Binding Modes Of Inhibitors Research Articles

Discovery of 1-Benzoyl 4-Phenoxypiperidines as Small-Molecule Inhibitors of the β-Catenin/B-Cell Lymphoma 9 Protein-Protein Interaction.

Structure-based design and optimization were performed to develop small-molecule β-catenin/B-cell lymphoma 9 (BCL9) inhibitors and improve their inhibitory activities. Compound ZL3138 with a novel 1-benzoyl 4-phenoxypiperidine scaffold was discovered to disrupt the β-catenin/BCL9 protein-protein interaction (PPI) with a Ki of 0.96 μM in AlphaScreen competitive inhibition assays and displayed good selectivity for β-catenin/BCL9 over β-catenin/E-cadherin PPIs. The binding mode of new inhibitors was characterized by structure-activity relationship and site-directed mutagenesis studies. Protein pull-down assays indicate that this series of compounds directly binds with β-catenin. Cellular target engagement and co-immunoprecipitation experiments demonstrate that ZL3138 binds with β-catenin and disrupts the β-catenin/BCL9 interaction without affecting the β-catenin/E-cadherin interaction in living cells. Further cell-based studies show that ZL3138 selectively suppresses transactivation of Wnt/β-catenin signaling, regulates transcription and expression of Wnt target genes, and inhibits the growth of Wnt/β-catenin-dependent cancer cells.

Progress toward B-Cell Lymphoma 6 BTB Domain Inhibitors for the Treatment of Diffuse Large B-Cell Lymphoma and Beyond.

B-cell lymphoma 6 (BCL6) is a master regulator of germinal center formation that produce antibody-secreting plasma cells and memory B-cells for sustained immune responses. The BTB domain of BCL6 (BCL6BTB) forms a homodimer that mediates transcriptional repression by recruiting its corepressor proteins to form a biologically functional transcriptional complex. The protein-protein interaction (PPI) between the BCL6BTB and its corepressors has emerged as a therapeutic target for the treatment of DLBCL and a number of other human cancers. This Perspective provides an overview of recent advances in the development of BCL6BTB inhibitors from reversible inhibitors, irreversible inhibitors, to BCL6 degraders. Inhibitor design and medicinal chemistry strategies for the development of novel compounds will be provided. The binding mode of new inhibitors to BCL6BTB are highlighted. Also, the in vitro and in vivo assays used for the evaluation of new compounds will be discussed.

Design, synthesis and biological evaluation of isoxazole-containing biphenyl derivatives as small-molecule inhibitors targeting the programmed cell death-1/ programmed cell death-ligand 1 immune checkpoint.

Monoclonal antibodies targeting the programmed cell death-1/ programmed cell death-ligand 1 (PD-1/PD-L1) immune checkpoint have achieved enormous success in cancer immunotherapy. But the antibody-based immunotherapies carry a number of unavoidable deficiencies such as poor pharmacokinetic properties and immunogenicity. Small-molecule PD-1/PD-L1 inhibitors offer the superiority of complementarity with monoclonal antibodies and represent an appealing alternative. A novel series of isoxazole-containing biphenyl compounds were designed, synthesized and evaluated as PD-1/PD-L1 inhibitors in this paper. The structure-activity relationship of the novel synthesized compounds indicated that the ring-closure strategy of introducing isoxazole could be employed and the 3-cyanobenzyl group was significant for the inhibitory activity against the PD-1/PD-L1 protein-protein interactions. Molecular docking studies were performed to help understand the binding mode of the small-molecule inhibitor with the PD-L1 dimer. In particular, compound II-12 was a promising anti-PD-1/PD-L1 inhibitor with the IC50 value of 23.0nM, providing valuable information for future drug development.

Levofloxacin and sulfa drugs linked via Schiff bases: Exploring their urease inhibition, enzyme kinetics and in silico studies

Involvement of urease in various pathological conditions specifically in gastric and peptic ulcers make it an important therapeutic target. In the present study urease inhibition was investigated by newly designed Schiff bases of levofloxacin. Structure elucidation of these compounds were done by spectral studies such as IR, 1H NMR and 13C NMR and elemental analysis. In vitro urease enzyme inhibition assay revealed the compounds LS01, LS06 and LS07 were found to be the most potent and showed comparable IC50 values 0.58±0.11, 0.45±0.21 µM and 0.52±0.28 µM respectively. The compound LS06 was competitive inhibitor with Ki value 1.13 µM while the compounds LS01 and LS07 were mixed type of inhibitors with Ki values 3.40 and 6.03 µM respectively. Plausible binding mode of competitive inhibitor was predicted using molecular docking studies. Ancillary to synthetic studies, density functional theory (DFT) at B3LYP/6–31G(d,p) basis sets in ground state is utilized in order to gain optimized geometries of LS01-LS09 molecules. Different geometric parameters like molecular electrostatic potential analysis, alignment of HOMO and LUMO levels, natural bonding orbital (NBO) analysis and global descriptor of reactivity were performed in support of experimental findings. All DFT based computed results showed best agreement with experimental finding and suggest that all synthesized compounds are chemically stable.

Chalcones and their B-aryl analogues as myeloperoxidase inhibitors: In silico, in vitro and ex vivo investigations

In the present study, a series of chalcones and their B-aryl analogues were prepared and evaluate as inhibitors of myeloperoxidase (MPO) chlorinating activity, using in vitro and ex vivo assays. Among these, B-thiophenyl chalcone (analogue 9) demonstrated inhibition of in vitro and ex vivo MPO chlorinating activity, exhibiting IC50 value of 0.53 and 19.2 µM, respectively. Potent ex vivo MPO inhibitors 5, 8 and 9 were not toxic to human neutrophils at 50 µM, as well as displayed weak 2,2-diphenyl-1-pycrylhydrazyl radical (DPPH•) and hypochlorous acid (HOCl) scavenger abilities. Docking simulations indicated binding mode of MPO inhibitors, evidencing hydrogen bonds between the amino group at 4′position (ring A) of chalcones with Gln91, Asp94, and Hys95 MPO residues. In this regard, the efficacy and low toxicity promoted aminochalcones and arylic analogues to the rank of hit compounds in the search for new non-steroidal anti-inflammatory compounds.

Inhibition Mechanism of Antimalarial Drugs Targeting the Cytochrome bc1 Complex

Plasmodium falciparum (P. falciparum) is the main parasite known to cause malaria in humans. The antimalarial drug atovaquone is known to inhibit the Qo-site of the cytochrome bc1 complex of P. falciparum, which ultimately blocks ATP synthesis, leading to cell death. Through the years, mutations of the P. falciparum cytochrome bc1 complex, causing resistance to atovaquone, have emerged. The present investigation applies molecular dynamics (MD) simulations to study how the specific mutations Y279S and L282V, known to cause atovaquone resistance in malarial parasites, affect the inhibition mechanism of two known inhibitors. Binding free energy estimates were obtained through free energy perturbation calculations but were unable to confidently resolve the effects of mutations due to the great complexity of the binding environment. Meanwhile, basic mechanistic considerations from the MD simulations provide a detailed characterization of inhibitor binding modes and indicate that the Y279S mutation weakens the natural binding of the inhibitors, while no conclusive effect of the L282V mutation could be observed.

Inhibitory mechanism of O-methylated quercetins, highly potent β-secretase inhibitors isolated from Caragana balchaschensis (Kom.) Pojark

Ethnopharmacological relevanceCaragana has a standing history of implementation in Traditional Chinese Medicine (TCM). Most species of this genus have been explored for multi-functional purposes, such as promoting blood circulation and curing neuralgia, fatigue, migraine, arthritis, and vascular hypertension (Meng et al., 2009). Among them, the well-known species C. sinica showed the most promising potential to increase the expression of ADAM10 among 313 tested medicinal plants, which is one of the promising approach for the treatment of Alzheimer's disease (AD). (Schuck et al., 2015). Aim of this studyThe aim of this work is to explore β-secretase inhibitory activity of compounds isolated from the aerial part of endemic Caragana balchaschensis (Kom.) Pojark. We provided a full characterization of their inhibitory mechanisms, binding affinities, and binding modes. Materials and methodsThe isolation of quercetin derivatives was accomplished by various chromatographical approaches and their structures were annotated by spectroscopic analysis. The detailed kinetic behavior of β-secretase inhibitors was determined by estimation of kinetic parameters (Km, Vmax, KI, and KIS). Binding affinities (KSV) and binding modes of inhibitors were elucidated by fluorescence quenching and molecular docking studies, respectively. ResultsO-methylated quercetins (2-7) were significantly effective in β-secretase inhibition with IC50 ranging from 1.2 to 6.5 μM. The most active one (6) was 20-fold effective than the mother skeleton, quercetin. The O-methyl motif was a critical factor in β-secretase inhibition: tri-O-methylated (1.2 μM) > di-O-methylated (3.5 μM) > mono-O-methylated (6.5 μM) > quercetin (25.2 μM). In the kinetic study, all quercetins (1–7) showed a noncompetitive inhibition, but glucoside ones (8 and 9) were mixed type I inhibitors. The binding affinities (KSV) were agreed with inhibitory potencies. The O-methylated quercetins were annotated as the most natural abundant metabolites in the aerial part by LC-ESI-TOF/MS. Binding modes of inhibitors to enzyme were elucidated by molecular docking experiments. ConclusionThis study disclosed that most of the major phenolic metabolites of the aerial part of C. balchaschensis are O-methylated quercetins, which have a significant inhibitory effect on β-secretase, which is a critical factor for AD.

The natural-based optimization of kojic acid conjugated to different thio-quinazolinones as potential anti-melanogenesis agents with tyrosinase inhibitory activity

Melanin pigment and melanogenesis are a two-edged sword. Melanin has a radioprotection role while melanogenesis has undesirable effects. Targeting the melanogenesis pathway, a series of kojyl thioether conjugated to different quinazolinone derivatives were designed, synthesized, and evaluated for their inhibitory activity against mushroom tyrosinase. All the synthesized compounds were screened for their anti-tyrosinase activity and all derivatives displayed better potency than kojic acid as the positive control. In this regard, 5j and 5h as the most active compounds showed an IC50 value of 0.46 and 0.50 µM, respectively. In kinetic evaluation against tyrosinase, 5j depicted an uncompetitive inhibition pattern. Designed compounds also exhibited mild antioxidant capacity. Moreover, 5j and 5h achieved good potency against the B16F10 cell line to reduce the melanin content, whilst showing limited toxicity against malignant cells. The proposed binding mode of new inhibitors evaluated through molecular docking was consistent with the results of structure–activity relationship analysis.

What coronavirus 3C-like protease tells us: From structure, substrate selectivity, to inhibitor design.

The emergence of a variety of coronaviruses (CoVs) in the last decades has posed huge threats to human health. Especially, the ongoing pandemic of coronavirus disease 2019 (COVID‐19) caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has led to more than 70 million infections and over 1.6 million of deaths worldwide in the past few months. None of the efficacious antiviral agents against human CoVs have been approved yet. 3C‐like protease (3CLpro) is an attractive target for antiviral intervention due to its essential role in processing polyproteins translated from viral RNA, and its conserved structural feature and substrate specificity among CoVs in spite of the sequence variation. This review focuses on all available crystal structures of 12 CoV 3CLpros and their inhibitors, and intends to provide a comprehensive understanding of this protease from multiple aspects including its structural features, substrate specificity, inhibitor binding modes, and more importantly, to recapitulate the similarity and diversity among different CoV 3CLpros and the structure–activity relationship of various types of inhibitors. Such an attempt could gain a deep insight into the inhibition mechanisms and drive future structure‐based drug discovery targeting 3CLpros.

Prediction of kinase inhibitors binding modes with machine learning and reduced descriptor sets

Protein kinases are receiving wide research interest, from drug perspective, due to their important roles in human body. Available kinase-inhibitor data, including crystallized structures, revealed many details about the mechanism of inhibition and binding modes. The understanding and analysis of these binding modes are expected to support the discovery of kinase-targeting drugs. The huge amounts of data made it possible to utilize computational techniques, including machine learning, to help in the discovery of kinase-targeting drugs. Machine learning gave reasonable predictions when applied to differentiate between the binding modes of kinase inhibitors, promoting a wider application in that domain. In this study, we applied machine learning supported by feature selection techniques to classify kinase inhibitors according to their binding modes. We represented inhibitors as a large number of molecular descriptors, as features, and systematically reduced these features in a multi-step manner while trying to attain high classification accuracy. Our predictive models could satisfy both goals by achieving high accuracy while utilizing at most 5% of the modeling features. The models could differentiate between binding mode types with MCC values between 0.67 and 0.92, and balanced accuracy values between 0.78 and 0.97 for independent test sets.

Discovery and Biological Evaluation of N-Methyl-pyrrolo[2,3-b]pyridine-5-carboxamide Derivatives as JAK1-Selective Inhibitors.

Janus kinase 1 (JAK1) plays a key role in most cytokine-mediated inflammatory and autoimmune responses through JAK/STAT signaling; thus, JAK1 inhibition is a promising therapeutic strategy for several diseases. Analysis of the binding modes of current JAK inhibitors to JAK isoforms allowed the design of N-alkyl-substituted 1-H-pyrrolo[2,3-b] pyridine carboxamide as a JAK1-selective scaffold, and the synthesis of various methyl amide derivatives provided 4-((cis-1-(4-chlorobenzyl)-2-methylpiperidin-4-yl)amino)-N-methyl-1H-pyrrolo[2,3-b]pyridine-5-carboxamide (31g) as a potent JAK1-selective inhibitor. In particular, the (S,S)-enantiomer of 31g (38a) exhibited excellent potency for JAK1 and selectivity over JAK2, JAK3, and TYK2. On investigating the effect of 31g on hepatic fibrosis, it was found that it reduces the proliferation and fibrogenic gene expression of TGF-β-induced hepatic stellate cells (HSCs). Specifically, 31g significantly inhibited TGF-β-induced migration of HSCs at 0.25 μM in wound-healing assays.

A combination of solid-state NMR and MD simulations reveals the binding mode of a rhomboid protease inhibitor.

Intramembrane proteolysis plays a fundamental role in many biological and pathological processes. Intramembrane proteases thus represent promising pharmacological targets, but few selective inhibitors have been identified. This is in contrast to their soluble counterparts, which are inhibited by many common drugs, and is in part explained by the inherent difficulty to characterize the binding of drug-like molecules to membrane proteins at atomic resolution. Here, we investigated the binding of two different inhibitors to the bacterial rhomboid protease GlpG, an intramembrane protease characterized by a Ser–His catalytic dyad, using solid-state NMR spectroscopy. H/D exchange of deuterated GlpG can reveal the binding position while chemical shift perturbations additionally indicate the allosteric effects of ligand binding. Finally, we determined the exact binding mode of a rhomboid protease-inhibitor using a combination of solid-state NMR and molecular dynamics simulations. We believe this approach can be widely adopted to study the structure and binding of other poorly characterized membrane protein–ligand complexes in a native-like environment and under physiological conditions.

Molecular dynamics investigation on the interaction of human angiotensin-converting enzyme with tetrapeptide inhibitors.

Angiotensin-converting enzyme (ACE) is a well-known zinc metalloenzyme whose physiological functions are vital to blood pressure regulation and management of hypertension. The development of more efficient peptide inhibitors is of great significance for the prevention and treatment of hypertension. In this research, molecular dynamics (MD) simulations were implemented to study the specific binding mechanism and interaction between human ACE (hACE) and tetrapeptides, YIHP, YKHP, YLVR, and YRHP. The calculation of relative binding free energy on the one hand verified that YLVR, an experimentally identified inhibitor, has a stronger inhibitory effect and, on the other hand, indicated that YRHP is the "best" inhibitor with the strongest binding affinity. Inspection of atomic interactions discriminated the specific binding mode of each tetrapeptide inhibitor with hACE and explained the difference of their affinity. Moreover, in-depth analysis of the MD production trajectories, including clustering, principal component analysis, and dynamic network analysis, determined the dynamic correlation between tetrapeptides and hACE and obtained the communities' distribution of a protein-ligand complex. The present study provides essential insights into the binding mode and interaction mechanism of the hACE-peptide complex, which paves a path for designing effective anti-hypertensive peptides.

Roles of Integration of Multiple Simulation Technologies in Insights into Binding Mechanism of Inhibitors to BRD Family

Insights into binding mechanism of inhibitors to targets are expected to provide a meaningfully theoretical guidance for design and development of effective inhibitors inhibiting of the activity of targets. It is well known that the bromodomain (BRD) family has been thought as a promising target utilized for treating various human diseases, such as inflammatory disorders, malignant tumors, acute myelogenous leukemia (AML), bone diseases, etc. In this work, we summarize the roles of integration of multiple simulation technologies in exploring atomic-level dynamics changes of the BRD family because of inhibitor bindings. Molecular dynamics (MD) simulations, binding free energy calculations, calculations of dynamics cross-correlation maps (DCCMs), and principal component (PC) analysis are integrated together to uncover binding modes of inhibitors to BRDs. The results obtained from binding free energy calculations can measure binding ability of inhibitors to BRDs, and explore the main driving forces of the binding of inhibitors to BRDs. The information stemming from PC analysis can reveal the changes in conformations, internal dynamics and movement patterns of BRDs due to inhibitor associations. Residue-based free energy decomposition method is wielded to unveil contributions of separate residues to inhibitor bindings, and explore the decisive factors that affect the bindings of inhibitors to BRDs.

A selectivity study of polysubstituted pyridinylimidazoles as dual inhibitors of JNK3 and p38α MAPK based on 3D-QSAR, molecular docking, and molecular dynamics simulation

Within the mitogen-activated protein kinase (MAPK) 1 family, the two kinases of c-Jun N-terminal kinase 3 (JNK3) and p38α MAPK have emerged in the last decades as particularly attractive therapeutic targets due to their implication in several neurodegenerative pathologic conditions. In this study, the structure and activity relationship of 60 dual JNK3/p38α MAPK inhibitors was explored; three-dimensional quantitative structure-activity relationship (3D-QSAR), including comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis fields (CoMSIA), was performed. From the data we got, the 3D-QSAR model (CoMFAJNK3 with q2 is 0.642, r2 is 0.958; CoMSIAJNK3 with q2 is 0.660, r2 is 0.963; CoMFAp38α with q2 is 0.605, r2 is 0.980; CoMSIAp38α with q2 is 0.608, r2 is 0.970) had good predictability. Molecular docking further revealed the binding mode of inhibitors to JNK3/p38α MAPK. The results of 3D-QSAR, molecular docking, and molecular dynamics simulation also provided guidance for the discovery of new dual inhibitors of JNK3 and p38α MAPK. Finally, 10 novel compounds with good potential activity and ADME/T profile were designed. Molecular dynamics simulation results validated that Met149/Lys 93/Gln 155 (JNK3) and Met109/Lys53 (p38α) located in the active site play a key role for novel dual inhibitors.

DDRE-31. EVALUATION OF HIGHLY POTENT AND SELECTIVE SINGLE-MOLECULE DUAL EGFR/PI3K INHIBITORS IN ADULT AND PEDIATRIC HIGH-GRADE GLIOMA

Abstract High grade gliomas (HGG) carry poor prognosis with median survival rates under 15 months post diagnosis. Due to dysregulation of kinase signaling pathways within these tumors, targeted kinase inhibition has been considered as a promising clinical strategy. However in HGG, many single-agent inhibitors of EGFR or PI3K have shown limited response due to activation of compensatory signaling. Single-molecule multikinase inhibitors may decrease resistance, present a single pharmacokinetic dosage profile, and reduce risks of multi-agent toxicities, supporting this strategy over dual drug combination approaches. To test this concept, a panel of inhibitors exploiting known binding modes of structurally-related ATP binding site inhibitors of EGFR/PI3K were synthesized and characterized. Of these, MTX-241 was least likely to act as a substrate for drug efflux proteins such as P-glycoprotein. Treatment of MTX-241 in a panel of eight human adult and pediatric HGG lines showed strong cytotoxic potency measured by growth inhibitory activity, with IC50s in the < 10 µM range. Tumor selectivity of MTX-241 was observed, with normal human astrocytes (NHA) nearly insensitive to MTX-241 even at >100 µM. MTX-241 was significantly more potent than clinically relevant inhibitors targeting EGFR/RTKs (gefitinib, lapatinib, dasatinib, imatinib) or PI3K (alpelisib, idelalisib). Synthesis and evaluation of a new series of compounds based on MTX-241 structure, but optimized for improved stability is ongoing. Our data suggests that a dual inhibitor of EGFR and PI3K, represents a viable therapeutic strategy in adult and pediatric HGG. Future studies will focus on evaluation of in vivo efficacy in tumor bearing mouse models.

Large domain movements through the lipid bilayer mediate substrate release and inhibition of glutamate transporters.

Glutamate transporters are essential players in glutamatergic neurotransmission in the brain, where they maintain extracellular glutamate below cytotoxic levels and allow for rounds of transmission. The structural bases of their function are well established, particularly within a model archaeal homolog, sodium, and aspartate symporter GltPh. However, the mechanism of gating on the cytoplasmic side of the membrane remains ambiguous. We report Cryo-EM structures of GltPh reconstituted into nanodiscs, including those structurally constrained in the cytoplasm-facing state and either apo, bound to sodium ions only, substrate, or blockers. The structures show that both substrate translocation and release involve movements of the bulky transport domain through the lipid bilayer. They further reveal a novel mode of inhibitor binding and show how solutes release is coupled to protein conformational changes. Finally, we describe how domain movements are associated with the displacement of bound lipids and significant membrane deformations, highlighting the potential regulatory role of the bilayer.

Malleability of the SARS-CoV-2 3CL Mpro Active-Site Cavity Facilitates Binding of Clinical Antivirals

The COVID-19 pandemic caused by SARS-CoV-2 requires rapid development of specific therapeutics and vaccines. The main protease of SARS-CoV-2, 3CL Mpro, is an established drug target for the design of inhibitors to stop the virus replication. Repurposing existing clinical drugs can offer a faster route to treatments. Here, we report on the binding mode and inhibition properties of several inhibitors using room temperature X-ray crystallography and in vitro enzyme kinetics. The enzyme active-site cavity reveals a high degree of malleability, allowing aldehyde leupeptin and hepatitis C clinical protease inhibitors (telaprevir, narlaprevir, and boceprevir) to bind and inhibit SARS-CoV-2 3CL Mpro. Narlaprevir, boceprevir, and telaprevir are low-micromolar inhibitors, whereas the binding affinity of leupeptin is substantially weaker. Repurposing hepatitis C clinical drugs as COVID-19 treatments may be a useful option to pursue. The observed malleability of the enzyme active-site cavity should be considered for the successful design of specific protease inhibitors.

Integrating Machine Learning-Based Virtual Screening With Multiple Protein Structures and Bio-Assay Evaluation for Discovery of Novel GSK3β Inhibitors.

Glycogen synthase kinase-3β (GSK3β) is associated with various key biological processes, and it has been considered as a critical therapeutic target for the treatment of many diseases. However, it is a big challenge to develop ATP-competition GSK3β inhibitors because of the high sequence homology with other kinases. In this work, a novel parallel virtual screening strategy based on multiple GSK3β protein structures, integrating molecular docking, complex-based pharmacophore, and naive Bayesian classification, was developed to screen a large chemical database, the 50 compounds with top-scores then underwent a luminescent kinase assay, which led to the discovery of two GSK3β inhibitor hits. The high screening enrichment rate indicates the reliability and practicability of the integrated protocol. Finally, molecular docking and molecular dynamics simulation were employed to investigate the binding modes of the GSK3β inhibitors, and some “hot residues” critical to GSK3β affinity were highlighted. The present study may provide some valuable guidance for the development of novel GSK3β inhibitors.

Biochemical, cellular and structural characterization of novel and selective ERK3 inhibitors

Triazolo[4,5-d]pyrimidin-5-amines were identified from kinase selectivity screening as novel ERK3 inhibitors with sub-100 nanomolar potencies in a biochemical assay using MK5 as substrate and with an attractive kinase selectivity profile. ERK3 crystal structures clarified the inhibitor binding mode in the ATP pocket with impact on A-loop, GC-loop and αC-helix conformations suggesting a potential structural link towards MK5 interaction via the FHIEDE motif. The inhibitors also showed sub-100 nM potencies in a cellular ERK3 NanoBRET assay and with excellent correlation to the biochemical IC50s. This novel series provides valuable tool compounds to further investigate the biological function and activation mechanism of ERK3.