A class of potent nonpeptidic inhibitors of human immunodeficiency virus protease has been designed by using the three-dimensional structure of the enzyme as a guide. By employing iterative protein cocrystal structure analysis, design, and synthesis the binding affinity of the lead compound was incrementally improved by over four orders of magnitude. An inversion in inhibitor binding mode was observed crystallographically, providing information critical for subsequent design and highlighting the utility of structural feedback in inhibitor optimization. These inhibitors are selective for the viral protease enzyme, possess good antiviral activity, and are orally available in three species.The human immunodeficiency virus (HIV) protease (HIV Pr) is currently one of the more promising therapeutic targets for the treatment of AIDS because of the essential role this enzyme plays in virus maturation and the general lack of efficacy observed with other lines of therapy (1). A large number of crystal structures have been determined of this enzyme, providing a wealth of structural information about interactions between HIV Pr and inhibitors (2). The HIV Pr has a unusual homodimeric C-2 symmetric structure in the absence of any bound ligand, and the extended substrate binding site is bisected by the symmetry axis. Each monomer contributes one catalytic aspartic residue and one twofold related (3-hairpin loop or "flap." The flaps cover the active site and participate in the binding of substrates and inhibitors. In addition, a characteristic bound water molecule forms a hydrogen bonding network between the flaps and bound inhibitors.Guided by the successful development of potent renin inhibitors (3), the identification of very potent peptide-based inhibitors of HIV Pr has been relatively straightforward (4). Properties inherent to peptide-based compounds, such as poor oral availability and short half-life, have remained a problem with this class of compounds. It is with this understanding that we began a program aimed at identifying completely nonpeptidic HIV Pr inhibitors by using the three-dimensional structure of the enzyme as a guide. In this report we describe the protein structure-based design of a series of potent nonpeptide inhibitors of HIV Pr which have good oral availability in several animal species. MATERIALS AND METHODSCrystallization of HIV-1 Protease, Data Collection, and Structure Solution. The details of the cloning, expression, and purification of HIV-1 protease have been described previously (5). Crystals of HIV Pr-inhibitor complexes were grown by the The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.hanging drop vapor diffusion method from 1.2-1.6 M ammonium sulfate/5% (vol/vol) dimethyl sulfoxide/3% (vol/vol) isopropyl alcohol/0.050 M citrate-phosphate buffer at a pH of 5.8. The crystals belong to space group P212121 with cell dimensions ...

To develop novel lipophilic thymidylate synthase (TS) inhibitors, the X-ray structure of Escherichia coli TS in ternary complex with FdUMP and the inhibitor 10-propargyl-5,8-dideazafolic acid (CB3717) was used as a basis for structure-based design. A total of 31 novel lipophilic TS inhibitors, lacking a glutamate residue, were synthesized; 26 of them had in common a N-((3,4-dihydro-2-methyl-6-quinazolinyl)methyl)-N-prop-2-ynylaniline+ ++ structure in which the aniline was appropriately substituted with simple lipophilic substituents either in position 3 or 4, or in both. Compounds were tested for their inhibition of E. coli TS and human TS and also for their inhibition of the growth in tissue culture of a murine leukemia, a human leukemia, and a thymidine kinase-deficient human adenocarcinoma. The crystal structures of five inhibitors complexed with E. coli TS were determined. Five main conclusions are drawn from this study. (i) A 3-substituent such as CF(3), iodo, or ethynyl enhances binding by up to 1 order of magnitude and in the case of CF(3) was proven to fill a nearby pocket in the enzyme. (ii) A simple strongly electron-withdrawing substituent such as NO(2) or CF(3)SO(2) in the 4-position enhances binding by 2 orders of magnitude; it is hypothesized that the transannular dipole so induced interacts favorably with the protein. (iii) Attempts to combine the enhancements of i and ii in the same molecule were generally unsuccessful (iv) A 4-C(6)H(5)SO(2) substituent provided both electron withdrawal and a van der Waal's interaction of the phenyl group with a hydrophobic surface at the mouth of the active site. The inhibition (K(is) = 12 nM) of human TS by this compound, 7n, showed that C(6)H(5)SO(2) provided virtually as much binding affinity as the CO-glutamate which it had replaced. (v) The series of compounds were poorly water soluble, and also the potent TS inhibition shown by several of them did not translate into good cytotoxicity. Compounds with large cyclic groups linked to position 4 by an SO or SO(2) group did, however, have IC(50)'s in the range 1-5 microM. Of these, 4-(N-((3,4-dihydro-2-methyl-6-quinazolinyl)methyl)-N-prop-2-ynylamino )phenyl phenyl sulfone, 7n, had IC(50)'s of about 1 microM and was chosen for further elaboration.

A series of potent nonpeptide inhibitors of the HIV protease have been identified. Using the structure of compound 3 bound to the HIV protease, bis tertiary amide inhibitor 9 was designed and prepared. Compound 9 was found to be about 17 times more potent than 3, and the structure of the protein-ligand complex of 9 revealed the inhibitor binds in an inverted binding mode relative to 3. Examination of the protein-ligand complex of 9 suggested several modifications in the P1 and P1' pockets. Through these modifications it was possible to improve the activity of the inhibitors another 100-fold, highlighting the utility of crystallographic feedback in inhibitor design. These compounds were found to have good antiviral activity in cell culture, were selective for the HIV protease, and were orally available in three animal models.

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