosteric activation and inhibition of mitochondrial glutaminase

Abstract The hydrolysis of glutamine to glutamate plus ammonia, catalyzed in the mitochondria by the enzyme glutaminase (Gls), plays a crucial role in the metabolism of glutamine by cancer cells. Recently, we discovered a novel class of allosteric inhibitors of a splice variant of Gls, known as glutaminase C (GAC), which inhibits the growth of Rho-GTPase transformed cells without affecting their normal cellular counterparts, with the lead compound being the bromo-benzophenanthridinone 968. Here, we took advantage of mouse embryonic fibroblasts (MEFs) transformed by the induced expression of an oncogenic guanine nucleotide exchange factor (GEF) named Dbl, which hyper-activates the Rho GTPases Rac, Rho, and CDC42, together with 13C-labeled glutamine and stable-isotope tracing methods, to establish that 968 selectively blocks the enhancement in glutaminolysis. We then determined how 968 inhibits the catalytic activity of GAC using biophysical approaches on purified GAC enzyme. First, we developed a FRET assay to examine the effects of 968 on the ability of GAC to undergo the dimer-to-tetramer transition necessary for enzyme activation. We next demonstrated how the fluorescence of a reporter group attached to GAC provides a direct read-out of the binding of 968 and related compounds to the enzyme. By combining these fluorescence assays, together with novel GAC mutants trapped in either the monomeric or dimeric state, we show that 968 has the highest affinity for monomeric GAC, and that the dose-dependent binding directly matches its dose-dependent inhibition of enzyme activity and cellular transformation. We then investigated the 968 structure-activity relationship, and to this end, characterized a robust real-time direct binding and inhibition assay for testing a panel of 968 derivatives. Additionally, we have developed novel 968-derivatives with unique functional moieties, such as fluorescent and photo-cross linking groups, to further elucidate the mechanism of GAC inhibition using recombinant GAC in vitro as well as in cancer cells. Together, these findings represent recent advances in our understanding of the regulation of GAC activity both in vitro and in cells, and highlight the potential future of targeting glutamine metabolism with novel small molecule therapeutics. Citation Format: Clint Stalnecker, Scott Ulrich, Jon Erickson, Sekar Ramachandran, Ralph DeBerardinis, Rick Cerione. Regulation of glutamine metabolism: Allosteric activation and inhibition of mitochondrial glutaminase. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1155. doi:10.1158/1538-7445.AM2015-1155

Abstract PR08: Drugging MYCN protein stability through an allosteric transition in Aurora kinase A

Abstract MYC family proteins represent a critical node in oncogenesis across a wide range of pediatric and adult cancers. This is particularly true in neuroblastoma, where amplification of MYCN confers an especially poor prognosis. Proteolytic degradation of MYCN protein is regulated in part by a kinase-independent function of Aurora Kinase A. We describe a class of allosteric inhibitors that disrupts the native conformation of Aurora A and causes degradation of MYCN protein across MYCN-expressing neuroblastoma cell lines. We compare co-crystal structures with structure-activity relationships across multiple inhibitors and chemotypes to delineate an Aurora A conformation-specific effect on proteolytic degradation of MYCN protein, which is driven markedly by conformation disrupting inhibitors, and impacted more modestly by current clinical Aurora Kinase A inhibitors, which disrupt the kinase structure less efficiently. Using functional assays of apoptosis, cell cycle, and proliferation we further define a distinct MYCN-specific effect distinguishing it from simple inhibition of mitosis by conventional Aurora kinase inhibitors. This new class of conformation disrupting inhibitors, which block stabilizing interactions between Aurora A and MYCN, represents a novel strategy to target MYCN-driven cancers. This abstract is also presented as Poster B46. Citation Format: W. Clay Gustafson, Justin G. Meyerowitz, Erin A. Nekritz, Elise Charron, Katherine K. Matthay, Nicholas T. Hertz, Martin Eilers, Kevan M. Shokat, William A. Weiss. Drugging MYCN protein stability through an allosteric transition in Aurora kinase A. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr PR08.

Conopeptide ρ-TIA Defines a New Allosteric Site on the Extracellular Surface of the α1B-Adrenoceptor

The G protein-coupled receptor (GPCR) superfamily is an important drug target that includes over 1000 membrane receptors that functionally couple extracellular stimuli to intracellular effectors. Despite the potential of extracellular surface (ECS) residues in GPCRs to interact with subtype-specific allosteric modulators, few ECS pharmacophores for class A receptors have been identified. Using the turkey β(1)-adrenergic receptor crystal structure, we modeled the α(1B)-adrenoceptor (α(1B)-AR) to help identify the allosteric site for ρ-conopeptide TIA, an inverse agonist at this receptor. Combining mutational radioligand binding and inositol 1-phosphate signaling studies, together with molecular docking simulations using a refined NMR structure of ρ-TIA, we identified 14 residues on the ECS of the α(1B)-AR that influenced ρ-TIA binding. Double mutant cycle analysis and docking confirmed that ρ-TIA binding was dominated by a salt bridge and cation-π between Arg-4-ρ-TIA and Asp-327 and Phe-330, respectively, and a T-stacking-π interaction between Trp-3-ρ-TIA and Phe-330. Water-bridging hydrogen bonds between Asn-2-ρ-TIA and Val-197, Trp-3-ρ-TIA and Ser-318, and the positively charged N terminus and Glu-186, were also identified. These interactions reveal that peptide binding to the ECS on transmembrane helix 6 (TMH6) and TMH7 at the base of extracellular loop 3 (ECL3) is sufficient to allosterically inhibit agonist signaling at a GPCR. The ligand-accessible ECS residues identified provide the first view of an allosteric inhibitor pharmacophore for α(1)-adrenoceptors and mechanistic insight and a new set of structural constraints for the design of allosteric antagonists at related GPCRs.

A Symmetric Region of the HIV-1 Integrase Dimerization Interface Is Essential for Viral Replication

HIV-1 integrase (IN) is an important target for contemporary antiretroviral drug design research. Historically, efforts at inactivating the enzyme have focused upon blocking its active site. However, it has become apparent that new classes of allosteric inhibitors will be necessary to advance the antiretroviral field in light of the emergence of viral strains resistant to contemporary clinically used IN drugs. In this study we have characterized the importance of a close network of IN residues, distant from the active site, as important for the obligatory multimerization of the enzyme and viral replication as a whole. Specifically, we have determined that the configuration of six residues within a highly symmetrical region at the IN dimerization interface, composed of a four-tiered aromatic interaction flanked by two salt bridges, significantly contributes to proper HIV-1 replication. Additionally, we have utilized a quantitative luminescence assay to examine IN oligomerization and have determined that there is a very low tolerance for amino acid substitutions along this region. Even conservative residue substitutions negatively impacted IN multimerization, resulting in an inactive viral enzyme and a non-replicative virus. We have shown that there is a very low tolerance for amino acid variation at the symmetrical dimeric interface region characterized in this study, and therefore drugs designed to target the amino acid network detailed here could be expected to yield a significantly reduced number of drug-resistant escape mutations compared to contemporary clinically-evaluated antiretrovirals.

Abstract B165: Abl kinase myristate-site inhibitor, mechanism and application

Abstract We recently reported a new class of allosteric inhibitors, exemplified by GNF-2, that selectively inhibit the proliferation of Bcr-Abl dependent cells. Here we demonstrate, using selection for resistant Bcr-Abl clones, site-directed mutagenesis, affinity chromatography, and steady-state kinetics, that GNF-2 inhibits Bcr-Abl kinase activity by binding to the Abl myristate binding pocket and stabilizes the auto-inhibited conformation. We demonstrate that the 2-hydroxyethyl amide analog of GNF-2, GNF-5, in combination with ATP-competitive inhibitors such as nilotinib and dasatinib can overcome the T315I “gatekeeper” mutant of Bcr-Abl which is resistant to all clinically approved Bcr-Abl inhibitors. These studies demonstrate that targeting of the Abl myristate binding site can provide an important pharmacological means to overcome mutations that cause resistance to ATP-competitive inhibitors. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B165.