Improved methods for 1H-3H heteronuclear shift correlation.

A better understanding of the structure of complex 3H-labeled molecules can be obtained by complete assignment of their 1H and 3H solution-state NMR spectra. The assignment process is aided by the detection of heteronuclear chemical shift correlations between 1H and 3H nuclei. Heteronuclear correlation (HETCOR) experiments previously applied to this task exhibit several drawbacks caused by the nature of both the pulse sequences and 1H-3H spin systems. The range of J-couplings involved in 1H-3H coupling networks make it challenging to perform correlation experiments using methods that rely on coherences created during free precession periods and interrupted by transfer pulses. Two alternative HETCOR experiments are demonstrated for 1H-3H systems in the present work and are shown to have advantages over earlier methods. The first experiment is known as hetero-TOCSY and correlates heteronuclear chemical shifts using J-cross polarization. This experiment achieves both homonuclear and heteronuclear mixing and connects the chemical shifts of all 1H and 3H nuclei in a coupling network. A second HETCOR experiment uses the heteronuclear Overhauser effect to obtain through-space correlations between nearby nuclei. The 1H-3H HETCOR experiments are phase sensitive and typically contain more correlations than other methods, which is beneficial for assignment purposes, while being sensitive enough to be applicable to routine analytical samples. The experiments were used to analyze 3H incorporation in sub-milligram quantities of 3H-labeled pharmaceutical derivatives with complex labeling schemes.

Inhibition of wild-type and mutant human immunodeficiency virus type 1 proteases by GW0385 and other arylsulfonamides.

The arylsulfonamide derivatives described herein were such potent inhibitors of human immunodeficiency virus type 1 (HIV-1) protease (enzyme, E) that values for the inhibition constants (K(i)) could not be determined by conventional steady-state kinetic techniques (i.e., the minimal enzyme concentration usable for the activity assay was much greater than the value of the dissociation constant). Consequently, two alternative methods were developed for estimation of K(i) values. The first method employed kinetic determinations of values for k(1) and k(-1), from which K(i) was determined (k(-1)/k(1)). The second method was a competitive displacement assay used to determine binding affinities of other inhibitors relative to that of GW0385. In these assays, the inhibitor of unknown affinity was used to displace [(3)H]GW0385 from E.[(3)H]GW0385. From the concentration of E.[(3)H]GW0385 at equilibrium, the concentrations of enzyme-bound and free inhibitors were calculated, and the ratio of the K(i) value of the unknown to that of GW0385 was determined (K(i,unknown)/K(i,GW0385)). The values of k(1) were calculated from data in which changes in the intrinsic protein fluorescence of the enzyme associated with inhibitor binding were directly or indirectly monitored. In the case of saquinavir, the fluorescence changes associated with complex formation were large enough to monitor directly. The value of k(1) for saquinavir was 62 +/- 2 microM(-1) s(-1). In the case of GW0385, the fluorescence changes associated with complex formation were too small to monitor directly. Consequently, the value of k(1) was estimated from a competition experiment in which the effect of GW0385 on the binding of E to saquinavir was determined. The value of k(1) for GW0385 was estimated from these experiments to be 137 +/- 4 microM(-1) s(-1). Because E.[(3)H]GW0385 was stable in the standard buffer at room temperature for greater than 33 days, the value of the first-order rate constant for dissociation of E.[(3)H]GW0385 (k(-1)) could be estimated from the time-course for exchange of E.[(3)H]GW0385 with excess unlabeled GW0385. The value of k(-1) calculated from these data was (2.1 +/- 0.1) x10(-6) s(-1) (t(1/2) = 91 h). The K(i) value of wild-type HIV-1 protease for GW0385, calculated from these values for k(1) and k(-1), was 15 +/- 1 fM. Three multidrug resistant enzymes had K(i) values for GW0385 that were less than 5 pM.