Molecular dynamics and structural analysis of the binding of COP1 E3 ubiquitin ligase to ß-catenin and TRIB pseudokinases.

Tribbles pseudokinases, Tribbles homolog 1 (TRIB1), Tribbles homolog 2 (TRIB2), and Tribbles homolog 3 (TRIB3), bind to constitutive photomorphogenesis protein 1 (COP1) E3 ligase to mediate the regulation of ß-catenin expression. The interaction mechanism between COP1 E3 ligase and ß-catenin has not been addressed to date. Based on the functional presence of TRIBs in wingless-related integration site (WNT) signaling, we analyzed their interaction patterns with ß-catenin and COP1. Here, through in silico approaches, we ascribe the COP1 binding pattern against TRIBs and ß-catenin. TRIB1 (355-DQIVPEY-361), TRIB2 (326-DQLVPDV-332), and TRIB3 (333-AQVVPDG-339) peptides revealed a shallow binding pocket at the COP1 interface to accommodate the V-P sequence motif. Reinvigoration of the comparative binding pattern and subtle structural analysis via docking, molecular dynamics simulations, molecular mechanics Poisson-Boltzmann surface area, topological, and tunnel analysis revealed that both ß-catenin phosphodegron (DSGXXS) and TRIB (D/E/AQXVPD/E) motifs occupied a common COP1 binding site. Current study suggests a structural paradigm of TRIB homologs bearing a conserved motif that may compete with ß-catenin phosphodegron signature for binding to WD40 domain of COP1. Thorough understanding of the structural basis for TRIB-mediated regulation of WNT/ß-catenin signaling may help in devising more promising therapeutic strategy for liver and colorectal cancers.

Structural basis of Klotho binding to VEGFR2 and TRPC1 and repurposing calcium channel blockers as TRPC1 antagonists for the treatment of age-related cardiac hypertrophy.

Cardiac hypertrophy results in the higher rate of heart failures among aged groups. Klotho is an anti-aging protein that is involved in the regulation of VEGF-mediated Ca2+ entry by direct interaction with Vascular endothelial growth factor receptor 2 (VEGFR2) and transient receptor potential canonical Ca2+ channel 1 (TRPC1). Here, in this study, through in silico analysis, we modeled TRPC1 3-dimensional structure and followed by its optimization, characterized the interaction pattern of TRPC1, Klotho and VEGFR2. Subsequent molecular dynamics (MD) simulation analysis revealed that Klotho-specific (P520-N630) region exhibited interaction with VEGFR2, while its C-terminal region (I822-A931) demonstrated binding to the 3rd extracellular loop of TRPC1 that is adjacent to pore region. Through TRPC1 homotetramer formation, the residues in the periphery of pore region were carefully evaluated. In order to scrutinize known Ca2+ channel blockers for their ability to bind at the pore region of TRPC1, 31 known compounds were tested through docking runs and three hits, named as diltiazem impurity B (b3), diltiazem (b5) and felodipine (b6) were selected for detailed binding analysis through MD runs. Evidently, inhibitor-bound TRPC1 pore area was more constricted (8.6 Å2, 25.1 Å2 and 18.8 Å2, respectively) than apo-TRPC1 (60 Å2). These findings suggest that Ca+2 channel blockers may serve as promising agents to impair the TRPC1 functional store-operated calcium channel (SOCC) activity in the old patients lacking Klotho expression. Thus, pore region of homotetrameric TRPC1 may be blocked via repurposing of known Ca+2 blockers to antagonize TRPC signaling for the treatment of cardiac hypertrophy.

Structural-based design of HD-TAC7 PROteolysis TArgeting chimeras (PROTACs) candidate transformations to abrogate SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for about 672 million infections and 6.85 million deaths worldwide. Upon SARS-CoV-2 infection, Histone deacetylases (HDACs) hyperactivate the pro-inflammatory response resulting in stimulation of Acetyl-Coenzyme A and cholesterol for viral entry. HDAC3 inhibition results in the anti-inflammatory activity and reduction of pro-inflammatory cytokines that may restrict COVID-19 progression. Here, we have designed 44 conformational ensembles of previously known HD-TAC7 by enumerating torsions of dihedral angles tested for their binding preferences against HDAC3. Through scrutinizing their placements at active site and binding affinities, three hits were isolated. Cereblon (CRBN) is a well-known E3 ligase that facilitates Proteolysis Targeting Chimeras (PROTACs) targeting. Three entities, including HDAC3-binding moiety (4-acetamido-N-(2-amino-4 fluorophenyl) benzamide), a 6-carbon linker, and CRBN binding ligand (pomalidomide) were assembled to design 4 PROTACs followed by energy minimization and docking against HDAC3 and CRBN, respectively. Subsequent molecular dynamics (MD) and free energy analyses corroborated similar binding trends and favorable energy values. Among all cases, Met88, GLu106, Pro352, Trp380 and Trp388 residues of CRBN, and Pro23, Arg28, Lys194, Phe199, Leu266, Thr299 and Ile346 residues of HDAC3 were engaged in PROTAC binding. Thus, conformational dynamics of both HDAC3 and CRBN moieties are essential for the placement of PROTAC, resulting in target degradation. Overall, the proposed bifunctional small molecules may effectively target HDAC3, stimulating innate immune response to restrict COVID-19 hyperinflammation. This study supports the basis for designing new PROTACs by limiting the conformational search space that may prove more efficient for targeting the protein of interest.Communicated by Ramaswamy H. Sarma.

CYP2C19*17 association with higher plasma 4-hydroxy tamoxifen in Pakistani (estrogen-positive) breast cancer patients.

Breast cancer (BC) continues to be the most common cancer in the women worldwide. Since estrogen receptor (ER)-positive BC accounts for the majority of newly diagnosed cases, endocrine therapy is advised to utilize either tamoxifen (Tam) or aromatase inhibitors. The use of Tam as a monotherapy or in conjunction with an aromatase inhibitor following two or three years of endocrine therapy has long been recommended. When used adjuvantly, Tam medication reduces BC mortality and relapses, while it extends survival times in metastatic BC when used in conjunction with other treatments. Unfortunately, the efficiency of Tam varies considerably. This study was conducted to explore the influence of genetic polymorphisms in CYP2C19 gene on Tam's pharmacogenetics and pharmacokinetics in estrogen-positive BC patients. Data from healthy, unrelated individuals (n = 410; control group) and ER-positive BC patients (n = 430) receiving 20 mg of Tam per day were recruited. Steady-state plasma concentrations of Tam and its three metabolites were quantified using the high-performance liquid chromatography in the patients. The CYP2C19 polymorphisms were genotyped using an Amplification Refractory Mutation System-Polymerase Chain Reaction (ARMS-PCR) approach. More than 65% of healthy individuals were extensive metabolizers (*1/*1) for CYP2C19, whereas more than 70% of ER-positive BC patients were rapid and ultrarapid metabolizers (*1/17*, *17/17*). The polymorphism CYP2C19*17 is significantly associated with higher 4-hydroxytamoxifen (4-OH-Tam). Patients with the *17/*17 genotype exhibited 1- to 1.5-fold higher 4-OH-Tam, which was also high in patients with the *1/*2 and *2/*2 genotypes.

E2UbcH5B-derived peptide ligands target HECT E3-E2 binding site and block the Ub-dependent SARS-CoV-2 egression: A computational study.

Homologous to E6AP carboxyl-terminus (HECT)-type E3 ligase performs ubiquitin (Ub)-proteasomal protein degradation via forming a complex with E2∼Ub. Enveloped viruses including SARS-CoV-2 escape from the infected cells by harnessing the E-class vacuolar protein-sorting (ESCRT) machinery and mimic the cellular system through PPAY motif-based linking to HECT Ub ligase activity. In the present study, we have characterized the binding pattern of E2UbcH5B to HECT domains of NEDD4L, WWP1, WWP2, HECW1, and HECW2 through in silico analysis to isolate the E2UbcH5B-specific peptide inhibitors that may target SARS-CoV-2 viral egression. Molecular dynamics analysis revealed more opening of E2UbcH5B-binding pocket upon binding to HECTNEDD4L, HECTWWP1, HECTWWP2, HECTHECW1, and HECTHECW2. We observed similar binding pattern for E2UbcH5B and mentioned HECT domains as previously reported for HECTNEDD4L where Trp762, Trp709, and Trp657 residues of HECTNEDD4L, HECTWWP1, and HECTWWP2 are involved in making contacts with Ser94 residue of E2UbcH5B. Similarly, corresponding to HECTNEDD4L Tyr756 residue, HECTWWP1, HECTWWP2, HECTHECW1, and HECTHECW2-specific Phe703, Phe651, Phe1387, and Phe1353 residues execute interaction with E2UbcH5B. Our analysis suggests that corresponding to Cys942 of HECTNEDD4L, Cys890, Cys838, Cys1574, and Cys1540 residues of HECTWWP1, HECTWWP2, HECTHECW1, and HECTHECW2, respectively are involved in E2-to-E3 Ub transfer. Furthermore, MM-PBSA free energy calculations revealed favorable energy values for E2UbcH5B-HECT complexes along with the individual residue contributions. Subsequently, two E2UbcH5B-derived peptides (His55-Phe69 and Asn81-Ala96) were tested for their binding abilities against HECT domains of NEDD4L, WWP1, WWP2, HECW1, and HECW2. Their binding was validated through substitution of Phe62, Pro65, Ile84, and Cys85 residues into Ala, which revealed an impaired binding, suggesting that the proposed peptide ligands may selectively target E2-HECT binding and Ub-transfer. Collectively, we propose that peptide-driven blocking of E2-to-HECT Ub loading may limit SARS-CoV-2 egression and spread in the host cells.