Designing of potent anti-diabetic molecules by targeting SIK2 using computational approaches.

Diabetes mellitus (DM) is one of the major health problems worldwide. WHO have estimated that 439 million people may have DM by the year 2030. Several classes of drugs such as sulfonylureas, meglitinides, thiazolidinediones etc. are available to manage this disease, however, there is no cure for this disease. Salt inducible kinase 2 (SIK2) is expressed several folds in adipose tissue than in normal tissues and thus SIK2 is one of the attractive targets for DM treatment. SIK2 inhibition improves glucose homeostasis. Several analogues have been reported and experimentally proven against SIK for DM treatment. But, identifying potential SIK2 inhibitors with improved efficacy and good pharmacokinetic profiles will be helpful for the effective treatment of DM. The objective of the present study is to identify selective SIK2 inhibitors with good pharmacokinetic profiles. Due to the unavailability of SIK2 structure, the modeled structure of SIK2 will be an important to understand the atomic level of SIK2 inhibitors in the binding site pocket. In this study, different molecular modeling studies such as Homology Modeling, Molecular Docking, Pharmacophore-based virtual screening, MD simulations, Density Functional Theory calculations and WaterMap analysis were performed to identify potential SIK2 inhibitors. Five molecules from different databases such as Binding_4067, TosLab_837067, NCI_349155, Life chemicals_ F2565-0113, Enamine_7623111186 molecules were identified as possible SIK2 inhibitors.

Water Mapping and Scoring Approaches to Predict the Role of Hydration Sites in the Binding Affinity of PAK1 Inhibitors.

AIM: This study aims to develop and establish a computational model that can identify potent molecules for p21-activating kinase 1 (PAK1) Background: PAK1 is a well-established drug target that has been explored for various therapeutic interventions. Control of this protein requires an indispensable inhibitor to curb the structural changes and subsequent activation of signalling effectors responsible for the progression of diseases, such as cancer, inflammatory, viral, and neurological disorders. OBJECTIVE: The study aims to establish a computational model that could identify active molecules which will further provide a platform for developing potential PAK1 inhibitors. METHODS: A congeneric series of 27 compounds were considered for this study, with Ki (nm) covering a minimum of 3 log range. The compounds were developed based on a previously reported Group-I PAK inhibitor, namely G-5555. The 27 compounds were subjected to the SP and XP mode of docking to understand the binding mode, its conformation and interaction patterns. To understand the relevance of biological activity from computational approaches, the compounds were scored against generated water maps to obtain WM/MM ΔG binding energy. Moreover, molecular dynamics analysis was performed for the highly active compound to understand the conformational variability and stability of the complex. We then evaluated the predictable binding pose obtained from the docking studies. RESULTS: From the SP and XP modes of docking, the common interaction pattern with the amino acid residues Arg299 (cation-π), Glu345 (Aromatic hydrogen bond), hinge region Leu347, salt bridges Asp393 and Asp407 was observed, among the congeneric compounds. The interaction pattern was compared with the co-crystal inhibitor FRAX597 of the PAK1 crystal structure (PDB id: 4EQC). The correlation with different docking parameters in the SP and XP modes was insignificant and thereby revealed that the SP and XP's scoring functions could not predict the active compounds. This was due to the limitations in the docking methodology that neglected the receptor flexibility and desolvation parameters. Hence, to recognise the desolvation and explicit solvent effects, as well as to study the Structure-Activity Relationships (SARs) extensively, WaterMap (WM) calculations were performed on the congeneric compounds. Based on displaceable unfavourable hydration sites (HS) and their associated thermodynamic properties, the WM calculations facilitated in understanding the significance of correlation in the folds of activity of highly active (19 and 17), moderately active (16 and 21) and less active (26 and 25) compounds. Furthermore, the scoring function from WaterMap, namely WM/MM, led to a significant R2 value of 0.72 due to a coupled conjunction with MM treatment and displaced unfavourable waters at the binding site. To check the "optimal binding conformation", molecular dynamics simulation was carried out with the highly active compound 19 to explain the binding mode, stability, interactions, solvent-accessible area, etc., which could support the predicted conformation with bioactive conformation. CONCLUSION: This study determined the best scoring function, established SARs and predicted active molecules through a computational model. This will contribute to the development of the most potent PAK1 inhibitors.

WaterMap and Molecular Dynamic Simulation-Guided Discovery of Potential PAK1 Inhibitors Using Repurposing Approaches.

p21-Activated kinase 1 (PAK1) is positioned at the nexus of several oncogenic signaling pathways. Currently, there are no approved inhibitors for disabling the transfer of phosphate in the active site directly, as they are limited by lower affinity, and poor kinase selectivity. In this work, a repurposing study utilizing FDA-approved drugs from the DrugBank database was pursued with an initial selection of 27 molecules out of ∼2162 drug molecules, based on their docking energies and molecular interaction patterns. From the molecules that were considered for WaterMap analysis, seven molecules, namely, Mitoxantrone, Labetalol, Acalabrutinib, Sacubitril, Flubendazole, Trazodone, and Niraparib, ascertained the ability to overlap with high-energy hydration sites. Considering many other displaced unfavorable water molecules, only Acalabrutinib, Flubendazole, and Trazodone molecules highlighted their prominence in terms of binding affinity gains through ΔΔG that ranges between 6.44 and 2.59 kcal/mol. Even if Mitoxantrone exhibited the highest docking score and greater interaction strength, it did not comply with the WaterMap and molecular dynamics simulation results. Moreover, detailed MD simulation trajectory analyses suggested that the drug molecules Flubendazole, Niraparib, and Acalabrutinib were highly stable, observed from their RMSD values and consistent interaction pattern with Glu315, Glu345, Leu347, and Asp407 including the hydrophobic interactions maintained in the three replicates. However, the drug molecule Trazodone displayed a loss of crucial interaction with Leu347, which was essential to inhibit the kinase activity of PAK1. The molecular orbital and electrostatic potential analyses elucidated the reactivity and strong complementarity potentials of the drug molecules in the binding pocket of PAK1. Therefore, the CADD-based reposition efforts, reported in this work, helped in the successful identification of new PAK1 inhibitors that requires further investigation by in vitro analysis.

Structural insights on binding mechanism of CAD complexes (CPSase, ATCase and DHOase).

Pyrimidine biosynthetic pathway enzymes constitute an important target for the development of antitumor drugs. To understand the role of binding mechanisms underlying the inborn errors of pyrimidine biosynthetic pathway, structure and function of enzymes have been analyzed. Pyrimidine biosynthetic pathway is initiated by CAD enzymes that harbor the first three enzymatic activities facilitated by Carbamoyl Phosphate Synthetase (CPSase), Aspartate Transcarbamoylase (ATCase) and Dihydroorotase (DHOase). While being an attractive therapeutic target, the lack of data driven us to study the CPSase (CarA and CarB) and its mode of binding to ATCase and DHOase which are the major limitation for its structural optimization. Understanding the binding mode of CPSase, ATCase and DHOase could help to identify the potential interface hotspot residues that favor the mechanism behind it. The mechanistic insight into the CAD complexes were achieved through Molecular modeling, Protein-Protein docking, Alanine scanning and Molecular dynamics (MD) Studies. The hotspot residues present in the CarB region of carboxy phosphate and carbamoyl phosphate synthetic domains are responsible for the assembly of CAD (CPSase-ATCase-DHOase) complexes. Overall analysis suggests that the identified hotspot residues were confirmed by alanine scanning and important for the regulation of pyrimidine biosynthesis. MD simulations analysis provided the prolonged stability of the interacting complexes. The present study reveals the novel hotspot residues such as Glu134, Glu147, Glu154, Asp266, Lys269, Glu274, Asp333, Trp459, Asp526, Asp528, Glu533, Glu544, Glu546, Glu800, Val855, Asp877, Tyr884 and Gln919 which could be targeted for structure-based inhibitor design to potentiate the CAD mediated regulation of aggressive tumors.Communicated by Ramaswamy H. Sarma.

Identification of Pak1 inhibitors using water thermodynamic analysis.

p21-activated kinases (Paks) play an integral component in various cellular diverse processes. The full activation of Pak is dependent upon several serine residues present in the N-terminal region, a threonine present at the activation loop, and finally the phosphorylation of these residues ensure the complete activation of Pak1. The present study deals with the identification of novel potent candidates of Pak1 using computational methods as anti-cancer compounds. A diverse energy based pharmacophore (e-pharmacophore) was developed using four co-crystal inhibitors of Pak1 having pharmacophore features of 5 (DRDRR), 6 (DRHADR), and 7 (RRARDRP and DRRDADH) hypotheses. These models were used for rigorous screening against e-molecule database. The obtained hits were filtered using ADME/T and molecular docking to identify the high affinity binders. These hits were subjected to hierarchical clustering using dendritic fingerprint inorder to identify structurally diverse molecules. The diverse hits were scored against generated water maps to obtain WM/MM ΔG binding energy. Furthermore, molecular dynamics simulation and density functional theory calculations were performed on the final hits to understand the stability of the complexes. Five structurally diverse novel Pak1 inhibitors (4835785, 32198676, 32407813, 76038049, and 32945545) were obtained from virtual screening, water thermodynamics and WM/MM ΔG binding energy. All hits revealed similar mode of binding pattern with the hinge region residues replacing the unstable water molecules in the binding site. The obtained novel hits could be used as a platform to design potent drugs that could be experimentally tested against cancer patients having increased Pak1 expression.

Design of novel PhMTNA inhibitors, targeting neurological disorder through homology modeling, molecular docking, and dynamics approaches.

Vanishing white matter (VWM) is a hereditary human disease, mostly prevalent in childhood caused by the defects in the eukaryotic initiation factor beta subunits. It is the first disease involved in the translation initiation factor, eIF2B. There is no specific treatment for VWM which mainly affect the brain and ovaries. The gray matter remains normal in all characteristics while the white matter changes texture, coming to the pathophysiology, many initiation factors are involved in the initiation of translation of mRNAs into polypeptides. In this study, the three-dimensional structure of PhMTNA protein was modeled and the stability ascertained through Molecular dynamic simulation (MDS) for 100 ns. The active site residues are conserved with the reported BsMTNA structure which is also confirmed through sitemap prediction. Through virtual screening and induced fit docking, top five leads against PhMTNA protein was identified based on their binding mode and affinity. ADME properties and DFT (Density Functional Theory) studies of these compounds were studied. In addition to that, computational mutagenesis studies were performed to identify the hotspot residues involved in the protein-ligand interactions. Overall analysis showed that the compound NCI_941 has a highest binding energy of -46.256 kcal mol-1 in the Arg57Ala mutant. Thus, the results suggest that NCI_941 would act as a potent inhibitor against PhMTNA protein.

In silico analysis suggests that PH0702 and PH0208 encode for methylthioribose-1-phosphate isomerase and ribose-1,5-bisphosphate isomerase, respectively, rather than aIF2Bß and aIF2Bδ.

The overall process of protein biosynthesis across all domains of life is similar; however, detailed insights reveal a range of differences in the proteins involved. For decades, the process of protein translation in archaea has been considered to be closer to eukaryotes than to bacteria. In archaea, however, several homologues of eukaryotic proteins involved in translation initiation have not yet been identified; one of them being the initiation factor eIF2B consisting of five subunits (α, ß, γ, δ and ε). Three open reading frames (PH0440, PH0702 and PH0208) in Pyrococcus horikoshii have been proposed to encode for the α-, ß- and δ-subunits of aIF2B, respectively. The crystal structure of PH0440 shows similarity toward the α-subunit of eIF2B. However, the capability of PH0702 and PH0208 to function as the ß- and δ-subunits of eIF2B, respectively, remains uncertain. In this study, we have taken up the task of annotating PH0702 and PH0208 using bioinformatics methods. The phylogenetic analysis of protein sequences belonging to IF2B-like family along with PH0702 and PH0208 revealed that PH0702 belonged to methylthioribose-1-phosphate isomerase (MTNA) group of proteins, whereas, PH0208 was found to be clustered in the group of ribose-1,5-bisphosphate isomerase (R15PI) proteins. A careful analysis of protein sequences and structures available for eIF2B, MTNA and R15PI confirms that PH0702 and PH0208 contain residues essential for the enzymatic activity of MTNA and R15PI, respectively. Additionally, the protein PH0208 comprises of the residues required for the dimer formation which is essential for the biological activity of R15PI. This prompted us to examine all eIF2B-like proteins from archaea and to annotate their function. The results reveal that majority of these proteins are homologues of the α-subunit of eIF2B, even though they lack the residues essential for their functional activity. A better understanding of the mechanism of GTP exchange during translation initiation in archaea is henceforth required.

Mechanical insights of oxythiamine compound as potent inhibitor for human transketolase-like protein 1 (TKTL1 protein).

Transketolase is a connecting link between glycolytic and pentose phosphate pathway, which is considered as the rate-limiting step due to synthesis of large number of ATP molecule and it can be proposed as a plausible target facilitating the growth of cancerous cells suggesting its potential role in cancer. Oxythiamine, an antimetabolite has been proved to be an efficient anticancerous compound in vitro, but its structural elucidation of the inhibitory mechanism has not yet been done against the human transketolase-like 1 protein (TKTL1). The three-dimensional (3D) structure of TKTL1 protein was modeled and subjected for refinement, stability and validation. Based on the reported homologs of transketolase (TKT), the active site residues His46, Ser49, Ser52, Ser53, Ile56, Leu82, Lys84, Leu123, Ser125, Glu128, Asp154, His160, Thr216 and Lys218 were identified and considered for molecular-modeling studies. Docking studies reveal the H-bond interactions with residues Ser49 and Lys218 that could play a major role in the activity of TKTL1. Molecular dynamics (MD) simulation study was performed to reveal the comparative stability of both native and complex forms of TKTL1. MD trajectory at 30 ns, confirm the role of active site residues Ser49, Lys84, Glu128, His160 and Lys218 in suppressing the activity of TKTL1. Glu128 is observed to be the most important residue for deprotonation state of the aminopyrimidine moiety and preferred to be the site of inhibitory action. Thus, the proposed mechanism of inhibition through in silico studies would pave the way for structure-oriented drug designing against cancer.

Molecular mechanism of anti-cancer activity of phycocyanin in triple-negative breast cancer cells.

BACKGROUND: Triple-negative breast cancers represent an important clinical challenge, as these cancers do not respond to conventional endocrine therapies or other available targeted agents. Phycocyanin (PC), a natural, water soluble and non-toxic molecule is shown to have potent anti-cancer property. METHODS: In this study, we determined the efficacy of PC as an anti-neoplastic agent in vitro on a series of breast cancer cell lines. We studied effects of PC in inducing DNA damage and apoptosis through western blot and qPCR. Also, anti-metastatic and anti-angiogenic properties were studied by classic wound healing and vasculogenic mimicry assays. RESULTS: We found that triple negative MDA-MB-231 cells were most sensitive to PC (IC50 : 5.98 ± 0.95 µM) as compared to other cells. They also showed decreased cell proliferation and reduced colony formation ability upon treatment with PC. Profile of Cell cycle analysis showed that PC caused G1 arrest which could be attributed to decreased mRNA levels of Cyclin E and CDK-2 and increased p21 levels. Mechanistic studies revealed that PC induced apoptosis as evident by increase in percentage of annexin positive cells, increase in γ-H2AX levels, and by changing the Bcl-2/Bax ratio followed by release of cytochrome C and increased Caspase 9 levels. MDA MB 231 cells treated with PC resulted in decreased cell migration and increased cell adhesive property and also showed anti-angiogenic effects. We also observed that PC suppressed cyclooxygenase-2 (COX-2) expression and prostaglandin E(2) production. All these biological effects of phycocyanin on MDA MB 231 cells could be attributed to decreased MAPK signaling pathway. We also observed that PC is non-toxic to non-malignant cells, platelets and RBC's. CONCLUSION: Taken together, these findings demonstrate, for the first time, that PC may be a promising anti-neoplastic agent for treatment of triple negative breast cancers.