RESUMO
Single nucleotide polymorphisms (SNPs) in the TUBB1 (ß-tubulin) gene have been implicated as the primary cause of macro thrombocytopenia. Therefore it is essential to identify the potential SNPs which are harmful to cause diseases such as macro thrombocytopenia. The impact caused by these variants on ß-tubulin is twofold, both structural and functional. Multiple in-silico tools were used to scrutinise the most deleterious nsSNPs (non-synonymous SNPs) via sequence and structure-based approaches. Further, the ß-tubulin protein model incorporating identified mutants was subjected to MD (molecular dynamic) simulations to analyse the impact on protein structure. A total of 2974 SNPs of TUBB1 were retrieved from various sources, and 32 nsSNPs were identified. By screening through sequence-based technique, 13 variants were detected as deleterious and further structure-based filtration was carried out to find thermally destabilising variants. Finally, three variants have been detected as highly destabilising by the mCSM server and chosen for the MD study. All three variants are present in the N-terminal, Intermediate, and C-terminal regions, breaking the spatial arrangement required for microtubule assembly. The spatial arrangement of these variants is in deviation with respect to WT (wild type) ß-tubulin. The protein model was subjected to a simulation period of 100 ns. The FEL analysis revealed multiple clusters with minor populations indicating the unstable conformation adapted by the ß-tubulin. The normal mode vector analysis exhibited high-intensity flexible motions at the C-terminal end, responsible for binding with MAPs (microtubule-associated proteins), an essential region in microtubule assembly. All these results reveal that the SNP's predicted eventually influence the spatial arrangement of ß-tubulin, which would disturb the stacking arrangement of αß tubulin dimer in microtubule assembly. The present study may set a path to cure the diseases like macro thrombocytopenia.Communicated by Ramaswamy H. Sarma.
RESUMO
BACKGROUND: Cereblon, an extensively studied multifunctional protein, is a Cullin 4-RING E3 ubiquitin ligase complex component. Cereblon is a well-known target of thalidomide and its derivatives. Cereblon is involved in multiple myeloma cell apoptosis. When ligands such as thalidomide and lenalidomide bind to cereblon, it recognizes various neosubstrates based on the ligand shape and properties. We have identified novel CRBN inhibitors, namely DHFO and its analogs, with structural features that are slightly different from thalidomide but stronger cereblon-binding affinity. We selected indanedione and indanone derivatives from the literature to understand and compare their cereblon-mediated substrate recognition potential. METHODS: Computational investigations of possible CRBN inhibitors were investigated by molecular docking with Autodock Vina and DockThor programs. The properties of the compounds' ADME/T and drug-likeness were investigated. A molecular dynamics study was carried out for four selected molecules, and the molecular interactions were analyzed using PCA-based FEL methods. The binding affinity was calculated using the MM/PBSA method. RESULTS: We conducted computational investigations on 68 indanedione and indanone derivatives binding with cereblon. Ten molecules showed better CRBN binding affinity than thalidomide. We studied the drug-likeness properties of the selected ten molecules, and four of the most promising molecules (DHFO, THOH, DIMS, and DTIN) were chosen for molecular dynamics studies. The MM/PBSA calculations showed that the DHFO, already shown to be a 5-LOX/COX2 inhibitor, has the highest binding affinity of - 163.16 kJ/mol with cereblon. CONCLUSION: The selected CRBN inhibitor DHFO has demonstrated the highest binding affinity with cereblon protein compared to other molecules. Thalidomide and its derivatives have a new substitute in the form of DHFO, which produces an interaction hotspot on the surface of the cereblon. Ease of chemical synthesis, low toxicity, versatile therapeutic options, and pleiotropism of DHFO analogs provide an opportunity for exploring clinical alternatives with versatile therapeutic potential for a new category of indanedione molecules as novel modulators of E3 ubiquitin ligases.
