Structural Basis For Inhibition Research Articles

Structural Aspects of the ROS1 Kinase Domain and Oncogenic Mutations

Protein kinases function as pivotal regulators in biological events, governing essential cellular processes through the transfer of phosphate groups from ATP molecules to substrates. Dysregulation of kinase activity is frequently associated with cancer, ocasionally arising from chromosomal translocation events that relocate genes encoding kinases. Fusion proteins resulting from such events, particularly those involving the proto-oncogene tyrosine-protein kinase ROS (ROS1), manifest as constitutively active kinases, emphasizing their role in oncogenesis. Notably, the chromosomal reallocation of the ros1 gene leads to fusion of proteins with the ROS1 kinase domain, implicated in various cancer types. Despite their prevalence, targeted inhibition of these fusion proteins relies on repurposed kinase inhibitors. This review comprehensively surveys experimentally determined ROS1 structures, emphasizing the pivotal role of X-ray crystallography in providing high-quality insights. We delve into the intricate interactions between ROS1 and kinase inhibitors, shedding light on the structural basis for inhibition. Additionally, we explore point mutations identified in patients, employing molecular modeling to elucidate their structural impact on the ROS1 kinase domain. By integrating structural insights with in vitro and in silico data, this review advances our understanding of ROS1 kinase in cancer, offering potential avenues for targeted therapeutic strategies.

Computational insight into structural basis of human ELOVL1 inhibition

Very long-chain fatty acids (VLCFAs) play a direct role in the development of a neurological disorder, X-linked adrenoleukodystrophy (X-ALD). Since ELOVL1 catalyzes the rate-limiting step of the synthesis of VLCFAs, it has emerged as an attractive target for the treatment of X-ALD. Recently two potent inhibitors, compound 22 (C22) and compound 27 (C27) have been reported to specifically inhibit human ELOVL1 but their structural basis of inhibition has not been explored. In the present study, we have used a homology model of human ELOVL1 to deduce the binding site and binding modes of C22 and C27. We have employed computational approaches to characterize the binding of C22 and C27. Initially, binding of hexacosanoyl-CoA (C26:0-CoA) to ELOVL1 was modelled and further validated by molecular dynamics (MD) simulation. We observed that the fatty acid tail of C26: CoA protrudes from a unique opening located at the occluded end of ELOVL1. Structural comparison of ELOVL1 with the crystal structure of ELOVL7 revealed that the unique opening was not present in human ELOVL7. Combined blind and focused molecular docking approaches revealed that C22 and C27 exhibit favourable binding in the same unique opening. Further, MD simulations and free binding energy calculations confirmed that C22 and C27 maintain the favourable binding in the unique opening of ELOVL1. Overall, our findings suggest that selective human ELOVL1 inhibitors block the binding of long tails of VLCFAs near the occluded end of ELOVL1. Present study will be helpful in the discovery and design of novel, selective and potent inhibitors of human ELOVL1.

Structural basis for inhibition and regulation of a chitin synthase from Candida albicans.

Chitin is one of the major components of the fungal cell wall. Chitin synthases (Chs's) catalyze chitin formation and translocation across the membrane. The detailed mechanism of chitin synthesis, translocation and assembly remains unknown due to the lack of high-resolution structures of chitin synthases. Chs's are targets of antifungal agents, including nikkomycin Z and polyoxin D. Nikkomycin Z has been clinically investigated to treat endemic fungal infectious disease (coccidioidomycosis). Polyoxin D has been widely used as a safe fungicide in organic agriculture. Lack of structural insights into the action of these inhibitors on Chs has hampered their further development for clinical usage because many fungal pathogens contain Chs's that are less sensitive to nikkomycins and polyoxins. We present the cryo-EM structures of Chs2 from the pathological fungi, Candida albicans (CaChs2), in the apo, substrate-bound, nikkomycin Z-bound, and polyoxin D-bound states. CaChs2 adopts a unique domain-swapped dimer configuration where a conserved motif in the domain swapped region controls enzyme activity. CaChs2 is also regulated by lipids. The chitin translocation tunnel is closed by the extracellular gate and plugged by a lipid molecule in the apo state to prevent non-specific leak. Analyses of substrate and inhibitor binding provide insights into the chemical logic of Chs inhibition, which can guide the optimization of Chs-targeted antifungal drugs.

Structural basis for inhibition and regulation of a chitin synthase from Candida albicans.

Chitin is an essential component of the fungal cell wall. Chitin synthases (Chss) catalyze chitin formation and translocation across the membrane and are targets of antifungal agents, including nikkomycin Z and polyoxin D. Lack of structural insights into the action of these inhibitors on Chs has hampered their further development to the clinic. We present the cryo-EM structures of Chs2 from Candida albicans (CaChs2) in the apo, substrate-bound, nikkomycin Z-bound, and polyoxin D-bound states. CaChs2 adopts a unique domain-swapped dimer configuration where a conserved motif in the domain-swapped region controls enzyme activity. CaChs2 has a dual regulation mechanism where the chitin translocation tunnel is closed by the extracellular gate and plugged by a lipid molecule in the apo state to prevent non-specific leak. Analyses of substrate and inhibitor binding provide insights into the chemical logic of Chs inhibition, which can guide Chs-targeted antifungal development.

Flavonoids from Alpinia officinarum as Potential Anti-Tubercular Agents Using Molecular Docking Studies

Bioguided isolation yielded three flavonoids galangin (1), kaempferide (2) and kaempferol (3) from methanol extract of Alpinia officinarum Hance. Galangin exhibited highest antitubercular activity, with MIC of < 4.0 μg/mL against M. bovis BCG (in vitro) and M. tuberculosis (ex vivo), than kaempferide. In vitro and ex vivo macrophage infection model assay revealed the inhibition of both active and dormant stage for M. tuberculosis H37Ra and M. bovis BCG on exposure to galangin. Molecular docking studies into the active site of DprE1 enzyme helped to understand the ligand-protein interactions and establish a structural basis for inhibition of M. tuberculosis. These flavonoids indicated their non-specificity towards M. tuberculosis by testing against Gram-positive and Gram-negative bacteria and also least cytotoxic up to 100 μg/mL on three human cancer cell lines THP-1, PANC-1 and A549, respectively. So these flavonoids are inhibitors against M. tuberculosis that can be explored further as potential antitubercular drugs.

Structural basis of inhibition of a transporter from Staphylococcus aureus, NorC, through a single-domain camelid antibody

Transporters play vital roles in acquiring antimicrobial resistance among pathogenic bacteria. In this study, we report the X-ray structure of NorC, a 14-transmembrane major facilitator superfamily member that is implicated in fluoroquinolone resistance in drug-resistant Staphylococcus aureus strains, at a resolution of 3.6 Å. The NorC structure was determined in complex with a single-domain camelid antibody that interacts at the extracellular face of the transporter and stabilizes it in an outward-open conformation. The complementarity determining regions of the antibody enter and block solvent access to the interior of the vestibule, thereby inhibiting alternating-access. NorC specifically interacts with an organic cation, tetraphenylphosphonium, although it does not demonstrate an ability to transport it. The interaction is compromised in the presence of NorC-antibody complex, consequently establishing a strategy to detect and block NorC and related transporters through the use of single-domain camelid antibodies.

Structural basis for inhibition of the AAA-ATPase Drg1 by diazaborine

The hexameric AAA-ATPase Drg1 is a key factor in eukaryotic ribosome biogenesis and initiates cytoplasmic maturation of the large ribosomal subunit by releasing the shuttling maturation factor Rlp24. Drg1 monomers contain two AAA-domains (D1 and D2) that act in a concerted manner. Rlp24 release is inhibited by the drug diazaborine which blocks ATP hydrolysis in D2. The mode of inhibition was unknown. Here we show the first cryo-EM structure of Drg1 revealing the inhibitory mechanism. Diazaborine forms a covalent bond to the 2′-OH of the nucleotide in D2, explaining its specificity for this site. As a consequence, the D2 domain is locked in a rigid, inactive state, stalling the whole Drg1 hexamer. Resistance mechanisms identified include abolished drug binding and altered positioning of the nucleotide. Our results suggest nucleotide-modifying compounds as potential novel inhibitors for AAA-ATPases.

Structure and Dynamics of Meprin β in Complex with a Hydroxamate-Based Inhibitor.

The astacin protease Meprin β represents an emerging target for drug development due to its potential involvement in disorders such as acute and chronic kidney injury and fibrosis. Here, we elaborate on the structural basis of inhibition by a specific Meprin β inhibitor. Our analysis of the crystal structure suggests different binding modes of the inhibitor to the active site. This flexibility is caused, at least in part, by movement of the C-terminal region of the protease domain (CTD). The CTD movement narrows the active site cleft upon inhibitor binding. Compared with other astacin proteases, among these the highly homologous isoenzyme Meprin α, differences in the subsites account for the unique selectivity of the inhibitor. Although the inhibitor shows substantial flexibility in orientation within the active site, the structural data as well as binding analyses, including molecular dynamics simulations, support a contribution of electrostatic interactions, presumably by arginine residues, to binding and specificity. Collectively, the results presented here and previously support an induced fit and substantial movement of the CTD upon ligand binding and, possibly, during catalysis. To the best of our knowledge, we here present the first structure of a Meprin β holoenzyme containing a zinc ion and a specific inhibitor bound to the active site. The structural data will guide rational drug design and the discovery of highly potent Meprin inhibitors.

Small Molecule HIV-1 Attachment Inhibitors: Discovery, Mode of Action and Structural Basis of Inhibition.

Viral entry into host cells is a critical step in the viral life cycle. HIV-1 entry is mediated by the sole surface envelope glycoprotein Env and is initiated by the interaction between Env and the host receptor CD4. This interaction, referred to as the attachment step, has long been considered an attractive target for inhibitor discovery and development. Fostemsavir, recently approved by the FDA, represents the first-in-class drug in the attachment inhibitor class. This review focuses on the discovery of temsavir (the active compound of fostemsavir) and analogs, mechanistic studies that elucidated the mode of action, and structural studies that revealed atomic details of the interaction between HIV-1 Env and attachment inhibitors. Challenges associated with emerging resistance mutations to the attachment inhibitors and the development of next-generation attachment inhibitors are also highlighted.

Structural Basis of Inhibition of Human Insulin-Regulated Aminopeptidase (IRAP) by Benzopyran-Based Inhibitors.

Inhibition of the insulin-regulated aminopeptidase (IRAP) improves memory and cognition in animal models. The enzyme has recently been crystallized and several series of inhibitors reported. We herein focused on one series of benzopyran-based inhibitors of IRAP known as the HFI series, with unresolved binding mode to IRAP, and developed a robust computational model to explain the structure-activity relationship (SAR) and potentially guide their further optimization. The binding model here proposed places the benzopyran ring in the catalytic binding site, coordinating the Zn2+ ion through the oxygen in position 3, in contrast to previous hypothesis. The whole series of HFI compounds was then systematically simulated, starting from this binding mode, using molecular dynamics and binding affinity estimated with the linear interaction energy (LIE) method. The agreement with experimental affinities supports the binding mode proposed, which was further challenged by rigorous free energy perturbation (FEP) calculations. Here, we found excellent correlation between experimental and calculated binding affinity differences, both between selected compound pairs and also for recently reported experimental data concerning the site directed mutagenesis of residue Phe544. The computationally derived structure-activity relationship of the HFI series and the understanding of the involvement of Phe544 in the binding of this scaffold provide valuable information for further lead optimization of novel IRAP inhibitors.

Medicinal Au(I) compounds targeting urease as prospective antimicrobial agents: unveiling the structural basis for enzyme inhibition.

A few gold compounds were recently found to show antimicrobial properties in vitro, holding great promise for the discovery of new drugs to overcome antibiotic resistance. Here, the inhibition of the bacterial virulence factor urease by four Au(I)-compounds, namely Au(PEt3)Cl, Au(PEt3)Br, Au(PEt3)I and [Au(PEt3)2]Cl, obtained from the antiarthritic Au(I)-drug Auranofin and earlier reported to act as antimicrobials, is investigated. The three monophosphino Au(I) complexes showed IC50 values in the 30-100 nM range, while the diphosphino Au(I) complex, though being less active, still showed a IC50 value of 7 μM. The structural basis for this inhibition was provided by solving the crystal structures of urease co-crystallized with Au(PEt3)I and [Au(PEt3)2]Cl: at least two Au(I) ions bind the enzyme in a flap domain involved in the catalysis, thus obliterating enzyme activity. Peculiar changes observed in the two structures reveal implications for the mechanism of soft metal binding and enzyme inactivation.

Validating the 1,2-Difluoro Motif As a Hybrid Bioisostere of CF3 and Et Using Matrix Metalloproteinases As Structural Probes.

Matrix metalloproteinases (MMPs) are involved in a spectrum of physiological processes, rendering them attractive targets for small-molecule drug discovery. Strategies to achieve selective inhibition continue to be intensively pursued, facilitated by advances in structural biology. Herein, we harness MMPs 2, 8, 9, and 13 to validate the vicinal difluoro motif as a hybrid bioisostere of CF3 and Et (BITE) in a series of modified barbiturate inhibitors. Crystallographic analyses of representative structures reveal conformations of the vicinal difluoro motif that manifest stabilizing hyperconjugative interactions consistent with the stereoelectronic gauche effect. Detailed docking studies of a potent difluorinated probe with MMP-9 are also disclosed and indicate that the structural basis of inhibition is a consequence of the anisotropic nature of the motif. Significant selectivity of MMP 13 versus MMP-2 can be achieved by subtle chain contraction in a BITE-modified inhibitor.

D-101 Structural Basis of Inhibition and Resistance Mechanism to EFdA, a highly potent NRTI

D-101 Structural Basis of Inhibition and Resistance Mechanism to EFdA, a highly potent NRTI

The JAK1/2 Inhibitor Momelotinib Does Not Inhibit Thiamine Uptake in Vitro

Abstract The clinical development of a selective JAK2 inhibitor, fedratinib, was terminated due to cases of Wernicke's encephalopathy, a disease related to thiamine insufficiency. Furthermore, a recent publication reported potent inhibition of the thiamine transporter 2 (THTR2; SLC19A3) by fedratinib1. This study aimed to determine the potential for other JAK inhibitors to inhibit thiamine transport and to better elucidate the structural basis for inhibition. The effect of the aminopyrimidine containing JAK inhibitors fedratinib, AZD1480, momelotinib, and XL-019 on thiamine transport in Caco-2 cells and HEK293 cells transfected with human THTR1 (SLC19A2) and 2 was assessed. Consistent with the prior publication, fedratinib inhibited thiamine uptake in Caco-2 cells (IC50=0.94 ± 0.08 μM) and THTR2 transfected cells (IC50=1.6 ± 0.52 μM). We also observed inhibition of THTR1 by fedratinib (IC50=7.9 ± 0.97 μM). Another JAK inhibitor, AZD1480, also inhibited thiamine transport, but to a lesser degree (Caco-2, IC50=8.1 ± 0.1 μM). In contrast, momelotinib and XL-019 did not inhibit thiamine transport in any of the systems tested. A pharmacophore model derived from the minimized structure of thiamine suggests that the 2,4-diaminopyrimidine containing JAK inhibitors fedratinib and AZD1480 can adopt a conformation matching several key features of thiamine, while inhibitors lacking the 4-amino group and/or an additional hydrogen bond donor poorly align with thiamine. In summary, these results suggest that not all JAK inhibitors have the potential to inhibit thiamine transport. In particular, momelotinib, currently in Phase 3 clinical trials for the treatment of pancreatic cancer and myelofibrosis, showed no potential to affect thiamine levels. Disclosures Giacomini: Gilead Sciences: Employment. Hao:Gilead Sciences: Employment. Chandrasekhar:Gilead Sciences: Employment. Twelves:Gilead Sciences: Employment. Whitney:Gilead Sciences: Employment. Lepist:Gilead Sciences: Employment. Ray:Gilead Sciences: Employment.

Abstract 1672: Structural basis for inhibition of ligand-dependent and -independent ErbB3 activation by KTN3379

Abstract ErbB3 signaling plays an important role in the progression of a number of solid tumor types, and is the target of several therapeutic monoclonal antibodies. In cancer, ErbB3 activation can be driven by its ligand Neuregulin, or in its absence, through high overexpression of its co-receptor ErbB2. Here, we report that the ErbB3 monoclonal antibody KTN3379, currently in phase 1 clinical development, robustly inhibits both mechanisms of ErbB3 activation in different tumor model settings. The crystal structure of a KTN3379 Fab in complex with the full ErbB3 extracellular domain reveals that the antibody binds with very high affinity to a novel and unique epitope in the boundary of domains 2 and 3, and locks the receptor in an inactive conformation. Thus, KTN3379 interferes with the first step of ErbB3 activation, which is required for both ligand-dependent and ligand-independent signaling. The mechanism of inhibition described here should be also applicable to EGFR and ErbB4. Citation Format: Diego Alvarado, Sangwon Lee, Etienne Greenlee, Gwenda F. Ligon, Jay S. Lillquist, Edward J. Natoli, Theresa M. LaVallee, Joseph Schlessinger. Structural basis for inhibition of ligand-dependent and -independent ErbB3 activation by KTN3379. [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 1672. doi:10.1158/1538-7445.AM2015-1672

277 The discovery and pre-clinical development of the first clinical stage EZH2-inhibitor, EPZ-6438 (E7438)

genes that govern progression through the cell cycle, the initiation of DNA repair, and apoptosis. The activity of p53 is tightly regulated by the MDM2 protein, in an auto-regulatory feedback loop. Inhibition of the MDM2−p53 protein–protein complex by small molecule inhibitors has been shown to reactivate normal p53 pathways in cells overexpressing MDM2, and so exert an anti-cancer effect. A number of series of potent small-molecule inhibitors have been developed as far as clinical trials. Understanding of the structural basis for inhibition from ligand-bond protein X-ray structures has been important in the optimisation of most of these series. We have developed a class of inhibitors of the MDM2−p53 interaction, based on an isoindolinone scaffold [J. Med. Chem. 2006, 49, 6209–6221, Bioorg. Med. Chem. Letters 2011, 21, 5916– 5919]. NU8406A, (R)-4-chloro-3-(4-chlorophenyl)-3-((1-(hydroxymethyl)cyclopropyl)methoxy)-2-(4-nitrobenzyl)isoindolin-1-one (IC50 = 43.8±6.2 nM) shows comparable in vitro activity to Nutlin-3a. Recently, we reported the MDM2 co-crystal structure of Nutlin-3a using a surface-entropy reduction mutant [Acta Cryst 2013, D69, 1358–1366]. We have applied these MDM2 mutants to solve the co-crystal structure of NU8406A. The structure shows that the binding mode of the isoindolinone is comparable to that for Nutlin-3a, and confirms the (R)-stereochemistry of the ligand. Preliminary structure–activity studies have revealed significant differences between the two series in the way that the Phe19 pocket is occupied.

407 Structural basis for inhibition of ligand-dependent and -independent ErbB3 activation by KTN3379

407 Structural basis for inhibition of ligand-dependent and -independent ErbB3 activation by KTN3379

Chloroquine Binding Reveals Flavin Redox Switch Function of Quinone Reductase 2

Quinone reductase 2 (NQO2) is an FAD-linked enzyme and the only known human target of two antimalarial drugs, primaquine (PQ) and chloroquine (CQ). The structural differences between oxidized and reduced NQO2 and the structural basis for inhibition by PQ and CQ were investigated by x-ray crystallography. Structures of oxidized NQO2 in complex with PQ and CQ were solved at 1.4 Å resolution. CQ binds preferentially to reduced NQO2, and upon reduction of NQO2-CQ crystals, the space group changed from P2(1)2(1)2(1) to P2(1), with 1-Å decreases in all three unit cell dimensions. The change in crystal packing originated in the negative charge and 4-5º bend in the reduced isoalloxazine ring of FAD, which resulted in a new mode of CQ binding and closure of a flexible loop (Phe(126)-Leu(136)) over the active site. This first structure of a reduced quinone reductase shows that reduction of the FAD cofactor and binding of a specific inhibitor lead to global changes in NQO2 structure and is consistent with a functional role for NQO2 as a flavin redox switch.

α-Amylase Inhibition: Some Practical Considerations

Enzyme inhibitors are important tools of nature for regulating the activity of enzymes in case of emergency. Proteinaceous inhibitors are the best studied in this group. Comparatively less is known about the inhibitors of α-amylase, which might be an attractive candidate having potentials in various fields, from the treatment of diabetes to crop protection. The area has benefited from the recent determination of structures of α-amylases, inhibitors and complexes. Besides, discussing various types of α-amylase inhibitors and their structural basis of inhibition, the biotechnological approaches for exploitation of its inhibitory function have been outlined in this minireview. Keywords: Amylase, inhibitor, acarbose, starch, diabetes, Proteinaceous inhibitors, α-amylase, phytopathogenic microorganisms, transgenic plants, Eleusine coracana, porcine pancreas (PPA), Streptomyces, Sorghum, Trichoderma resei, 3-D structure, hyperglycemia, acarviosine-glucose, α-amylase inhibitor, Apostolidis, γ-purothionin, thaumatin, Tribolium castaneum, bruchids, pig pancreatic α-amylase, Ragi bifunctional inhibitor, sorghum inhibitor, Ragi α-amylase/trypsin inhibitor, Barley α-amylase/subtilisin inhibitor

AV411 (Ibudilast) and AV1013 are non-competitive inhibitors of macrophage migration inhibitory factor: a novel induced-fit allosteric inhibition mechanism (133.11)

Abstract AV411 (Ibudilast) is an anti-inflammatory drug that was initially developed for the treatment of bronchial asthma but which also has been used for cerebrovascular and ocular indications. It is a non-selective inhibitor of various phosphodiesterases (PDEs) and has varied anti-inflammatory activity. More recently, AV411 has been studied as a possible therapeutic for the treatment of neuropathic pain and opioid withdrawal through its actions on glial cells. As described herein, the PDE inhibitors rolipram, theophylline, isobutylmethylxanthine (IBMX), cilostazol, arofylline, AV411 and its PDE-inhibition compromised analog AV1013 inhibit the catalytic and chemotactic functions of the pro-inflammatory protein, macrophage migration inhibitory factor (MIF). Enzymatic analysis indicates that these compounds are non-competitive inhibitors of the p-hydroxyphenylpyruvate (HPP) tautomerase activity of MIF. The allosteric binding site of AV411 and AV1013 is identified by NMR and X-ray crystallography, and the structural basis of inhibition is reported using the crystal structure of the MIF/AV1013/HPP ternary complex.