GSK3732394 is a multi-specific biologic inhibitor of HIV entry currently under clinical evaluation. A key component of this molecule is an Adnectin that binds to CD4 and inhibits downstream actions of gp160. Studies were performed to help elucidate the binding site of the Adnectin on CD4 and understand the mechanism of inhibition.
Hydrogen-deuterium exchange mass spectrometry (HDX) was used to examine comparative deuteration rates of amide backbone protons of CD4, either in the absence or presence of saturating amounts of Adnectin. In addition, crystal structures of CD4 bound to both the Adnectin and a Fab subunit of ibalizumab were solved at a 3.7Å resolution. Cryo-EM studies of Adnectin bound to soluble CD4 were also generated. Finally, mutagenic analyses on CD4 were performed to confirm and extend these findings.
Using HDX, CD4 peptides at the N-terminus of D2 and in D3 showed differential rates of deuteration (both enhanced and slowed) in the presence of the Adnectin that mapped predominantly to the D2-D3 interface. The structure of the ibalizumab Fab/CD4 D1-D4/Adnectin complex revealed an extensive interface between the Adnectin and residues on CD4 domains D2-D4 that stabilize a novel T-shaped CD4 conformation. A cryo-EM map of the gp140/CD4/combinectin complex clearly shows the bent conformation for CD4 while bound to gp140. Mutagenic analyses on CD4 confirmed that amino acid F202 forms a key interaction with the Adnectin. In addition, amino acid L151 was shown to be a critical determinant of the specificity for binding to human CD4 protein over related primate CD4 molecules. Mutation of L151 to R (the residue present in cynomolgus monkey CD4) abrogated Adnectin binding to human CD4, while the reverse mutation (R151L) restored binding to cynomolgus monkey CD4.
The significant conformational change of CD4 upon Adnectin binding brings the D1 domain of CD4 in proximity to the host cell membrane surface and provides a potential explanation for the ability of the CD4-bound Adnectin to inhibit HIV-1 infection. In addition, mutations of D2-D3-interface residues, specifically F202 and L151, dramatically impacted Adnectin binding to human and primate CD4, providing a rationale for the observed species specificity of the Adnectin.