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. 2010 Aug 1;429(3):527-32.
doi: 10.1042/BJ20091645.

Identification of broad-based HIV-1 protease inhibitors from combinatorial libraries

Max W Chang  1 , Michael J Giffin, Rolf Muller, Jeremiah Savage, Ying C Lin, Sukwon Hong, Wei Jin, Landon R Whitby, John H Elder, Dale L Boger, Bruce E Torbett
Affiliations

Identification of broad-based HIV-1 protease inhibitors from combinatorial libraries

Max W Chang et al. Biochem J. .

Abstract

Clinically approved inhibitors of the HIV-1 protease function via a competitive mechanism. A particular vulnerability of competitive inhibitors is their sensitivity to increases in substrate concentration, as may occur during virion assembly, budding and processing into a mature infectious viral particle. Advances in chemical synthesis have led to the development of new high-diversity chemical libraries using rapid in-solution syntheses. These libraries have been shown previously to be effective at disrupting protein-protein and protein-nucleic acid interfaces. We have screened 44000 compounds from such a library to identify inhibitors of the HIV-1 protease. One compound was identified that inhibits wild-type protease, as well as a drug-resistant protease with six mutations. Moreover, analysis of this compound suggests an allosteric non-competitive mechanism of inhibition and may represent a starting point for an additional strategy for anti-retroviral therapy.

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Figures

Figure 1
Figure 1. Representative group of chemical substituents that are used in the reaction in the context of the compound scaffold
Each substituent is found at circle site labeled A, in this case generating 10 different compounds. For additional information see Materials and Methods and [18, 19].
Figure 2
Figure 2. Compounds used in this study, (A) compound 1 and (B) compound 2
Compound 1 was identified through protease – substrate screens of the original chemical library, see text, whereas compound 2 is a derivative of compound 1 with the Boc and methyl groups removed from the ends of the compound.
Figure 3
Figure 3. IC50 titration of compound 1 against wild type and the 6X multi-drug resistant proteases
Foreground, evaluation of compound 1, log [I], against wild type (●) and 6X multi-drug resistant (▼) proteases. Inset: For comparison, titration of TL-3, log[TL-3], a protease inhibitor which is effective against the wild type (●) protease, but not the multi-drug resistant 6X protease mutant [14] (▼), is shown. Results from nonlinear regression indicate that the IC50s are within a factor of 2 of each other for wild type and multi-drug resistant 6X protease mutant. IC50 curve fitting was performed as described in the Materials and Methods. ± values indicate the standard error.
Figure 4
Figure 4. Michaelis-Menten kinetics of compound 1 and 2 against wild type protease
(A) Compound 1 was used at 0 (●), 3 (■), 10 (▲), and 30 (▼) µM and (B) compound 2 was used as 0 (■), 10 (▲), 15 (▼), and 20 (●) µM over a range of µM substrate concentrations [S] for determination of the Michaelis-Menten kinetics. This is a representative result from 1 of 3 experiments. (C) Nonlinear regression of Vmax as a function of compound 1 (■) or 2 (▲) concentration. Shown is a representative experiment and standard errors for individual points varied less then 10% of the mean and curve fitting is described in the Materials and Methods. ± values indicate the standard error.
Figure 5
Figure 5. Yonetani and Theorell plot of v/vi versus concentration of Pepstatin A and compound 1
0 µM of compound 1 (▼); 20 µM of compound 1 (▲); 30 µM of compound 1 (■); 45 µM of compound 1 (●) with varying concentrations of Pepstatin A. Each point was in triplicate and this is a representative result from 1 of 4 experiments. Assay conditions and curve fitting described in Materials and Methods.
Figure 6
Figure 6. IC50 titrations of compound 1 against wild type and tethered dimer proteases
Compound 1 [I] demonstrates similar inhibitory efficacy against wild type (●) and the tethered protease dimer (■). Standard errors for individual points varied less then 10% of the mean and shown is a representative experiment. Assay conditions described in the Materials and Methods
Figure 7
Figure 7. Docked conformation showing compound 1 bound outside of the HIV-1 protease (2HS1) active site
A space-filling rendering of the exo site showing the location of the solvent exposed cleft and binding of compound 1. The exo site is a feature of the protease altered by movement of the flaps. Insert of protease shows the area that is magnified. The predicted binding energy of this conformation was −7.2 kcal/mol, equivalent to a Ki of 5.2 µM.
See this image and copyright information in PMC

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