Amide hydrogen exchange in HIV–1 subtype B and C proteases – insights into reduced drug susceptibility and dimer stability

Abstract

Since its identification, HIV has continued to have a detrimental impact on the lives of millions of people throughout the world. The protease of HIV is a major target in antiviral treatment. The South African HIV–1 subtype C (C–SA) protease displays weaker binding affinity for some clinically approved protease inhibitors in comparison with the HIV–1 subtype B protease. The heavy HIV burden in sub‐Saharan Africa, where subtype C HIV–1 predominates, makes this disparity a topic of great interest. In light of this, the enzyme activity and affinity of protease inhibitors for the subtype B and C–SA proteases were determined. The relative vitality, indicating the selective advantage of polymorphisms, of the C–SA protease relative to the subtype B protease in the presence of ritonavir and darunavir was four‐ and tenfold greater, respectively. Dynamic differences that contribute to the reduced drug susceptibility of the C–SA protease were investigated by performing hydrogen–deuterium exchange/mass spectrometry (HDX/MS) on unbound subtype B and C–SA proteases. The reduced propensity to form the E35–R57 salt bridge, and alterations in the hydrophobic core of the C–SA protease, are proposed to affect the anchoring of the flexible flaps, resulting in an increased proportion of the fully open flap conformation. HDX/MS data suggested that the N–terminus of both proteases is less stable than the C–terminus of the proteases, thus explaining the increased efficacy of dimerization inhibitors targeted toward the C–terminus of HIV proteases. As far as we are aware, this is the first report on assessment of HIV protease dynamics using HDX/MS.

Exploring the flap dynamics of the South African HIV subtype C protease in presence of FDA‐approved inhibitors: MD study

“…In the current analysis, ATV attained flap tip distance values of slightly less than 8 Å and DRV‐ and LPV‐complexes showed higher tendency to remain in semi‐open conformation (Supporting Information Figure S3) while TPV displayed even smaller flap tip distances. Interestingly, all these drug complexes rarely achieved full closed conformations (5–6 Å) which has been reported as an important characteristic of C‐SA protease by experimental evidence . The drug complexes displayed flap‐tip distances of 7–9 Å and did not achieve 5–6 Å attained by Apo as shown in Supporting Information Figure S3A.…”

“…Among different subtypes of HIV‐1, subtype C (C‐SA), is mostly prevalent in this region and this subtype accounts for approximately 50% of all HIV‐1 infections globally . C‐SA protease has eight point mutations compared to subtype B and these are T12S, I15V, L19I, M36I, R41K, H69K, L89M and I93L . In subtype B, the hinge and cantilever regions (loops) slide over I15 and this interaction with the hydrophobic core allows anchoring of the flaps .…”

“…This core has 19 amino acid residues with hydrophobic side chains (5L, 11V, 13I, 15I, 22A, 24L, 33L, 36M, 38L, 62I, 64I, 66I, 75V, 77V, 85I, 89L, 90L, 93I and 97L) found in each monomer of HIV‐1 protease. These amino acid residues exchange hydrophobic interactions which are essential to maintaining conformational flexibility of the proteases including flap dynamics . Furthermore, four of these residues are mutated in C‐SA protease and these are I15V, M36I and L89M and I93L.…”

“…Furthermore, four of these residues are mutated in C‐SA protease and these are I15V, M36I and L89M and I93L. In the C‐SA HIV‐1 protease, these polymorphisms lead to perturbed hydrophobic sliding that are likely to be one of the major reasons for drug resistance …”

“…This is as a result of the conformational change that is associated with inhibitor or substrate binding . Furthermore, experimental results revealed that the reduced drug susceptibility of C‐SA protease may be attributed to altered dynamics around the flaps (hydrophobic core) of the C‐SA protease as well as reduced probability of Glu35–Arg57 salt bridge formation. Both of these factors are assumed to have a strong correlation with the region that allows for flap opening and closure .…”