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-Δ^12,14-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.     

Validation of an Allosteric Binding Site of Src Kinase Identified by Unbiased Ligand Binding Simulations

Allostery plays a primary role in regulating protein activity, making it an important mechanism in human disease and drug discovery. Identifying allosteric regulatory sites to explore their biological significance and therapeutic potential is invaluable to drug discovery; however, identification remains a challenge. Allosteric sites are often “cryptic” without clear geometric or chemical features. Since allosteric regulatory sites are often less conserved in protein kinases than the orthosteric ATP binding site, allosteric ligands are commonly more specific than ATP competitive inhibitors. We present a generalizable computational protocol to predict allosteric ligand binding sites based on unbiased ligand binding simulation trajectories. We demonstrate the feasibility of this protocol by revisiting our previously published ligand binding simulations using the first identified viral proto-oncogene, Src kinase, as a model system. The binding paths for kinase inhibitor PP1 uncovered three metastable intermediate states before binding the high-affinity ATP-binding pocket, revealing two previously known allosteric sites and one novel site. Herein, we validate the novel site using a combination of virtual screening and experimental assays to identify a V-type allosteric small-molecule inhibitor that targets this novel site with specificity for Src over closely related kinases. This study provides a proof-of-concept for employing unbiased ligand binding simulations to identify cryptic allosteric binding sites and is widely applicable to other protein–ligand systems.     

Protein Allostery and Ligand Design: Computational Design Meets Experiments to Discover Novel Chemical Probes

Herein we examine the determinants of the allosteric inhibition of the mitochondrial chaperone TRAP1 by a small molecule ligand. The knowledge generated is harnessed into the design of novel derivatives with interesting biological properties.TRAP1 is a member of the Hsp90 family of proteins, which work through sequential steps of ATP processing coupled to client-protein remodeling. Isoform selective inhibition of TRAP1 can provide novel information on the biomolecular mechanisms of molecular chaperones, as well as new insights into the development of small molecules with therapeutic potential.Our analysis of the interactions between an active first-generation allosteric ligand and TRAP1 shows how the small molecule induces long-range perturbations that influence the attainment of reactive poses in the active site. At the same time, the dynamic adaptation of the allosteric binding pocket to the presence of the first-generation compound sets the stage for the design of a set of second-generation ligands: the characterization of the formation/disappearance of pockets around the allosteric site that is used to guide optimize the ligands’ fit for the allosteric site and improve inhibitory activities. The effects of the newly designed molecules are validated experimentally in vitro and in vivo. We discuss the implications of our approach as a promising strategy towards understanding the molecular determinants of allosteric regulation in chemical and molecular biology, and towards speeding up the design of allosteric small molecule modulators.     

Discovery of isoquinolinone and naphthyridinone-based inhibitors of poly(ADP-ribose) polymerase-1 (PARP1) as anticancer agents: Structure activity relationship and preclinical characterization

The exploitation of GLU988 and LYS903 residues in PARP1 as targets to design isoquinolinone (I & II) and naphthyridinone (III) analogues is described. Compounds of structure I have good biochemical and cellular potency but suffered from inferior PK. Constraining the linear propylene linker of structure I into a cyclopentene ring (II) offered improved PK parameters, while maintaining potency for PARP1. Finally, to avoid potential issues that may arise from the presence of an anilinic moiety, the nitrogen substituent on the isoquinolinone ring was incorporated as part of the bicyclic ring. This afforded a naphthyridinone scaffold, as shown in structure III. Further optimization of naphthyridinone series led to identification of a novel and highly potent PARP1 inhibitor 34, which was further characterized as preclinical candidate molecule. Compound 34 is orally bioavailable and displayed favorable pharmacokinetic (PK) properties. Compound 34 demonstrated remarkable antitumor efficacy both as a single-agent as well as in combination with chemotherapeutic agents in the BRCA1 mutant MDA-MB-436 breast cancer xenograft model. Additionally, compound 34 also potentiated the effect of agents such as temozolomide in breast cancer, pancreatic cancer and Ewing’s sarcoma models.     

Discovery and structure activity relationships of 7-benzyl triazolopyridines as stable,selective, and reversible inhibitors of myeloperoxidase

Myeloperoxidase (MPO) is a heme peroxidase found in neutrophils, monocytes and macrophages that efficiently catalyzes the oxidation of endogenous chloride into hypochlorous acid for antimicrobial activity. Chronic MPO activation can lead to indiscriminate protein modification causing tissue damage, and has been associated with chronic inflammatory diseases, atherosclerosis, and acute cardiovascular events. Triazolopyrimidine 5 is a reversible MPO inhibitor; however it suffers from poor stability in acid, and is an irreversible inhibitor of the DNA repair protein methyl guanine methyl transferase (MGMT). Structure-based drug design was employed to discover benzyl triazolopyridines with improved MPO potency, as well as acid stability, no reactivity with MGMT, and selectivity against thyroid peroxidase (TPO). Structure-activity relationships, a crystal structure of the MPO-inhibitor complex, and acute in vivo pharmacodynamic data are described herein.     

Scaffold-hopping identifies furano[2,3-d]pyrimidine amides as potent Notum inhibitors

The carboxylesterase Notum is a key negative regulator of the Wnt signaling pathway by mediating the depalmitoleoylation of Wnt proteins. Our objective was to discover potent small molecule inhibitors of Notum suitable for exploring the regulation of Wnt signaling in the central nervous system. Scaffold-hopping from thienopyrimidine acids 1 and 2, supported by X-ray structure determination, identified 3-methylimidazolin-4-one amides 20–24 as potent inhibitors of Notum with activity across three orthogonal assay formats (biochemical, extra-cellular, occupancy). A preferred example 24 demonstrated good stability in mouse microsomes and plasma, and cell permeability in the MDCK-MDR1 assay albeit with modest P-gp mediated efflux. Pharmacokinetic studies with 24 were performed in vivo in mouse with single oral administration of 24 showing good plasma exposure and reasonable CNS penetration. We propose that 24 is a new chemical tool suitable for cellular studies to explore the fundamental biology of Notum.     

Optimisation of estrogen receptor subtype-selectivity of a 4-Aryl-4H-chromene scaffold previously identified by virtual screening

4-Aryl-4H-Chromene derivatives have been previously shown to exhibit anti-proliferative, apoptotic and anti-angiogenic activity in a variety of tumor models in vitro and in vivo generally via activation of caspases through inhibition of tubulin polymerisation. We have previously identified by Virtual Screening (VS) a 4-aryl-4H-chromene scaffold, of which two examples were shown to bind Estrogen Receptor α and β with low nanomolar affinity and <20-fold selectivity for α over β and low micromolar anti-proliferative activity in the MCF-7 cell line. Thus, using the 4-aryl-4H-chromene scaffold as a starting point, a series of compounds with a range of basic arylethers at C-4 and modifications at the C3-ester substituent of the benzopyran ring were synthesised, producing some potent ER antagonists in the MCF-7 cell line which were highly selective for ERα (compound 35; 350-fold selectivity) or ERβ (compound 42; 170-fold selectivity).     

Inhibition of Mycobacterium tuberculosis InhA: Design,synthesis and evaluation of new di-triclosan derivatives

Multi-drug resistant tuberculosis (MDR-TB) represents a growing problem for global healthcare systems. In addition to 1.3 million deaths in 2018, the World Health Organisation reported 484,000 new cases of MDR-TB. Isoniazid is a key anti-TB drug that inhibits InhA, a crucial enzyme in the cell wall biosynthesis pathway and identical in Mycobacterium tuberculosis and M. bovis. Isoniazid is a pro-drug which requires activation by the enzyme KatG, mutations in KatG prevent activation and confer INH-resistance. ‘Direct inhibitors’ of InhA are attractive as they would circumvent the main clinically observed resistance mechanisms. A library of new 1,5-triazoles, designed to mimic the structures of both triclosan molecules uniquely bound to InhA have been synthesised. The inhibitory activity of these compounds was evaluated using isolated enzyme assays with 2 (5-chloro-2-(4-(5-(((4-(4-chloro-2-hydroxyphenoxy)benzyl)oxy)methyl)-1H-1,2,3-triazol-1-yl)phenoxy)phenol) exhibiting an IC₅₀ of 5.6 µM. Whole-cell evaluation was also performed, with 11 (5-chloro-2-(4-(5-(((4-(cyclopropylmethoxy)benzyl)oxy)methyl)-1H-1,2,3-triazol-1-yl)phenoxy)phenol) showing the greatest potency, with an MIC₉₉ of 12.9 µM against M. bovis.     

Explaining and Predicting Allostery with Allosteric Database and Modern Analytical Techniques

Allostery is a phenomenon that the protein activity is regulated when a non-functional site on it is bounded. This phenomenon is important in life process and disease therapy. However, it is difficult to study allostery due to the lack of knowledge. Facing this demand, we have created Allosteric Database (ASD) 10 years before to collect numerous kinds of allosteric data. In this review, we will introduce the 4 categories of data in ASD. For each category, we further reviewed how researchers applied ASD data to conduct studies. We focused on their research topics, analytical methods and conclusions. Several discoveries of new drug targets and allosteric modulators driven by ASD are also summarized. We hope this review could inspire researchers with new utilities of ASD data.     

Neutralizing Antibodies Against Allosteric Proteins: Insights From a Bacterial Adhesin

Allosteric proteins transition between ‘inactive’ and ‘active’ states. In general, such proteins assume distinct conformational states at the level of secondary, tertiary and/or quaternary structure. Different conformers of an allosteric protein can be antigenically dissimilar and induce antibodies with a highly distinctive specificities and neutralizing functional effects. Here we summarize studies on various functional types of monoclonal antibodies obtained against different allosteric conformers of the mannose-specific bacterial adhesin FimH – the most common cell attachment protein of Escherichia coli and other enterobacterial pathogens. Included are types of antibodies that activate the FimH function via interaction with ligand-induced binding sites or by wedging between domains as well as antibodies that inhibit FimH through orthosteric, parasteric, or novel dynasteric mechanisms. Understanding the molecular mechanism of antibody action against allosteric proteins provides insights on how to design antibodies with a desired functional effect, including those with neutralizing activity against bacterial and viral cell attachment proteins.     

Allostery and Missense Mutations as Intermittently Linked Promising Aspects of Modern Computational Drug Discovery

Drug research and development is a multidisciplinary field with its own successes. Yet, given the complexity of the process, it also faces challenges over the long development stages and even includes those that develop once a drug is marketed, i.e. drug toxicity and drug resistance. Better success can be achieved via well designed criteria in the early drug development stages. Here, we introduce the concepts of allostery and missense mutations, and argue that incorporation of these two intermittently linked biological phenomena into the early computational drug discovery stages would help to reduce the attrition risk in later stages of the process. We discuss the individual or in concert mechanisms of actions of mutations in allostery. Design of allosteric drugs is challenging compared to orthosteric drugs, yet they have been gaining popularity in recent years as alternative systems for the therapeutic regulation of proteins with an action-at-a-distance mode and non-invasive mechanisms. We propose an easy-to-apply computational allosteric drug discovery protocol which considers the mutation effect, and detail it with three case studies focusing on (1) analysis of effect of an allosteric mutation related to isoniazid drug resistance in tuberculosis; (2) identification of a cryptic pocket in the presence of an allosteric mutation of falcipain-2 as a malarial drug target; and (3) deciphering the effects of SARS-CoV-2 evolutionary mutations on a potential allosteric modulator with changes to allosteric communication paths.     

Synthesis of novel 2-pyrrolidinone and pyrrolidine derivatives and study of their inhibitory activity against autotaxin enzyme

Autotaxin (ATX), a glycoprotein (~125 kDa) isolated as an autocrine motility factor from melanoma cells, belongs to a seven-membered family of ectonucleotide pyrophosphatase/phosphodiesterase (ENPP), and exhibits lysophospholipase D activity. ATX is responsible for the hydrolysis of lysophosphatidylcholine (LPC) to produce the bioactive lipid lysophosphatidic acid (LPA), which is upregulated in a variety of pathological inflammatory conditions, including fibrosis, cancer, liver toxicity and thrombosis. Given its role in human disease, the ATX-LPA axis is an interesting target for therapy, and the development of novel potent ATX inhibitors is of great importance. In the present work a novel class of ATX inhibitors, optically active derivatives of 2-pyrrolidinone and pyrrolidine heterocycles were synthesized. Some of them exhibited interesting in vitro activity, namely the hydroxamic acid 16 (IC₅₀ 700 nM) and the carboxylic acid 40b (IC₅₀ 800 nM), while the boronic acid derivatives 3k (IC₅₀ 50 nM), 3l (IC₅₀ 120 nM), 3 m (IC₅₀ 180 nM) and 21 (IC₅₀ 35 nM) were found to be potent inhibitors of ATX.     

Structural Basis of the Allosteric Inhibition of Human ABCG2 by Nanobodies

ABCG2 is an ATP-binding cassette transporter that exports a wide range of xenobiotic compounds and has been recognized as a contributing factor for multidrug resistance in cancer cells. Substrate and inhibitor interactions with ABCG2 have been extensively studied and small molecule inhibitors have been developed that prevent the export of anticancer drugs from tumor cells. Here, we explore the potential for inhibitors that target sites other than the substrate binding pocket of ABCG2. We developed novel nanobodies against ABCG2 and used functional analyses to select three inhibitory nanobodies (Nb8, Nb17 and Nb96) for structural studies by single particle cryo-electron microscopy. Our results showed that these nanobodies allosterically bind to different regions of the nucleotide binding domains. Two copies of Nb8 bind to the apex of the NBDs preventing them from fully closing. Nb17 binds near the two-fold axis of the transporter and interacts with both NBDs. Nb96 binds to the side of the NBD and immobilizes a region connected to key motifs involved in ATP binding and hydrolysis. All three nanobodies prevent the transporter from undergoing conformational changes required for substrate transport. These findings advance our understanding of the molecular basis of modulation of ABCG2 by external binders, which may contribute to the development of a new generation of inhibitors. Furthermore, this is the first example of modulation of human multidrug resistance transporters by nanobodies.     

Rational design,synthesis and biological evaluation of novel multitargeting anti-AD iron chelators with potent MAO-B inhibitory and antioxidant activity

A series of (3-hydroxypyridin-4-one)-coumarin hybrids were developed and investigated as potential multitargeting candidates for the treatment of Alzheimer's disease (AD) through the incorporation of iron-chelating and monoamine oxidase B (MAO-B) inhibition. This combination endowed the hybrids with good capacity to inhibit MAO-B as well as excellent iron-chelating effects. The pFe³⁺ values of the compounds were ranging from 16.91 to 20.16, comparable to more potent than the reference drug deferiprone (DFP). Among them, compound 18d exhibited the most promising activity against MAO-B, with an IC₅₀ value of 87.9 nM. Moreover, compound 18d exerted favorable antioxidant activity, significantly reversed the amyloid-β_1-42 (Aβ_1-42) induced PC12 cell damage. More importantly, 18d remarkably ameliorated the cognitive dysfunction in a scopolamine-induced mice AD model. In brief, a series of hybrids with potential anti-AD effect were successfully obtained, indicating that the design of iron chelators with MAO-B inhibitory and antioxidant activities is an attractive strategy against AD progression.     

Discovery of ASP5286: A novel non-immunosuppressive cyclophilin inhibitor for the treatment of HCV

Evidence indicates that hepatitis C virus (HCV) utilizes cellular cyclophilin proteins in its replication, and cyclophilin inhibitors represent a new class of anti-HCV agents. We have established an efficient synthetic methodology to generate FR901459 derivatives via N, O-acyl migration reaction while avoiding total synthesis. Through a detailed structure–activity relationship study, we improved anti-HCV activity while decreasing immunosuppressive activity. Additionally, we discovered the importance of substitution at the 3 position for not only improving anti-HCV activity but also pharmacokinetic profile. Finally, by striking an appropriate balance between potency, solubility, and permeability, we discovered ASP5286 (13) as a potential clinical candidate for anti-HCV therapy.     

Development of an efficient semisynthetic modification of FR901459 via a novel regioselective N,O-acyl migration

HCV utilizes cellular protein cyclophilins in the virus replication cycle and cyclophilin inhibitors have become a new class of anti-HCV agents. In our screening of natural products, we identified a unique cyclosporin analogue, FR901459, as a cyclophilin inhibitor with potent anti-HCV activity. In this work, we developed an efficient synthetic methodology to prepare FR901459 derivatives via an N, O-acyl migration reaction. This method allows us to efficiently manipulate the amino acid residues at the 3 position while avoiding lengthy total synthesis for each compound. By using this methodology, we discovered 4, which has superior anti-HCV activity and decreased immunosuppressive activity compared to FR901459.     

The MDMX Acidic Domain Uses Allovalency to Bind Both p53 and MDMX

Autoinhibition of p53 binding to MDMX requires two short-linear motifs (SLiMs) containing adjacent tryptophan (WW) and tryptophan-phenylalanine (WF) residues. NMR spectroscopy was used to show the WW and WF motifs directly compete for the p53 binding site on MDMX and circular dichroism spectroscopy was used to show the WW motif becomes helical when it is bound to the p53 binding domain (p53BD) of MDMX. Binding studies using isothermal titration calorimetry showed the WW motif is a stronger inhibitor of p53 binding than the WF motif when they are both tethered to p53BD by the natural disordered linker. We also investigated how the WW and WF motifs interact with the DNA binding domain (DBD) of p53. Both motifs bind independently to similar sites on DBD that overlap the DNA binding site. Taken together our work defines a model for complex formation between MDMX and p53 where a pair of disordered SLiMs bind overlapping sites on both proteins.     

Novel alkoxyamide-based histone deacetylase inhibitors reverse cisplatin resistance in chemoresistant cancer cells

Although histone deacetylase inhibitors (HDACi) have shown promising antitumor effects in specific types of blood cancer, their effects on solid tumors are limited. Previously, we developed LMK235 (5), a class I and class IIb preferential HDACi with chemosensitizing effects on breast cancer, ovarian cancer and HNSCC. Based on its promising effects on solid tumor cells, we modified the cap group of 5 to improve its anticancer activity. The tri- and dimethoxy-phenyl substituted compounds 13a and 13d turned out to be the most potent HDAC inhibitors of this study. The isoform profiling revealed a dual HDAC2/HDAC6 inhibition profile, which was confirmed by the acetylation of α-tubulin and histone H3 in Cal27 and Cal27CisR. In combination with cisplatin, both compounds enhanced the cisplatin-induced cytotoxicity via caspase-3/7 activation. The effect was more pronounced in the cisplatin resistant subline Cal27CisR. The pretreatment with 13d resulted in a complete resensitisation of Cal27CisR with IC₅₀ values in the range of the parental cell line. Therefore, 13d may serve as an epigenetic tool to analyze and modulate the cisplatin resistance of solid tumors.