Role of E3 Ligases in Cancer Therapeutics

Abstract

Tribbles pseudokinases, Tribbles homologs (TRIB1-3) bind to constitutive photomorphogenesis protein 1 (COP1) E3 ligase to mediate the regulation of β-catenin expression. The interaction mechanism between COP1 E3 ligase and β-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 β-catenin and COP1. Here, through in silico approaches, we ascribe the COP1 binding patterns against TRIBs and β-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 β-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 β-catenin phosphodegron signature for binding to WD40 domain of COP1. A thorough understanding of the structural basis for TRIB-mediated regulation of WNT/β-catenin signaling may help in devising a more promising therapeutic strategy for targeting liver and colorectal cancers. 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. Current study focuses on characterizing the binding pattern of E2UbcH5B to HECT domains of NEDD4L (NEDD4 Like E3 Ubiquitin Protein Ligase), WWP1 (WW Domain Containing E3 Ubiquitin Protein Ligase 1), WWP2 (WW Domain Containing E3 Ubiquitin Protein Ligase 2), HECW1 (HECT, C2 and WW Domain Containing E3 Ubiquitin Protein Ligase 1), and HECW2 (HECT, C2 and WW Domain Containing E3 Ubiquitin Protein Ligase 2) 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 HECT proteins. We observed a similar binding pattern for E2UbcH5B and mentioned DRSML QAU Abstract xiii HECT domains as previously reported for HECTNEDD4L , where Trp762, Trp709, and Trp657 residues of HECTNEDD4L, HECTWWP1, and HECTWWP2 were involved in making contacts with Ser94 residue of E2UbcH5B. Similarly, corresponding to HECTNEDD4L Tyr756 residue, other HECT 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. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for more than 30 million infections and 0.9 million deaths worldwide. Upon SARS-CoV-2 infection, Histone deacetylases (HDACs) hyperactivate the pro-inflammatory response resulting in the 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 the active site and binding affinities, 3 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 their energy minimization and docking against HDAC3 and CRBN, respectively. Subsequent molecular dynamics and free energy analyses corroborated comparable binding trends and favorable energy values. Among all cases, Met88, GLu106, Pro352, Trp380 and Trp388 residues of CRBN, and Pro23, Arg28, Lys194, DRSML QAU Abstract xiii 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 the target degradation. Overall, the proposed bifunctional small molecules may effectively target HDAC3 and stimulate the innate immune response to restrict COVID-19 hyperinflammation. Additionally, the current 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.