Leveraging shape screening and molecular dynamics simulations to optimize PARP1-Specific chemo/radio-potentiators for antitumor drug design.

PARP1 plays a pivotal role in DNA repair within the base excision pathway, making it a promising therapeutic target for cancers involving BRCA mutations. Current study is focused on the discovery of PARP inhibitors with enhanced selectivity for PARP1. Concurrent inhibition of PARP1 with PARP2 and PARP3 affects cellular functions, potentially causing DNA damage accumulation and disrupting immune responses. In step 1, a virtual library of 593 million compounds has been screened using a shape-based screening approach to narrow down the promising scaffolds. In step 2, hierarchical docking approach embedded in Schrödinger suite was employed to select compounds with good dock score, drug-likeness and MMGBSA score. Analysis supplemented with decomposition energy, molecular dynamics (MD) simulations and hydrogen bond frequency analysis, pinpointed that active site residues; H862, G863, R878, M890, Y896 and F897 are crucial for specific binding of ZINC001258189808 and ZINC000092332196 with PARP1 as compared to PARP2 and PARP3. The binding of ZINC000656130962, ZINC000762230673, ZINC001332491123, and ZINC000579446675 also revealed interaction involving two additional active site residues of PARP1, namely N767 and E988. Weaker or no interaction was observed for these residues with PARP2 and PARP3. This approach advances our understanding of PARP-1 specific inhibitors and their mechanisms of action, facilitating the development of targeted therapeutics.

Identification of selective inhibitors for phosphodiesterase 5A using e-pharmacophore modelling and large-scale virtual screening-based structure guided drug discovery approaches.

The inhibition of Phosphodiesterase 5A (PDEA5) has the potential to modulate pulmonary arterial hypertension and cardiovascular diseases. Exploring the cross-reactivity of clinically available PDE5A therapeutics with PDE6A is intriguing in order to develop highly selective PDE5A compounds in cardiovascular arena. In the current study, we leveraged e-pharmacophore based screening and molecular dynamics (MD) simulation to discover more selective PDE5A inhibitors as compared to the PDE6A catalytic domain. e-Pharmacophore based mapping of the CoCoCo database (7 million compounds: ∼ 150,000,000 conformers), followed by Glide docking, MM-GBSA, and protein-inhibitor interaction analysis, revealed 1536427, 4832637 and 6788240 as stable, tight binders of PDE5A instead of PDE6A. These compounds adhere to Lipinski Rule of Five (RO5) and ADME/Tox criteria. MD simulations analysis showed that 1536427 stays stable and tightly binds to catalytic (Q-region) core of PDE5A catalytic domain as compared to sildenafil. Pronounced inward motions of the hydrophobic (H-region) and Lid region indicate the closure of PDE5A-1536427 complex, whereas this region in PDE6A-1536427 is more open. Significant differences in the interactions, stability, and dynamics of 1536427 were observed in the catalytic domain of PDE6A, demonstrating less specificity for PDE6A in comparison to PDE5A. After lead optimization and therapeutic interventions, this proposed lead may emerge as a promising PDE5A selective inhibitor.Communicated by Ramaswamy H. Sarma.

A recursive molecular docking coupled with energy-based pose-rescoring and MD simulations to identify hsGC ßH-NOX allosteric modulators for cardiovascular dysfunctions.

Modulating the activity of human soluble guanylate cyclase (hsGC) through allosteric regulation of the ßH-NOX domain has been considered as an immediate treatment for cardiovascular disorder (CVDs). Currently available ßH-NOX domain-specific agonists including cinaciguat are unable to deal with the conundrum raised due to oxidative stress in the case of CVDs and their associated comorbidities. Therefore, the idea of investigating novel compounds for allosteric regulation of hsGC activation has been rekindled to circumvent CVDs. Current study aims to identify novel ßH-NOX domain-specific compounds that can selectively turn on sGC functions by modulating the conformational dynamics of the target protein. Through a comprehensive computational drug-discovery approach, we first executed a target-based performance assessment of multiple docking (PLANTS, QVina, LeDock, Vinardo, Smina) scoring functions based on multiple performance metrices. QVina showed the highest capability of selecting true-positive ligands over false positives thus, used to screen 4.8 million ZINC15 compounds against ßH-NOX domain. The docked ligands were further probed in terms of contact footprint and pose reassessment through clustering analysis and PLANTS docking, respectively. Subsequently, energy-based AMBER rescoring of top 100 low-energy complexes, per-residue energy decomposition analysis, and ADME-Tox analysis yielded the top three compounds i.e. ZINC000098973660, ZINC001354120371, and ZINC000096022607. The impact of three selected ligands on the internal structural dynamics of the ßH-NOX domain was also investigated through molecular dynamics simulations. The study revealed potential electrostatic interactions for better conformational dialogue between ßH-NOX domain and allosteric ligands that are critical for the activation of hsGC as compared to the reference compound.

E-pharmacophore based virtual screening for identification of dual specific PDE5A and PDE3A inhibitors as potential leads against cardiovascular diseases.

The need of circumventing life-threatening cardiovascular disorders (CVDs) and pulmonary hypertension (PHT) worldwide prompts researchers to develop effective therapeutic agents. Crucial role of cyclic nucleotide phosphodiesterase-5 (PDE5A) and cyclic nucleotide phosphodiesterase-3 (PDE3A) in cardiovascular signaling makes them potential drug targets for the treatment of CVDs and PHT. In this study, one-drug-multiple-target strategy has been employed to screen inhibitors exhibiting dual specificity through Phase-generated and statistically validated e-pharmacophore models of PDE5A and PDE3A. An extensive CoCoCo database of 7 million compounds with ∼150,000,000 conformations was virtually screened by sequential e-pharmacophore mapping followed by Lipinski Rule of Five (RO5) evaluation and hierarchical docking simulations. Finally, docked hits were subjected to rigorous MMGBSA analysis to estimate the relative spatial affinity of the drug-like compounds. The hits (354 and 366 ligands against PDE5A and PDE3A, respectively) were further optimized through 2D clustering followed by a comprehensive 2D and 3D interaction analysis. Five structurally diverse hits mapped equally well with the e-pharmacophore models and showed promising inhibitory interactions with conserved four catalytic features of PDE5A and PDE3A, thus exhibiting dual specificity. Proposed lead compounds exhibited the lowest MMGBSA binding energies and were found to be in agreement with Lipinski Rule of Five (RO5) and ADME/Tox criteria as compared to sildenafil. The proposed dual inhibitors could thus provide promising outcomes for the discovery of dual as well as multipotent drug like compounds after lead optimization and primary therapeutic interventions.

Exploring the structural basis of conformational heterogeneity and autoinhibition of human cGMP-specific protein kinase Iα through computational modelling and molecular dynamics simulations.

Protein kinase Iα (PKGIα) is a pivotal cyclic guanosine monophosphate (cGMP) signalling protein. Major steps related to the structural plasticity of PKGIα have been inferred but the structural aspects of the auto-inhibition and multidomain tertiary organization of human PKGIα in active and inactive form are not clear. Here we combine computational comparative modelling, protein-protein docking and molecular dynamics (MD) simulations to investigate structural details of the repressed state of the catalytic domain of PKGIα. Exploration of the potential inhibitory conformation of the auto-inhibitory domain (AI) within the catalytic cleft reveals that the pseudo-substrate motif binds with residues of the glycine rich loop and substrate-binding lobe. Dynamic changes as a result of coupling of the catalytic and AI domains are also investigated. The three-dimensional homodimeric models of PKGIα in the active and inactive state indicate that PKGIα in its inactive-state attains a compact globular structure where cyclic nucleotide binding (CNB-A/B) domains are buried, whereas the catalytic domains are inaccessible with their substrate-binding pockets facing the N-terminal of CNB-A. Contrary to this, the active-state model of PKGIα shows an extended conformation where CNB-A/B domains are slightly rearranged and the catalytic domains of homodimer flanking the C-terminal with their substrate binding lobes free to entrap downstream proteins. These findings are consistent with previously reported static images of the multidomain organization of PKGIα. Structural insights pertaining to the conformational heterogeneity and auto-inhibition of PKGIα provided in this study may help to understand the dynamics-driven effective regulation of PKGIα.

Probing the Structural Dynamics of the Catalytic Domain of Human Soluble Guanylate Cyclase.

In the nitric oxide (NO) signaling pathway, human soluble guanylate cyclase (hsGC) synthesizes cyclic guanosine monophosphate (cGMP); responsible for the regulation of cGMP-specific protein kinases (PKGs) and phosphodiesterases (PDEs). The crystal structure of the inactive hsGC cyclase dimer is known, but there is still a lack of information regarding the substrate-specific internal motions that are essential for the catalytic mechanism of the hsGC. In the current study, the hsGC cyclase heterodimer complexed with guanosine triphosphate (GTP) and cGMP was subjected to molecular dynamics simulations, to investigate the conformational dynamics that have functional implications on the catalytic activity of hsGC. Results revealed that in the GTP-bound complex of the hsGC heterodimer, helix 1 of subunit α (α:h1) moves slightly inwards and comes close to helix 4 of subunit ß (ß:h4). This conformational change brings loop 2 of subunit ß (ß:L2) closer to helix 2 of subunit α (α:h2). Likewise, loop 2 of subunit α (α:L2) comes closer to helix 2 of subunit ß (ß:h2). These structural events stabilize and lock GTP within the closed pocket for cyclization. In the cGMP-bound complex, α:L2 detaches from ß:h2 and establishes interactions with ß:L2, which results in the loss of global structure compactness. Furthermore, with the release of pyrophosphate, the interaction between α:h1 and ß:L2 weakens, abolishing the tight packing of the binding pocket. This study discusses the conformational changes induced by the binding of GTP and cGMP to the hsGC catalytic domain, valuable in designing new therapeutic strategies for the treatment of cardiovascular diseases.

Cryo-EM density map fitting driven in-silico structure of human soluble guanylate cyclase (hsGC) reveals functional aspects of inter-domain cross talk upon NO binding.

The human soluble Guanylate Cyclase (hsGC) is a heterodimeric heme-containing enzyme which regulates many important physiological processes. In eukaryotes, hsGC is the only known receptor for nitric oxide (NO) signaling. Improper NO signaling results in various disease conditions such as neurodegeneration, hypertension, stroke and erectile dysfunction. To understand the mechanisms of these diseases, structure determination of the hsGC dimer complex is crucial. However, so far all the attempts for the experimental structure determination of the protein were unsuccessful. The current study explores the possibility to model the quaternary structure of hsGC using a hybrid approach that combines state-of-the-art protein structure prediction tools with cryo-EM experimental data. The resultant 3D model shows close consistency with structural and functional insights extracted from biochemistry experiment data. Overall, the atomic-level complex structure determination of hsGC helps to unveil the inter-domain communication upon NO binding, which should be of important usefulness for elucidating the biological function of this important enzyme and for developing new treatments against the hsGC associated human diseases.

The Molecular Organization of Human cGMP Specific Phosphodiesterase 6 (PDE6): Structural Implications of Somatic Mutations in Cancer and Retinitis Pigmentosa.

In the cyclic guanosine monophosphate (cGMP) signaling pathway, phosphodiesterase 6 (PDE6) maintains a critical balance of the intracellular concentration of cGMP by catalyzing it to 5' guanosine monophosphate (5'-GMP). To gain insight into the mechanistic impacts of the PDE6 somatic mutations that are implicated in cancer and retinitis pigmentosa, we first defined the structure and organization of the human PDE6 heterodimer using computational comparative modelling. Each subunit of PDE6αß possesses three domains connected through long α-helices. The heterodimer model indicates that the two chains are likely related by a pseudo two-fold axis. The N-terminal region of each subunit is comprised of two allosteric cGMP-binding domains (Gaf-A & Gaf-B), oriented in the same way and interacting with the catalytic domain present at the C-terminal in a way that would allow the allosteric cGMP-binding domains to influence catalytic activity. Subsequently, we applied an integrated knowledge-driven in silico mutation analysis approach to understand the structural and functional implications of experimentally identified mutations that cause various cancers and retinitis pigmentosa, as well as computational saturation mutagenesis of the dimer interface and cGMP-binding residues of both Gaf-A, and the catalytic domains. We studied the impact of mutations on the stability of PDE6αß structure, subunit-interfaces and Gaf-cGMP interactions. Further, we discussed the changes in interatomic interactions of mutations that are destabilizing in Gaf-A (R93L, V141 M, F162 L), catalytic domain (D600N, F742 L, F776 L) and at the dimer interface (F426A, F248G, F424 N). This study establishes a possible link of change in PDE6αß structural stability to the experimentally observed disease phenotypes.

Dynamic Characterization of the Human Heme Nitric Oxide/Oxygen (HNOX) Domain under the Influence of Diatomic Gaseous Ligands.

Soluble guanylate cyclase (sGC) regulates numerous physiological processes. The ß subunit Heme Nitric Oxide/Oxygen (HNOX) domain makes this protein sensitive to small gaseous ligands. The structural basis of the activation mechanism of sGC under the influence of ligands (NO, O2, CO) is poorly understood. We examine the effect of different ligands on the human sGC HNOX domain. HNOX systems with gaseous ligands were generated and explored using Molecular Dynamics (MD). The distance between heme Fe2+ and histidine in the NO-ligated HNOX (NO-HNOX) system is larger compared to the O2, CO systems. NO-HNOX rapidly adopts the conformation of the five-group metal coordination system. Loops α, ß, γ and helix-f exhibit increased mobility and different hydrogen bond networks in NO-HNOX compared to the other systems. The removal of His from the Fe coordination sphere in NO-HNOX is assisted by interaction of the imidazole ring with the surrounding residues which in turn leads to the release of signaling helix-f and activation of the sGC enzyme. Insights into the conformational dynamics of a human sGC HNOX domain, especially for regions which are functionally critical for signal transduction, are valuable in the understanding of cardiovascular diseases.

Computational screening of medicinal plant phytochemicals to discover potent pan-serotype inhibitors against dengue virus.

Emergence of Dengue as one of the deadliest viral diseases prompts the need for development of effective therapeutic agents. Dengue virus (DV) exists in four different serotypes and infection caused by one serotype predisposes its host to another DV serotype heterotypic re-infection. We undertook virtual ligand screening (VLS) to filter compounds against DV that may inhibit inclusively all of its serotypes. Conserved non-structural DV protein targets such as NS1, NS3/NS2B and NS5, which play crucial role in viral replication, infection cycle and host interaction, were selected for screening of vital antiviral drug leads. A dataset of plant based natural antiviral derivatives was developed. Molecular docking was performed to estimate the spatial affinity of target compounds for the active sites of DV's NS1, NS3/NS2B and NS5 proteins. The drug likeliness of the screened compounds was followed by ADMET analysis whereas the binding behaviors were further elucidated through molecular dynamics (MD) simulation experiments. VLS screened three potential compounds including Canthin-6-one 9-O-beta-glucopyranoside, Kushenol W and Kushenol K which exhibited optimal binding with all the three conserved DV proteins. This study brings forth novel scaffolds against DV serotypes to serve as lead molecules for further optimization and drug development against all DV serotypes with equal effect against multiple disease causing DV proteins. We therefore anticipate that the insights given in the current study could be regarded valuable towards exploration and development of a broad-spectrum natural anti-dengue therapy.

A computational structural analysis of functional attributes of hypodermin A and B proteins: A way forward for vaccine development.

Hypodermosis is a parasitic disease of cattle. The pathogenicity of the disease is attributed to Hypodermin proteins (Hypodermin A, Hypodermin B and Hypodermin C). Studies suggest that Hypodermin proteins may be defined as Serine proteases and collagenases. The structure of both proteases Hypodermin A and Hypodermin B were modeled using the Swiss-model server followed by its validation using Procheck, Errat and Verify-3D. Afterwards, both Hypodermin A and Hypodermin B were docked against collagen in order to study its interaction with respective Hypodermin proteins. The structure of both Hypodermin A and Hypodermin B showed more bent towards hydrophobic nature as more beta sheets were present in them. Both structures were also superimposed to check out similarities and differences present between them. Serine, Aspartic acid, Histidine, Glutamic acid and Lysine are found as interacting residues that are involved in hydrogen bonding with collagen. The interactions are found in the active domain region of Hypodermin proteins. The interacting residues were present in the active region of the hypodermin proteins thus validating the docking studies. This study may help in the drug development against hypodermosis with least side effects.

Decreased serum PON1 arylesterase activity in familial hypercholesterolemia patients with a mutated LDLR gene.

Paraoxonase 1 (PON1) is a serum enzyme associated with high density lipoprotein (HDL) regulation through its paraoxonase and arylesterase activity. PON1 inhibits the oxidation of HDL and low density lipoprotein (LDL), and is involved in the pathogenesis of a variety of diseases including atherosclerosis. Conversely, mutations in the low density lipoprotein receptor (LDLR) result in failure of receptor mediated endocytosis of LDL leading to its elevated plasma levels and onset of familial hypercholesterolemia (FH). In the current study we investigated the role of PON1 polymorphisms rs662; c.575A > G (p.Gln192Arg) and rs854560; c.163T > A (p.Leu55Met) in a large family having FH patients harboring a functional mutation in LDLR. Genotypes were revealed by RFLP, followed by confirmation through Sanger sequencing. PON1 activity was measure by spectrophotometry. Our results show significantly reduced serum paraoxonase and arylesterase activities in FH patients compared with the healthy individuals of the family (p < 0.05). PON1 QQ192 genotype showed a significantly higher association with FH (p=0.0002). PON1 Q192 isoform was associated with reduced serum paraoxonase activity by in silico analysis and PON1 R192 exhibited higher serum paraoxonase and arylesterase activity than the other polymorphs. Our results highlight that the combination of LDLR mutations and PON1 MMQQ genotypes may lead to severe cardiac events.

Design, synthesis, characterization and computational docking studies of novel sulfonamide derivatives.

This study reports three novel sulfonamide derivatives 4-Chloro-N-[(4-methylphenyl) sulphonyl]-N-propyl benzamide (1A), N-(2-hydroxyphenyl)-4-methyl benzene sulfonamide (1B) and 4-methyl-N-(2-nitrophenyl) benzene sulfonamide (1C). The compounds were synthesised from starting material 4-methylbenzenesulfonyl chloride and their structure was studied through 1H-NMR and 13C-NMR spectra. Computational docking was performed to estimate their binding energy against bacterial p-amino benzoic acid (PABA) receptor, the dihydropteroate synthase (DHPS). The derivatives were tested in vitro for their antimicrobial activity against Gram+ and Gram- bacteria including E. coli, B. subtilis, B. licheniformis and B. linen. 1A was found active only against B. linen; 1B was effective against E. coli, B. subtilis and B. linen whereas 1C showed activity against E. coli, B. licheniformis and B. linen. 1C showed maximum activity with minimum inhibitory concentration (MIC) of 50, 100 and 150 µg/mL against E. coli, B. licheniformis and B. linen respectively. 1C exhibited maximum affinity to DHPS with binding free energy of -8.1 kcal/mol. It enriched in the top 0.5 % of a library of 7663 compounds, ranked in order of their binding affinity against DHPS. 1C was followed by 1B which showed a moderate to low level MIC of 100, 250 and 150 µg/mL against E. coli, B. subtilis and B. linen respectively, whereas 1A showed a moderate level MIC of 100 µg/mL but only against B. linen. These derivatives may thus serve as potential anti-bacterial alternatives against resistant pathogens.