Structural Explanations to Altered Drug Resistance Pathways in HIV-1 Non-Clade B Proteases

Abstract

The majority of human immunodeficiency virus-1 (HIV-1) infections across the world result from non-B clades. CRF01_AE is predominantly seen in Southeast Asia and the protease differs by ∼10% when compared to the clade B protease. Polymorphisms in CRF01_AE are often associated with drug resistance in the clade B protease. CRF01_AE protease has been observed to develop the unique N88S mutation in response to nelfinavir (NFV) therapy which is not commonly seen in clade B protease. We present here, structural and binding thermodynamic data on CRF01_AE protease in order to explain how sequence polymorphisms within CRF01_AE protease might affect its activity as well as to explain the altered NFV resistance pathway observed in CRF01_AE. The crystal structure of HIV-1 CRF01_AE N88S protease in complex with Darunavir (DRV) was determined to a resolution of 1.76 A and was compared with the clade B protease in complex with DRV. The CRF01_AE structure shows a significant change in the flap hinge region of the protease when compared to the clade B structure. The Ser88 side chain in the CRF01_AE structure is involved with a novel network of hydrogen bonds and interacts with the side chain of Asp30. This likely disrupts a critical hydrogen bond required for NFV binding. Binding constants and thermodynamic parameters for CRF01_AE and clade B protease were determined by isothermal titration calorimetry (ITC). Calorimetric data indicate that wild type CRF01_AE protease has ten fold and two fold weaker to NFV and DRV respectively when compared to clade B protease. This weakened affinity may permit the alternative pathway for NFV resistance via N88S, which is a mutation outside the active site.