Ranking mAb-excipient interactions in biologics formulations by NMR spectroscopy and computational approaches.

Excipients are added to biopharmaceutical formulations to enhance protein stability and enable the development of robust formulations with acceptable physicochemical properties, but the mechanism by which they confer stability is not fully understood. Here, we aimed to elucidate the mechanism through direct experimental evidence of the binding affinity of an excipient to a monoclonal antibody (mAb), using saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopic method. We ranked a series of excipients with respect to their dissociation constant (KD) and nonspecific binding constants (Ns). In parallel, molecular dynamic and site identification by ligand competitive saturation (SILCS)-Monte Carlo simulations were done to rank the excipient proximity to the proteins, thereby corroborating the ranking by STD NMR. Finally, the excipient ranking by NMR was correlated with mAb conformational and colloidal stability. Our approach can aid excipient selection in biologic formulations by providing insights into mAb-excipient affinities before conventional and time-consuming excipient screening studies are conducted.

Roles of variable linker length in dual acting virucidal entry inhibitors on HIV-1 potency via on-the-fly free energy molecular simulations.

The Dual-Acting Virolytic Entry Inhibitors, or DAVEI's, are a class of recombinant chimera fusion proteins consisting of a lectin, a flexible polypeptide linker, and a fragment of the membrane-proximal external region (MPER) of HIV-1 gp41. DAVEIs trigger virolysis of HIV-1 virions through interactions with the trimeric envelope glycoprotein complex (Env), though the details of these interactions are not fully determined as yet. The purpose of this work was to use structural modeling to rationalize a dependence of DAVEI potency on the molecular length of the linker connecting the two components. We used temperature accelerated molecular dynamics and on-the-fly parameterization to compute free energy versus end-to-end distance for two different linker lengths, DAVEI L0 (His6 ) and DAVEI L2 ([Gly4 Ser]2 His6 ). Additionally, an envelope model was created based on a cryo-electron microscopy-derived structure of a cleaved, soluble Env construct, with high-mannose glycans added which served as putative docking locations for the lectin, along with MPER added that served as a putative docking location for the MPER region of DAVEI (MPERDAVEI ). Using MD simulation, distances between the lectin C-terminus and Env gp41 MPER were measured. We determined that none of the glycans were close enough to gp41 MPER to allow DAVEI L0 to function, while one, N448, will allow DAVEI L2 to function. These findings are consistent with the previously determined dependence of lytic function on DAVEI linker lengths. This supports the hypothesis that DAVEI's engage Env at both glycans and the Env MPER in causing membrane poration and lysis.

Roles of conserved tryptophans in trimerization of HIV-1 membrane-proximal external regions: Implications for virucidal design via alchemical free-energy molecular simulations.

The Dual-Action Virolytic Entry Inhibitors, or "DAVEI's," are a class of recombinant fusions of a lectin, a linker polypeptide, and a 15-residue fragment from the membrane-proximal external region (MPER) of HIV-1 gp41. DAVEI's trigger rupture of HIV-1 virions, and the interaction site between DAVEI MPER and HIV-1 lies in the gp41 component of the envelope glycoprotein Env. Here, we explore the hypothesis that DAVEI MPER engages Env gp41 in a mode structurally similar to a crystallographic MPER trimer. We used alchemical free-energy perturbation to assess the thermodynamic roles of each of the four conserved tryptophan residues on each protomer of MPER3 . We found that a W666A mutation had a large positive ΔΔG for all three protomers, while W672A had a large positive ΔΔG for only two of the three protomers, with the other tryptophans remaining unimportant contributors to MPER3 stability. The protomer for which W672 is not important is unique in the placement of its W666 sidechain between the other two protomers. We show that the unique orientation of this W666 sidechain azimuthally rotates its protomer away from the orientation it would have if the trimer were symmetric, resulting in the diminished interaction of this W672 with the rest of MPER3 . Our findings are consistent with our previous experimental study of W-to-A mutants of DAVEI. This suggests that DAVEI MPER may engage HIV-1 Env to form a mixed trimer state in which one DAVEI MPER forms a trimer by displacing a more weakly interacting protomer of the endogenous Env MPER trimer.