Multitasking Pharmacophores Support Cabotegravir-Based Long-Acting HIV Pre-Exposure Prophylaxis (PrEP).

Cabotegravir is an integrase strand transfer inhibitor (INSTI) for HIV treatment and prevention. Cabotegravir-based long-acting pre-exposure prophylaxis (PrEP) presents an emerging paradigm for infectious disease control. In this scheme, a combination of a high efficacy and low solubility of anti-infection drugs permits the establishment of a pharmaceutical firewall in HIV-vulnerable groups over a long period. Although the structure-activity-relationship (SAR) of cabotegravir as an INSTI is known, the structural determinants of its low solubility have not been identified. In this work, we have integrated multiple experimental and computational methods, namely X-ray diffraction, solid-state NMR (SSNMR) spectroscopy, solution NMR spectroscopy, automated fragmentation (AF)-QM/MM and density functional theory (DFT) calculations, to address this question. The molecular organization of cabotegravir in crystal lattice has been determined. The combination of very-fast magic-angle-sample-spinning (VF MAS) SSNMR and solution NMR, as supported by AF-QM/MM and DFT calculations, permits the identification of structural factors that contribute to the low aqueous solubility of cabotegravir. Our study reveals the multitasking nature of pharmacophores in cabotegravir, which controls the drug solubility and, meanwhile, the biological activity. By unraveling these function-defining molecular features, our work could inspire further development of long-acting HIV PrEP drugs.

CP-MAS and Solution NMR Studies of Allosteric Communication in CA-assemblies of HIV-1.

Solution and solid-state NMR spectroscopy are highly complementary techniques for studying structure and dynamics in very high molecular weight systems. Here we have analysed the dynamics of HIV-1 capsid (CA) assemblies in presence of the cofactors IP6 and ATPγS and the host-factor CPSF6 using a combination of solution state and cross polarisation magic angle spinning (CP-MAS) solid-state NMR. In particular, dynamical effects on ns to µs and µs to ms timescales are observed revealing diverse motions in assembled CA. Using CP-MAS NMR, we exploited the sensitivity of the amide/Cα-Cß backbone chemical shifts in DARR and NCA spectra to observe the plasticity of the HIV-1 CA tubular assemblies and also map the binding of cofactors and the dynamics of cofactor-CA complexes. In solution, we measured how the addition of host- and co-factors to CA -hexamers perturbed the chemical shifts and relaxation properties of CA-Ile and -Met methyl groups using transverse-relaxation-optimized NMR spectroscopy to exploit the sensitivity of methyl groups as probes in high-molecular weight proteins. These data show how dynamics of the CA protein assembly over a range of spatial and temporal scales play a critical role in CA function. Moreover, we show that binding of IP6, ATPγS and CPSF6 results in local chemical shift as well as dynamic changes for a significant, contiguous portion of CA, highlighting how allosteric pathways communicate ligand interactions between adjacent CA protomers.

Structure and Dynamics Perturbations in Ubiquitin Adsorbed or Entrapped in Silica Materials Are Related to Disparate Surface Chemistries Resolved by Solid-State NMR Spectroscopy.

Protein immobilization on material surfaces is emerging as a powerful tool in the design of devices and active materials for biomedical and pharmaceutical applications as well as for catalysis. Preservation of the protein's biological functionality is crucial to the design process and is dependent on the ability to maintain its structural and dynamical integrity while removed from the natural surroundings. The scientific techniques to validate the structure of immobilized proteins are scarce and usually provide limited information as a result of poor resolution. In this work, we benchmarked the ability of standard solid-state NMR techniques to resolve the effects of binding to dissimilar silica materials on a model protein. In particular, the interactions between ubiquitin and the surfaces of MCM41, SBA15, and silica formed in situ were tested for their influence on the structure and dynamics of the protein. It is shown that the protein's globular fold in the free state is only slightly perturbed in the three silica materials. Local motions on a residue level that are quenched by immobilization or, conversely, that arise from the process are also detailed. NMR measurements show that these perturbations are unique to each silica material and can serve as reporters of the characteristic surface chemistry.