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Amide hydrogen exchange in HIV-1 subtype B and C proteases--insights into reduced drug susceptibility and dimer
Previn Naicker1, Stoyan Stoychev, Heini W Dirr
1Protein Structure-Function Research Unit, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, South Africa.
Insights
South African HIV-1 subtype C protease shows reduced susceptibility to inhibitors compared to subtype B. This is due to dynamic differences, including altered flap anchoring, affecting drug binding.
Area of Science:
- Biochemistry
- Virology
- Structural Biology
Background:
- Human Immunodeficiency Virus (HIV) protease is a key target for antiviral therapies.
- HIV-1 subtype C, prevalent in sub-Saharan Africa, exhibits reduced susceptibility to certain protease inhibitors compared to subtype B.
- Understanding these differences is critical for developing effective HIV treatments.
Purpose of the Study:
- To investigate the enzyme activity and binding affinity of HIV-1 subtype B and C-SA proteases with clinically approved protease inhibitors.
- To explore the dynamic differences between subtype B and C-SA proteases that contribute to reduced drug susceptibility.
- To provide insights into the structural basis for differential drug response.
Main Methods:
- Enzyme activity and protease inhibitor affinity assays were performed.
- Hydrogen-deuterium exchange mass spectrometry (HDX/MS) was used to analyze protease dynamics.
- Comparative analysis of unbound subtype B and C-SA proteases was conducted.
Main Results:
- The C-SA protease demonstrated significantly greater vitality (4-10 fold) in the presence of ritonavir and darunavir compared to subtype B.
- HDX/MS revealed reduced propensity to form the E35-R57 salt bridge and alterations in the hydrophobic core of C-SA protease.
- These structural changes likely affect flexible flap anchoring, leading to a higher proportion of the open flap conformation in C-SA protease.
- N-termini of both proteases were found to be less stable than C-termini, suggesting potential for C-terminus targeted dimerization inhibitors.
Conclusions:
- The study elucidates dynamic differences in HIV-1 subtype C protease contributing to reduced drug susceptibility.
- Altered salt bridge formation and hydrophobic core structure impact protease flap dynamics and inhibitor binding.
- Findings highlight the importance of considering protease dynamics in drug design for HIV-1 subtype C.
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.
