3rd Conference on Retroviruses and Opportunistic Infections Washington, DC - January 28-February 1, 1996

Conf Retroviruses Opportunistic Infect 1996 Jan 28-Feb 1; 3rd:80 (abstract no. 149)

Tummino PJ, Nouhan CJ, Graham M, Hagen SE, Tait BD, VaraPrasad JV, Lunney E, Hupe
Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, Ann Arbor, MI.

Nonpeptidic pyranones and 5,6 dihydropyranones have been optimized from low micromolar into low nanomolar HIV-1 protease inhibitors using structure-based design methods. The inhibitory activity against purified enzyme was initially determined at pH 4.7, close to the pH optimum of the viral protease. It was later found that some pyranones are less potent at a higher pH of 6.2, whole some of the dihydropyranones are relatively pH-insensitive. This pH-dependence may have relevance to cellular anti-HIV activity. Both a pyranone and dihydropyranone are highly potent at pH 4(K_i=3nM), but the pH-sensitive pyranone is inactive against HIV while the pH-insensitive dihydropyranone demonstrates micromolar cellular anti-HIV activity. X-ray crystallographic structural data indicated the 4-hydroxyl group common to all the inhibitors (found to have a pKa of 4-5) is within hydrogen bonding distance of the two catalytic Asps of the protease. Thus, it is possible that the loss of inhibitory activity at higher pH is due to deprotonation of the 4-hydroxyl group and loss of an important enzyme-inhibitor interaction. In order to more fully elucidate the pH dependence of inhibition by these compounds, we determined the Ki values as a function of pH from 3.2 to 6.7 for a 4-OH-pyranone, a 4-OH dihydropyranone, and a 4-NH₂-dihydropyranone. The data was fit to an evaluation that accounted for theK_i change as a protonation/deprotonation event. For the pryanone, the K_i change has a pKa =4.5, and an inactive deprotonated form. The dihydropyranone was found to have a pKa=5.6, higher than that of the structurally similar pyranone. Despite not possessing a group with a pKa within the experimental range, the 4-NH₂-dihydropyranone inhibitory activity decreased at higher pH, with a pKa =5.4. The results are discussed in terms of a protonation/deprotonation model for the enzyme and inhibitor, and in relation to structural data.

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