Planejamento in silico para a construção de nanocorpos (VHH) miméticos anti-integrina α4β1 (CD49d/CD29) humana

Abstract

Multiple Sclerosis (MS) is an autoimmune, inflammatory, and neurodegenerative disease and the leading cause of non-traumatic neurological disability in young adults. Its incidence ranges from over 100 cases per 100,000 inhabitants in North America and Western Europe to less than 30 cases per 100,000 inhabitants in equatorial regions, with an estimated 40,000 cases in Brazil. MS involves leukocytes, chemokines, and cytokines such as TNF-³, IFN-´, and IL-17, as well as ³4³1 integrin, which facilitates lymphocyte migration into the central nervous system (CNS) via the VCAM-1 receptor. Natalizumab (NTZ) inhibits this interaction and serves as an effective immunotherapy; however, its prolonged use compromises immune surveillance, increasing susceptibility to opportunistic infections and reactivation of the John Cunningham (JC) polyomavirus, leading to Progressive Multifocal Leukoencephalopathy (PML). Additionally, NTZ has side effects such as bleeding, dizziness, nausea, and muscle stiffness, along with high costs and a lack of exclusive selectivity for MS, as it also interacts with ³4³7 integrin, which is associated with Crohn9s disease. To overcome these limitations, nanobodies (VHHs) have emerged as a promising alternative driven by in silico tools and deep learning algorithms that enable faster and more cost-effective production. In this study, we developed an initial strategy to create VHHs that mimic NTZ while preserving its properties and specificity. Initially, SASA calculations (via FreeSASA) were used to characterize the VH/NTZ-³4 interface. Native VHHs (VHH-IN) were selected from the SAbDab database, prioritizing those with structurally similar CDR3 regions to VH/NTZ. Three grafting strategies were employed to transfer CDRs from VH/NTZ to VHHs: ENXSEQ, ENXSDR, and ENXEST. ENXSEQ unified the VH/NTZ CDRs into the frameworks (FR) of the VHHs by modeling in NanobodyBuilder2. The VHH/NTZ-³4 complexes were generated via molecular docking using HADDOCK 2.4, and the best complexes were selected based on the DockQ score. Although ENXSDR better preserved the properties of VHH-IN but had lower specificity for ³4, ENXEST exhibited intermediate conservation and greater VHH/NTZ-³4 complex formation. In contrast, ENXSEQ was the least conservative, but showed intermediate specificity for the antigen. These results indicate that hybrid VHHs maintain specificity for the ³4 subunit, varying according to the grafting strategy, with ENXSDR being the most promising. However, further optimization is required to enhance specificity.