Human immunodeficiency virus type 1 proteinase resistance to symmetric cyclic urea inhibitor analogs

Abstract

Resistant virus was isolated from virus propagated in cell culture in the presence of the human immunodeficiency virus type 1 (HIV-1) proteinase inhibitor DMP 323, Ro 31-8959, or A-75925. The proteinase gene of resistant virus was sequenced, and key mutations (G48V, V82A, I84V, L90M, and G48V/L90M) were introduced into clones used for the expression, purification, and further characterization of the enzyme. The mutant enzymes were all less active than the wild-type enzyme, as judged by k(cat) and k(cat)/Km values. L90M had a lower Km than the wild type, whereas the G48V/L90M double mutant had an increased Km compared with that of the wild type, contributing to a 10-fold reduction in the k(cat)/Km. Vitality values were used to show that the enzyme of the I84V mutant is the enzyme most resistant to the two cyclic urea inhibitors DMP 323 and AHA 008. Virus with the same mutation is also resistant, although the double mutation L10F/I84V confers even greater resistance. All of these mutants are more resistant to DMP 323 than to AHA 008. The resistance of the I84V mutant may be attributed to a loss of van der Waals interactions with the inhibitor, since the larger amino acid side chain involved in the interaction is replaced by a smaller side chain. This is supported by the lower level of resistance to AHA 008 that was observed. The phenyl groups of AHA 008 should protrude deeper into the S1 and S1' subsites than those of the smaller compound DMP 323, reducing the loss of interaction energy. These results reveal that small structural modifications of inhibitors that do not affect the inhibitory effect on wild-type virus can influence the inhibition of resistant strains. This is of importance for optimizing drugs with respect to their potency and resistance.

Predicting Drug‐Resistant Mutations of HIV Protease

“…Most significantly, the vitality values obtained in the present study reproduce the experimentally obtained values (Figure 3 c) and the corresponding drug resistance 1. 9…”

“…The corresponding results are depicted in Figure 3. The computed Δ G bind (drug) values of DMP323 in the native and the V82F and I84V mutants of HIV proteases are in excellent agreement with the measured values9, 10 (Figure 3 b). The present LRA/α computational values of Δ G bind (drug) are similar to the values reported in Ref.…”

“…However, in the absence of any observed drug‐resistant mutations we needed to validate our approach on the much more challenging cases of mutations of nonpolar residues. More specifically, since it has been observed that the resistance to DMP323 involves the V82F and I84V mutants,1, 9 we validated our approach by calculating the change in vitality associated with mutations at residues 82 and 84. The corresponding results are depicted in Figure 3.…”

Improved Structure−Activity Relationship Analysis of HIV-1 Protease Inhibitors Using Interaction Kinetic Data

“…The subgroup with all clinical inhibitors except amprenavir also contained the two most effective P2/P2′ analogues of B268 (B388 and B369) (group 1a). Note that B388 is the asymmetric combination of the 14,16 The Ki value of atazanavir was determined as described previously 18 and an estimated value for the Ki value of lopinavir was obtained by normalizing data from Sham et al 19 to this dataset using ritonavir as an internal reference. two symmetric compounds B268 and B369.…”

Symmetric fluoro‐substituted diol‐based HIV protease inhibitors

“…Enzyme activity/inhibition studies were performed as described by Nillroth et al . [34]. Briefly, a fluorimetric assay was used to determine the effects of the inhibitors on HIV‐1 protease.…”