Allosteric Binding Pocket Research Articles

Structural insights into endogenous ligand selectivity and activation mechanisms of FFAR1 and FFAR2

Free fatty acid receptors (FFARs) play critical roles in metabolic regulation and are potential therapeutic targets for metabolic and inflammatory diseases. A comprehensive understanding of the activation mechanisms and endogenous ligand selectivity of FFARs is essential for drug discovery. Here, we report two cryoelectron microscopy structures of the human FFAR1 bound to the endogenous ligand docosahexaenoic acid (DHA) and Gi1 protein as well as FFAR2 in complex with butyrate and Gi1 at 3.2Å and 3.3Å resolution, respectively. These structures highlight that distinct locations and sizes of the orthosteric ligand binding pockets are crucial determinants of the endogenous ligand selectivity of this receptor subfamily. Additionally, computational analysis reveals a potential allosteric ligand binding pocket in FFAR2. Furthermore, we observe that the upward movement of helix V upon endogenous ligand binding is responsible for receptor activation. These insights will significantly aid in the development of drugs targeting this receptor family.

Targeting SHP2 Cryptic Allosteric Sites for Effective Cancer Therapy.

SHP2, a pivotal component downstream of both receptor and non-receptor tyrosine kinases, has been underscored in the progression of various human cancers and neurodevelopmental disorders. Allosteric inhibitors have been proposed to regulate its autoinhibition. However, oncogenic mutations, such as E76K, convert SHP2 into its open state, wherein the catalytic cleft becomes fully exposed to its ligands. This study elucidates the dynamic properties of SHP2 structures across different states, with a focus on the effects of oncogenic mutation on two known binding sites of allosteric inhibitors. Through extensive modeling and simulations, we further identified an alternative allosteric binding pocket in solution structures. Additional analysis provides insights into the dynamics and stability of the potential site. In addition, multi-tier screening was deployed to identify potential binders targeting the potential site. Our efforts to identify a new allosteric site contribute to community-wide initiatives developing therapies using multiple allosteric inhibitors to target distinct pockets on SHP2, in the hope of potentially inhibiting or slowing tumor growth associated with SHP2.

Synthesis and Biological Evaluation of Novel MB327 Analogs as Resensitizers for Desensitized Nicotinic Acetylcholine Receptors after Intoxication with Nerve Agents

Poisoning with organophosphorus compounds, which can lead to a cholinergic crisis due to the inhibition of acetylcholinesterase and the subsequent accumulation of acetylcholine (ACh) in the synaptic cleft, is a serious problem for which treatment options are currently insufficient. Our approach to broadening the therapeutic spectrum is to use agents that interact directly with desensitized nicotinic acetylcholine receptors (nAChRs) in order to induce functional recovery after ACh overstimulation. Although MB327, one of the most prominent compounds investigated in this context, has already shown positive properties in terms of muscle force recovery, this compound is not suitable for use as a therapeutic agent due to its insufficient potency. By means of in silico studies based on our recently presented allosteric binding pocket at the nAChR, i.e. the MB327-PAM-1 binding site, three promising MB327 analogs with a 4-aminopyridinium ion partial structure (PTM0056, PTM0062 and PTM0063) were identified. In this study, we present the synthesis and biological evaluation of a series of new analogs of the aforementioned compounds with a 4-aminopyridinium ion partial structure (PTM0064-PTM0072), as well as hydroxy-substituted analogs of MB327 (PTMD90-0012 and PTMD90-0015) designed to substitute entropically unfavorable water clusters identified during molecular dynamics simulations. The compounds were characterized in terms of their binding affinity towards the aforementioned binding site by applying the UNC0642 MS Binding Assays and in terms of their muscle force reactivation in rat diaphragm myography. More potent compounds were identified compared to MB327, as some of them showed a higher affinity towards MB327-PAM-1 and also a higher recovery of neuromuscular transmission at lower compound concentrations. To improve the treatment of organophosphate poisoning, direct targeting of nAChRs with appropriate compounds is a key step, and this study is an important contribution to this research.

The LexA-RecA* structure reveals a cryptic lock-and-key mechanism for SOS activation.

The bacterial SOS response plays a key role in adaptation to DNA damage, including genomic stress caused by antibiotics. SOS induction begins when activated RecA*, an oligomeric nucleoprotein filament that forms on single-stranded DNA, binds to and stimulates autoproteolysis of the repressor LexA. Here, we present the structure of the complete Escherichia coli SOS signal complex, constituting full-length LexA bound to RecA*. We uncover an extensive interface unexpectedly including the LexA DNA-binding domain, providing a new molecular rationale for ordered SOS gene induction. We further find that the interface involves three RecA subunits, with a single residue in the central engaged subunit acting as a molecular key, inserting into an allosteric binding pocket to induce LexA cleavage. Given the pro-mutagenic nature of SOS activation, our structural and mechanistic insights provide a foundation for developing new therapeutics to slow the evolution of antibiotic resistance.

A Hybrid Approach Combining Shape-Based and Docking Methods to Identify Novel Potential P2X7 Antagonists from Natural Product Databases.

P2X7 is an ATP-activated purinergic receptor implicated in pro-inflammatory responses. It is associated with the development of several diseases, including inflammatory and neurodegenerative conditions. Although several P2X7 receptor antagonists have recently been reported in the literature, none of them is approved for clinical use. However, the structure of the known antagonists can serve as a scaffold for discovering effective compounds in clinical therapy. This study aimed to propose an improved virtual screening methodology for the identification of novel potential P2X7 receptor antagonists from natural products through the combination of shape-based and docking approaches. First, a shape-based screening was performed based on the structure of JNJ-47965567, a P2X7 antagonist, using two natural product compound databases, MEGx (~5.8 × 103 compounds) and NATx (~32 × 103 compounds). Then, the compounds selected by the proposed shape-based model, with Shape-Tanimoto score values ranging between 0.624 and 0.799, were filtered for drug-like properties. Finally, the compounds that met the drug-like filter criteria were docked into the P2X7 allosteric binding site, using the docking programs GOLD and DockThor. The docking poses with the best score values were submitted to careful visual inspection of the P2X7 allosteric binding site. Based on our established visual inspection criteria, four compounds from the MEGx database and four from the NATx database were finally selected as potential P2X7 receptor antagonists. The selected compounds are structurally different from known P2X7 antagonists, have drug-like properties, and are predicted to interact with key P2X7 allosteric binding pocket residues, including F88, F92, F95, F103, M105, F108, Y295, Y298, and I310. Therefore, the combination of shape-based screening and docking approaches proposed in our study has proven useful in selecting potential novel P2X7 antagonist candidates from natural-product-derived compounds databases. This approach could also be useful for selecting potential inhibitors/antagonists of other receptors and/or biological targets.

A comprehensive assessment of the cholinergic-supporting and cognitive-enhancing effects of Rosa damascena Mill. (Damask rose) essential oil on scopolamine-induced amnestic rats.

Alzheimer's disease (AD) is a neurodegenerative condition characterized by gradual loss of cognitive abilities (dementia) and is a major public health problem. Here, we aimed at investigating the effects of Rosa damascena essential oil (RDEO) on learning and memory functions in a rat model of amnesia induced by scopolamine, as well as on changes in acetylcholinesterase (AChE) activity, M1 muscarinic acetylcholine receptor (mAChR) expression, and brain-derived neurotrophic factor (BDNF) levels in the extracted brain tissues. The control, amnesia (scopolamine, 1mg/kg/i.p.) and treatment (RDEO, 100μL/kg/p.o. or galantamine, 1.5mg/kg/i.p.) groups were subjected to Morris water maze and new object recognition tests. AChE activity was assayed by ELISA, and M1 mAChR and BDNF concentration changes were determined by western blotting. Also, using computational tools, human M1 mAChR was modeled in an active conformation, and the major components of RDEO were docked onto this receptor. According to our behavioral tests, RDEO was able to mitigate the learning and memory impairments caused by scopolamine in vivo. Our in vitro assays showed that the observed positive effects correlated well with a decrease in AChE activity and an increase in M1 mAChR and BDNF levels in amnestic rat brains. We also demonstrated in an in silico setting that the major components of RDEO, specifically -citronellol, geraniol, and nerol, could be accommodated favorably within the allosteric binding pocket of active-state human M1 mAChR and anchored here chiefly by hydrogen-bonding and alkyl-π interactions. Our findings offer a solid experimental foundation for future RDEO-based medicinal product development for patients suffering from AD.

Gaining Insights into Key Structural Hotspots within the Allosteric Binding Pockets of Protein Kinases.

Protein kinases are essential regulators of cell function and represent one of the largest and most diverse protein families. They are particularly influential in signal transduction and coordinating complex processes like the cell cycle. Out of the 518 human protein kinases identified, 478 are part of a single superfamily sharing catalytic domains that are related in sequence. The dysregulation of protein kinases due to certain mutations has been associated with various diseases, including cancer. Although most of the protein kinase inhibitors identified as type I or type II primarily target the ATP-binding pockets of kinases, the structural and sequential resemblances among these pockets pose a significant challenge for selective inhibition. Therefore, targeting allosteric pockets that are beside highly conserved ATP pockets has emerged as a promising strategy to prevail current limitations, such as poor selectivity and drug resistance. In this article, we compared the binding pockets of various protein kinases for which allosteric (type III) inhibitors have already been developed. Additionally, understanding the structure and shape of existing ligands could aid in identifying key interaction sites within the allosteric pockets of kinases. This comprehensive review aims to facilitate the design of more effective and selective allosteric inhibitors.

Development of a Fluorescent Ligand for the Intracellular Allosteric Binding Site of the Neurotensin Receptor 1.

The membrane protein family of G protein-coupled receptors (GPCRs) represents a major class of drug targets. Over the last years, the presence of additional intracellular binding sites besides the canonical orthosteric binding pocket has been demonstrated for an increasing number of GPCRs. Allosteric modulators harnessing these pockets may represent valuable alternatives when targeting the orthosteric pocket is not successful for drug development. Starting from SBI-553, a recently discovered intracellular allosteric modulator for neurotensin receptor subtype 1 (NTSR1), we developed the fluorescent molecular probe 14. Compound 14 binds to NTSR1 with an affinity of 0.68 μM in the presence of the agonist NT(8-13). NanoBRET-based ligand binding assays with 14 were established to derive the affinity and structure-activity relationships for allosteric NTSR1 modulators in a direct and nonisotopic manner, thereby facilitating the search for and optimization of novel allosteric NTSR1 ligands. As a consequence of cooperativity between the ligands binding to the allosteric and orthosteric pocket, compound 14 can also be used to investigate orthosteric NTSR1 agonists and antagonists. Moreover, employing 14 as a probe in a drug library screening, we identified novel chemotypes as binders for the intracellular allosteric SBI-553 binding pocket of NTSR1 with single-digit micromolar affinity. These hits may serve as interesting starting points for the development of novel intracellular allosteric ligands for NTSR1 as a highly interesting yet unexploited drug target in the fields of pain and addiction disorder therapy.

Potential acridinedione derivatives for the development of a heterobifunctional PROTAC for targeted degradation of PCSK9 protein

Proprotein convertase substilisin-like/kexin type 9 (PCSK9) interacts with the low-density lipoprotein (LDL) receptor by protein-protein interaction (PPI). The inhibition of this PPI lowers the LDL cholesterol levels, thereby reducing the risks of cardiovascular diseases. We identified in-house synthesized acridinedione scaffolds as binders of PCSK9 through microsecond timescale conventional and biased molecular dynamics (MD) simulations. The free energy of binding was calculated by the Molecular Mechanics Poisson-Boltzmann Surface Area and umbrella sampling methods for acridinedione molecules and compared with reference molecules (co-crystallized inhibitors). Three acridinedione molecules (DSPD-2, DSPD-4, and DSPD-6) were identified as potential binders of the PCSK9 allosteric binding site from preliminary docking and steered MD simulations. The stability of molecules inside the allosteric pocket was established by the low values of root mean square deviations of backbone C-α atoms. The acridinedione derivatives also showed a shorter distance of hydrogen bonds as compared to the standard molecules. The molecule DSPD-6 showed the lowest binding free energy as compared to other selected and standard molecules. The binding of the selected molecules at the allosteric binding pocket had no effect on structural parameters of LDL receptor-binding site of PCSK9 protein. Finally, a proteolysis targeting chimera (PROTAC) was developed by attaching a proteasome recruiting tag to DSPD-6 (lowest binding affinity) to achieve ligand-induced protein degradation. These results qualify the development of the DSPD-6 molecule as a heterobifunctional PROTAC for targeted degradation of PCSK9 protein. Moreover, our computational strategy provides a framework for the identification of molecules against conventionally undruggable PPI targets.

A platform for predicting mechanism of action based on bacterial transcriptional responses identifies an unusual DNA gyrase inhibitor

In the search for much-needed new antibacterial chemical matter, a myriad of compounds have been reported in academic and pharmaceutical screening endeavors. Only a small fraction of these, however, are characterized with respect to mechanism of action (MOA). Here, we describe a pipeline that categorizes transcriptional responses to antibiotics and provides hypotheses for MOA. 3D-printed imaging hardware PFIboxes) profiles responses of Escherichia coli promoter-GFP fusions to more than 100 antibiotics. Notably, metergoline, a semi-synthetic ergot alkaloid, mimics a DNA replication inhibitor. Invitro supercoiling assays confirm this prediction, and a potent analog thereof (MLEB-1934) inhibits growth at 0.25μg/mL and is highly active against quinolone-resistant strains of methicillin-resistant Staphylococcus aureus. Spontaneous suppressor mutants map to a seldom explored allosteric binding pocket, suggesting a mechanism distinct from DNA gyrase inhibitors used in the clinic. In all, the work highlights the potential of this platform to rapidly assess MOA of new antibacterial compounds.

Abstract 3256: Novel SOX2-targeting bioPROTACs for the treatment of cancers

Abstract Targeted protein degradation (TPD) technology has been developed showing the therapeutic potential to modulate historically ‘undruggable’ disease-associated proteins. Currently, proteolysis targeting chimeras (PROTACs) using small-molecule binders represent the TPD field, and PROTAC-induced proximity between target proteins and E3 ligases enables specific degradation of target proteins. However, substantial amounts of clinically relevant cancer targets such as transcription factors lack active sites or allosteric binding pockets for small molecules which challenges the development of classical PROTACs for these targets. SOX2 is one of the pluripotency transcription factors and has been well-known for its crucial role in tumor development through promoting cancer cell survival, metastasis, stemness, and drug resistance. Numerous studies have also reported SOX2 amplification and overexpression in various tumor tissues. Thus, SOX2 has been validated as an attractive anticancer target, but directly targeting SOX2 has been proved to be difficult since SOX2 is an “undruggable” transcription factor. EPD Biotherapeutics develops EPDegTM, a next-generation engineered protein degrader platform in order to overcome the limitations of small molecule-based PROTAC. Here we report a series of SOX2-targeting bioPROTACs, being composed of a nanobody against SOX2 with high affinity and several ubiquitin E3 ligases with deletion of natural substrate binding domain to allow highly selective degradation of SOX2 protein. To evaluate the in vitro efficacy, SOX2 bioPROTACs were generated by mRNA IVT with CleanCap ® and modified uridine. First, robust degradation of SOX2 with a half-maximal degradation concentration (DC50) of the picomolar range was observed with the treatment of SOX2 bioPROTACs in multiple SOX2-overexpressing cancer cell lines. The degradation was ubiquitin- and proteosome-dependent manner, and neither nanobody nor ubiquitin E3 ligase alone affected SOX2 protein level. Then, SOX2 bioPROTACs led to significant cancer cell growth inhibition caused by apoptotic cell death with a half-maximal growth inhibition concentration (GI50) of the picomolar range. Notably, chemo-resistant cancer cell lines expressing SOX2 protein in high levels were sensitized to chemotherapy in combination with our SOX2 bioPROTACs. Furthermore, i.v. injection of one of our lead candidates formulated with LNP significantly inhibited tumor growth in the A549 xenograft model, proving the in vivo anti-tumor efficacy of SOX2 degraders for the first time in the world. Taken together, EPD’s SOX2 bioPROTACs show potent in vitro and in vivo anti-tumor efficacy as first-in-class SOX2 degraders and provide new insights for the therapeutic potential of transcription factor-degrading strategies. Citation Format: Jaehyun Choi, Saeyi Lim, Xuelian Bai, Hyerin Song, Yeonji Oh, Yeryoung Yong, Seunghyun Lee. Novel SOX2-targeting bioPROTACs for the treatment of cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3256.

Exploring transmembrane allostery in the MexB: DB08385 variant as a promising inhibitor-like candidate against Pseudomonas aeruginosa antibiotic resistance: a computational study.

Pseudomonas aeruginosa, a formidable pathogen renowned for its antimicrobial resistance, poses a significant threat to immunocompromised individuals. In this regard, the MexAB-OprM efflux pump acts as a pivotal line of defense by extruding antimicrobials from bacterial cells. The inner membrane homotrimeric protein MexB captures antibiotics and translocates them into the outer membrane OprM channel protein connected through the MexA adaptor protein. Despite extensive efforts, competitive inhibitors targeting the tight (T) protomer of the MexB protein have not received FDA approval for medical use. Over the past few years, allosteric inhibitors have become popular as alternatives to the classical competitive inhibitor-based approach because of their higher specificity, lower dosage, and reduced toxicological effects. Hence, in this study, we unveiled the existence of a transmembrane allosteric binding pocket of MexB inspired by the recent discovery of an important allosteric inhibitor, BDM88855, for the homolog AcrB protein. While repurposing BDM88855 proved ineffective in controlling the MexB loose (L) protomer, our investigation identified a promising alternative: a chlorine-containing variant of DB08385 (2-Cl DB08385 or Variant 1). Molecular dynamics simulations, including binding free energy estimation coupled with heterogeneous dielectric implicit membrane model (implicit-membrane MM/PBSA), interaction entropy (IE) analysis and potential of mean force (PMF) calculation, demonstrated Variant 1's superior binding affinity to the transmembrane pocket, displaying the highest energy barrier in the ligand unbinding process. To elucidate the allosteric crosstalk between the transmembrane and porter domain of MexB, we employed the 'eigenvector centrality' measure in the linear mutual information obtained from the protein correlation network. Notably, this study confirmed the presence of an allosteric transmembrane site in the MexB L protomer. In addition to this, Variant 1 emerged as a potent regulator of allosteric crosstalk, inducing an 'O-L intermediate state' in the MexB L protomer. This induced state might hold the potential to diminish substrate intake into the access pocket, leading to the ineffective efflux of antibiotics.

From Hit to Lead: Structure-Based Optimization of Novel Selective Inhibitors of Receptor-Interacting Protein Kinase 1 (RIPK1) for the Treatment of Inflammatory Diseases.

Receptor-interacting protein kinase 1 (RIPK1) is a key regulator of cellular necroptosis, which is considered as an important therapeutic target for necroptosis-related indications. Herein, we report the structural optimization and structure-activity relationship investigations of a series of eutectic 5-substituted-indole-3-carboxamide derivatives. The prioritized compound 10b exhibited low nanomolar IC50 values against RIPK1 and showed good kinase selectivity. Based on its eutectic structure, 10b occupied both the allosteric and ATP binding pockets of RIPK1, making it a potent dual-mode inhibitor of RIPK1. In vitro, 10b had a potent protective effect against necroptosis in cells. Compound 10b also provided robust protection in a TNFα-induced systemic inflammatory response syndrome (SIRS) model and imiquimod (IMQ)-induced psoriasis model. It also showed good pharmacokinetic properties and low toxicity. Overall, 10b is a promising lead compound for drug discovery targeting RIPK1 and warrants further study.

Discovery of KRB-456, a KRAS G12D Switch-I/II Allosteric Pocket Binder That Inhibits the Growth of Pancreatic Cancer Patient-derived Tumors.

There are no clinically approved drugs directly abrogating mutant KRAS G12D. Here, we discovered a small molecule, KRB-456, that binds a dynamic allosteric binding pocket within the switch-I/II region of KRAS G12D. KRB-456 inhibits P-MEK, P-AKT, and P-S6 levels in vivo and inhibits the growth of subcutaneous and orthotopic xenografts derived from patients with pancreatic cancer. This discovery warrants further advanced preclinical and clinical studies in pancreatic cancer.

A Comparative Study of Chemical Profiling and Bioactivities between Thai and Foreign Hemp Seed Species (Cannabis sativa L.) Plus an In-Silico Investigation.

Hemp (Cannabis sativa L.) is a plant widely used by humans for textiles, food, and medicine. Thus, this study aimed to characterize the chemical profiling of 12 hemp seed extracts from Thai (HS-TH) and foreign (HS-FS) samples using gas chromatography-mass spectrometry (GC-MS). Their antibacterial activity and α-glucosidase inhibitory activity were assayed. Linoleic acid (17.63-86.53%) was a major component presented in Thai hemp seed extracts, while α,β-gluco-octonic acid lactone (30.39%), clionasterol (13.42-29.07%), and glyceryl-linoleate (15.12%) were detected as the main metabolites found in foreign hemp seed extracts. Furthermore, eight extracts from both Thai and foreign hemp seed exhibited antibacterial activity against Staphylococcus aureus, Staphylococcus epidermidis, Methicillin-resistant Staphylococcus aureus, and Cutibacterium acnes, with MIC values ranging from 128 to 2048 µg/mL. Interestingly, the ethanol extract of Thai hemp seed (HS-TH-2-M-E) showed superior α-glucosidase inhibition (IC50 value of 33.27 ug/mL) over foreign species. The combination between Thai hemp species (HS-TH-2-M-E) and acarbose showed a synergistic effect against α-glucosidase. Furthermore, the docking investigation revealed that fatty acids had a greater impact on α-glucosidase than fatty acid esters and cannabinoids. The computational simulation predicts a potential allosteric binding pocket of guanosine on glucosidase and is the first description of gluco-octonic acid's anti-glucosidase activity in silico. The findings concluded that Thai hemp seed could be used as a resource for supplemental drugs or dietary therapy for diabetes mellitus.

Synthesis, molecular modeling Insights, and anticancer assessment of novel polyfunctionalized Pyridine congeners

The present study describes synthesizing a novel series of polyfunctionalized pyridine congeners 1–18 and assessed for cytotoxic efficacies versus HCT-116, MCF-7, and HepG-2 among one non-cancerous BJ-1 human normal cell. Most compounds were precisely potent anticancer candidate drugs. The molecular impact of the most active compounds 9, 10, 11, 13, 15, and 17 was evaluated after MCF-7 treatment. The gene expression of pro- and ant-apoptosis markers P53, Bax, Caspase-3 and Bcl-2 as well as VEGFR-2 and HER2 were determined. Compounds 13 and 15 induced upregulation of pro-apoptosis of P53, Bax, Caspase-3 and downregulation of anti-apoptosis Bcl-2 gene. However, compound 15 showed higher effect compared to 13 and respective control. Moreover, a slight reduction in HER2 gene expression was detected due to compound 15 treatment, while VEGFR-2 gene was upregulated. In agreement, the immunoblotting analysis showed higher accumulation of P53, Bax, Caspase-3 proteins and of decrease the Bcl-2 protein levels. Furthermore, docking studies united with molecular dynamic simulation exposed compounds 13 and 15 fitting in the middle of the active site at the interface linking the ATP binding site and the allosteric hydrophobic binding pocket. Finally, we performed Petra/Osiris/ Molinspiration (POM) analysis for the newly synthesized compounds. The evaluation of primary in silico parameters revealed significant differences among individual polyfunctionalized pyridine compounds, highlighting the most promising candidates. These preliminary results may help in coordinating and initiating other research projects focused on polyfunctionalized pyridine compounds, especially those with predicted bioactivity, low toxicity, optimal ADME parameters, and promising perspectives.

Non-Covalent Interactions in the Self-Assembly of Dihydropyridone Supramolecules and In Vitro Anti-Cancer Assessment in Human Lung Adenocarcinoma Cell Line (A549).

The synthesis of dihydropyridone derivatives has been reported by ring rearrangement of pyrans using iodine and formic acid as a catalyst separately. Dihydropyridones were crystallized subjected for single-crystal X-ray crystallography to acquire their structural parameters. The different non-covalent interactions involved within the supramolecular systems were studied and validated using Hirshfeld surface plot analysis. N-H⋅⋅⋅O interactions between the lactam group dominate. Still, other non-covalent interactions such as C-H⋅⋅⋅N, C-H⋅⋅⋅O, C-H⋅⋅⋅C, N-H⋅⋅⋅N, C-H⋅⋅⋅π, and lone pair⋅⋅⋅π systems act as the driving force in facilitating the self-assembly of the dihydropyridone supramolecules. The synthesized compounds were analyzed by in vitro techniques using human lung adenocarcinoma (A549) to evaluate their cytotoxic activities. Ethyl 4-(4-chlorophenyl)-5-cyano-2-methyl-6-oxo-1,4,5,6- tetrahydropyridine-3-carboxylate has shown the highest cytotoxicity among all the synthesized compounds. Molecular recognition properties of the dihydropyridone compounds were also studied, employing molecular docking tools to gain insight into the binding mode inside the allosteric binding pocket of the Eg5 protein through non-covalent interactions.

Quest for selective MMP9 inhibitors: a computational approach

Matrix Metalloproteinases-9 (MMP-9) is one of the important targets that play a vital role in various diseases such as cancer, Alzheimer’s, arthritis, etc. Traditionally, MMP-9 inhibitors have been unable to achieve selectivity to get around this target; thereby, novel mechanisms such as inhibition of activated MMP-9 zymogen (pro-MMP-9) have been discovered. The JNJ0966 was one of the few compounds that attained the requisite selectivity by inhibiting the activation of MMP-9 zymogen (pro-MMP-9). Since JNJ0966, no other small molecules have been identified. Herein, extensive in silico studies were called upon to bolster the prospect of exploring potential candidates. The key objective of this research is to identify the potential hits from the ChEMBL database via molecular docking and dynamics approach. Protein with PDB ID: 5UE4, having a unique inhibitor in an allosteric binding pocket of MMP-9, was chosen for the study. Structure-based virtual screening and MMGBSA binding affinity calculations were performed, and five potential hits were finalized. Detailed analysis of the best-scoring molecules was performed with ADMET analysis and molecular dynamics (MD) simulation. All five hits outperformed JNJ0966 in the docking assessment, ADMET analysis, and molecular dynamics simulation. Accordingly, our research findings imply that these hits can be investigated for in vitro and in vivo studies against proMMP9 and might be explored as potential anticancer drugs. The outcome of our research might contribute in expediting the exploration of drugs that inhibits proMMP-9. Communicated by Ramaswamy H. Sarma

Identification of the molecular determinants of antagonist potency in the allosteric binding pocket of human P2X4.

P2X receptors are a family of ATP-gated cation channels comprising seven subtypes in mammals, which play key roles in nerve transmission, pain sensation and inflammation. The P2X4 receptor in particular has attracted significant interest from pharmaceutical companies due to its physiological roles in neuropathic pain and modulation of vascular tone. A number of potent small-molecule P2X4 receptor antagonists have been developed, including the allosteric P2X4 receptor antagonist BX430, which is approximately 30-fold more potent at human P2X4 compared with the rat isoform. A single amino-acid difference between human and rat P2X4 (I312T), located in an allosteric pocket, has previously been identified as critical for BX430 sensitivity, implying that BX430 binds in this pocket. Using a combination of mutagenesis, functional assay in mammalian cells and in silico docking we confirmed these findings. Induced-fit docking, permitting the sidechains of the amino-acids of P2X4 to move, showed that BX430 could access a deeper portion of the allosteric pocket, and that the sidechain of Lys-298 was important for shaping the cavity. We then performed blind docking of 12 additional P2X4 antagonists into the receptor extracellular domain, finding that many of these compounds favored the same pocket as BX430 from their calculated binding energies. Induced-fit docking of these compounds in the allosteric pocket enabled us to show that antagonists with high potency (IC50 ≤ 100nM) bind deep in the allosteric pocket, disrupting a network of interacting amino acids including Asp-85, Ala-87, Asp-88, and Ala-297, which are vital for transmitting the conformational change following ATP binding to channel gating. Our work confirms the importance of Ile-312 for BX430 sensitivity, demonstrates that the allosteric pocket where BX430 binds is a plausible binding pocket for a series of P2X4 antagonists, and suggests a mode of action for these allosteric antagonists involving disruption of a key structural motif required for the conformational change induced in P2X4 when ATP binds.

Rationale design of novel PIEZO1 modulators.

Chemical modulators of mechanosensitive Piezo channels would help better understand physiology and could treat certain diseases. A promising small molecule to expand the currently limited Piezo channel pharmacome is Yoda1, a Piezo1-selective activator identified from blind high-throughput screening. We previously uncovered an allosteric Yoda1 binding pocket in Piezo1, sandwiched between two hydrophobic transmembrane domains called Repeats A and B. Here, we show that crosslinking these Repeats with disulfide bridges inhibits the effects of Yoda1, supporting a mechanism in which Yoda1 acts like a wedge by separating these two domains from each other. Using open and shut Piezo1 structural models and rigorous in silico ligand binding free energy calculations, we next show that Yoda1 interacts with this binding region with a higher affinity for the open state, as expected for an allosteric activator. Our free energy calculations further recapitulate structure activity relationships established for seven Yoda1 analogs. Among 155 purchasable compounds virtually selected against a 3D pharmacophore map of the Yoda1 binding site in the open state, several act as bona fide Piezo1 activators with chemical scaffolds distinct from Yoda1 or other known Piezo1 modulators. This work provides a structural and thermodynamic framework for the activation of Piezo1 by Yoda1 and demonstrates the possibility to computationally exploit this pharmacological region to rationally design novel molecules with potential clinical value.