Ashraf Brik

HIV-1 protease: mechanism and drug discovery

It has now been two decades since acquired immunodeficiency syndrome (AIDS) was first reported by the US Center for Diseases Control (CDC). A few years later, it was found that a retrovirus called human immunodeficiency virus (HIV) is the causative agent in AIDS. In a short time, AIDS increased to epidemic proportions throughout the world, affecting more than 40 million people today and killing so far more than 22 million (UNAIDS, 2001). Since the outbreak of the AIDS epidemic, tremendous efforts have been directed towards development of antiretroviral therapies that target HIV type 1 in particular (HIV-1). The identification of the HIV retrovirus and the accumulated knowledge about the role of the different elements in its life cycle led researchers around the world to develop inhibitors that target different steps in the life cycle of the virus. One of these targets is HIV-1 protease (HIV PR), an essential enzyme needed in the proper assembly and maturation of infectious virions. Understanding the chemical mechanism of this enzyme has been a basic requirement in the development of efficient inhibitors. In this review, we summarize studies conducted in the last two decades on the mechanism of HIV PR and the impact of their conclusions on the drug discovery processes.

1,2,3-triazole as a peptide surrogate in the rapid synthesis of HIV-1 protease inhibitors.

Given the ubiquitous nature of the peptide linkage in biological molecules, replacement of the amide bond with isosteres in potential drug candidates has been a continual goal of many laboratories. Successful replacements will provide improved stability, lipophilicity, and absorption. Many surrogates have been introduced already, yet the synthesis of many of these isosteres in a combinatorial way is difficult and requires several steps. Thus, the discovery of new peptide surrogates with easier syntheses is an important achievement that could open new opportunities for the study of amide-containing molecules and the development of inhibitors with novel physicochemical properties. We have used the copper(i)-catalyzed azide–alkyne [3+2] cycloaddition as a straightforward reaction for the preparation of inhibitor libraries. Over 100 compounds were synthesized in microtiter plates and screened in situ. Two of these compounds—AB2 (pdb-1zp8) and AB3 (pdb-1zpA)—showed the best activity against wild type and mutant HIV-1 proteases (Table 1). AB2 and AB3, were then computationally docked by using AutoDock3. The docking simulation produced two conformations of approximately equal energy. One conformation placed the triazole in the position normally adopted by the peptide unit—between P2’ and P1’—in peptidomimetic compounds. Furthermore, the central nitrogen of the triazole was perfectly positioned to form a hydrogen bond with the water molecule normally found under the protease flaps. This water molecule also formed a hydrogen bond with the sulfonamide as seen in the crystallographic structure of amprenavir when bound to HIV-1 protease. The other conformation positioned the compounds in a similar place, but with the triazole rotated by 180 8. This allowed for a slightly better fit of the triazole substituent but sacrificed the hydrogen bond with the water molecule. In this work we have solved the ambiguity in binding conformation by solving the crystal structure of two inhibitors derived from a library of triazole compounds with HIV-1 protease. Interestingly, the two structures show that the triazole ring is an effective amide surrogate that retains all hydrogen bonds in the active site (Figure 1). HIV-1 protease (3 mgmL 1 in 0.025m sodium acetate pH 5.4, 10 mm dithiothreitol, 1 mm EDTA) was combined with inhibitor (32 mm in 50% (v/v) dimethylsulfoxide and 2-methylpentane2,4-diol) at 4 8C to give a 2:1 molar ratio of inhibitor to protein, and the mixture was centrifuged to remove the precipitate. The complex was crystallized by the hanging-drop vapor-diffusion method by mixing 9.6 mL of protease solution with 4 mL of crystallization buffer (1.34m ammonium sulfate, 0.1m sodium acetate, pH 4.8–5.4). Plates were sealed at 20 8C for one to two weeks. Data were collected from frozen crystals at the Argonne National Laboratory SER-CAT beamline 22-ID and with a Rigaku Table 1. Binding constants of 1,2,3-triazole compounds to HIV-1 protease.

Rapid Diversity-Oriented Synthesis in Microtiter Plates for In Situ Screening of HIV Protease Inhibitors

Since the early days of the discovery of HIV-1 protease (HIV-1 PR), this enzyme has been selected as an important target for the inhibition of viral replication. The enormous effort over the past two decades to develop effective molecules that inhibit the HIV1 PR has resulted in the discovery of drugs that have dramatically improved the quality of life and survival of the patients infected with HIV-1. To date there are six different HIV-1 PR inhibitors (PI) that are commercially available. These drugs are administered in combination with the reverse transcriptase inhibitors in what is called TMhighly active anti-retroviral therapy (HAART)∫. Unfortunately, many drug-resistant and cross-resistant mutant HIV-1 PRs have been identified, thus hampering long term suppression of the virus and resulting in return of AIDS symptoms. Therefore, the development of new protease inhibitors, which are efficacious against both the wild type and drug resistant HIV-1 PR and less prone to development of resistance, is urgently needed. During the last decade, the number and throughput of biological assays of protease activity has notably increased. However, the high rates of HIV-1 PR mutation still outpace conventional drug discovery efforts, mostly because of limitations associated with identification of the lead structures and, to a greater extent, slow structure ± activity profiling. While the former can be improved by rational design and computational studies, rapid synthesis of diverse analogues and their optimization still remains a challenge. We have recently developed a new strategy to facilitate the drug discovery process: diversityoriented organic synthesis in microtiter plates followed by in situ screening without product isolation and protecting group manipulation. This strategy was demonstrated with the use of amide-forming reaction in a rapid identification of new potent HIV protease inhibitors. Click chemistry has emerged as a strategy for the rapid and efficient assembly of molecules with diverse functionality on both laboratory and production scales. Enabled by a few nearly perfect reactions, it guarantees reliable synthesis of the desired products in high yield and purity. Modularity, selectivity, and wide scope make click chemistry ideal for achieving diversity in just a few steps and with no need for further purification. Advantages of click chemistry in biological studies have recently been demonstrated in several applications: construction of fluorescent oligonucleotides for DNA sequencing, in situ assembly of acetylcholinesterase inhibitors, chemically orthogonal high fidelity bioconjugation, and activity-based protein profiling in whole proteomes. In principle, this type of chemistry is well suited for microscale synthesis and for biological screening in situ. To demonstrate its feasibility we have used the copper(I)-catalysed triazole formation for the synthesis of sugar arrays in the above mentioned microtiter plate format, followed by in situ screening of glycosyltransferase inhibitors and enzyme glycosylation. Herein, we report an expedient approach to the discovery of novel HIV-1 PR inhibitors based on the latest advance in the copper(I)-catalyzed 1,2,3-triazole synthesis. 11] This highly reliable process, which proceeds well in aqueous solvents and tolerates virtually all functional groups without the need for protection, made it possible to quickly generate the desired libraries of potential inhibitors and to screen them directly in microtiter plates, without any purification, against HIV-1 PR and its mutants. The efficacy of hydroxyethylamine isosteres as transition-state mimics and as backbone replacements of amide bonds in the P1/P1 position of aspartyl protease inhibitors has been well documented, most notably in incorporation in the structures of three commercially available drugs, amprenavir, nelfinavir, and saquinavir. We, therefore, envisioned a library of compounds which retained this core, while diversifying the P2/P2 residues to generate new inhibitors. Starting from the optically active epoxy amine 1, two different azide cores were prepared as summarized in Scheme 1. Epoxy amine 1 in H2O/EtOH was

Microtiter plate based chemistry and in situ screening: a useful approach for rapid inhibitor discovery

The use of libraries extracted from nature or constructed by combinatorial chemistry, have been widely appreciated in the drug discovery area. In this perspective, we present our contribution to the field of enzyme inhibitor discovery using a useful approach that allows diversification of a common core in a microtiter plate followed by in situ screening. Our method relies on an organic reaction that is highly selective, high yielding, amenable to the microscale and preferably can be performed in water. The core can be a designed molecule based on the structural and mechanistic information of the target, a compound with a weak binding affinity, or a natural product. Several reactions were found useful for this approach and were applied to the rapid discovery of potent inhibitors of representative enzymes.

Tetrabutylammonium fluoride-assisted rapid N9-alkylation on purine ring : Application to combinatorial reactions in microtiter plates for the discovery of potent sulfotransferase inhibitors in situ

n Abstractn n Tremendous efforts have been invested in the synthesis of purine libraries due to their importance in targeting various enzymes involved in different diseases and cellular processes. The synthesis of N9-alkylated purine scaffolds relied mostly on Mitsunobu conditions with a variety of alcohols or strong basic conditions with different organic halides. A more reliable and efficient way for the synthesis of N9-alkylated purine scaffolds is reported. This method uses tetrabutylammonium fluoride (TBAF) to assist such chemistry. In many cases, the reactions were completed within 10min and gave the desired product in high yield and selectivity. Moreover, these mild reaction conditions permitted its use in combinatorial reactions in microtiter plates followed by in situ screening for the discovery of potent sulfotransferase inhibitors.n n

Rapid discovery of potent sulfotransferase inhibitors by diversity-oriented reaction in microplates followed by in situ screening.

Rapid diversity‐oriented microplate library synthesis and in situ screening with a high‐throughput fluorescence‐based assay were used to develop potent inhibitors of β‐arylsulfotransferase IV (β‐AST‐IV). This strategy leads to facile inhibitor synthesis and study as it allows protecting‐group manipulation and product isolation from other library components to be avoided. Through repeated library formation, three aspects of inhibitor makeup, the identities of the two binding groups and the length of the linker between them, were independently optimized. Several potent inhibitors were obtained, one of which was determined to have an inhibition constant Ki of 5 nM. This compound is the most potent β‐AST‐IV inhibitor developed to date, with a Ki value more than five orders of magnitude lower than the Michaelis constant Km for the substrate whose binding it inhibits.

Tetrabutylammonium Fluoride‐Mediated Rapid Alkylation Reaction in Microtiter Plates for the Discovery of Enzyme Inhibitors in Situ

We have recently developed a new strategy for the rapid identification and optimization of enzymes inhibitors in microtiter plates. This approach relies on the use of high-yield organic reactions that can be carried out in water or water-miscible nontoxic solvents on microscales without protecting groups, so that the product can be assayed directly in situ without isolation or purification. Using this approach, one can quickly modify a lead compound with a small set of building blocks to identify a potent inhibitor. For example, using amideand triazole-forming reactions, we have discovered potent inhibitors against HIV protease, SARS 3CL protease, a-fucosidase, sulfotransferase, and a-1,3-fucosyltransferase. In order to expand the scope of this approach, we report here the development of tetrabutylammonium fluoride (TBAF)-mediated alkylation in microtiter plates for the identification of potent inhibitors in situ against several enzymes including cathepsin B, arylsulfotransferase, and HIV protease.

Design and Synthesis of Broad-Based Mono- and Bi- Cyclic Inhibitors of FIV and HIV Proteases

Based on the substrate transition state and our strategy to tackle the problem of drug resistance, a series of HIV/FIV protease (HIV /FIV PR) monocyclic inhibitors incorporating a 15- or 17-membered macrocycle with an equivalent P3 or P3 group and a unique unnatural amino acid, (2R, 3S)-3-amino-2-hydroxy-4-phenylbutyric acid, have been designed and synthesized. In addition, based on the structure of TL3 with small P3/P3 group, we have synthesized two conformationally restricted bicyclic inhibitors containing the macrocycle, which mimic the P1/P1-P3/P3 tripeptide [Phe-Val-Ala] of TL3. We have found that the contribution of the macrocycle in our monocyclic inhibitors is important to the overall activity, but the ring size does not affect the activity to a significant extent. Several inhibitors that were developed in this work, exhibit low nanomolar inhibitory activity against the wild-type HIV/FIV PR and found to be highly effective against some drug-resistant as well as TL3-resistant mutants of HIV PRs. Compound 15, in particular, is the most effective cyclic inhibitor in hand to inhibit FIV replication in tissue culture at a concentration of 1.0 micro g/mL (1.2 microM).

Mutants of 4-oxalocrotonate tautomerase catalyze the decarboxylation of oxaloacetate through an imine mechanism.

A designed single amino acid substitution can alter the catalytic activity and mechanism of 4‐oxalocrotonate tautomerase (4‐OT). While the wild‐type enzyme catalyzes only the tautomerization of oxalocrotonate, the Pro1Ala mutant (P1A) catalyzes two reactions—the original tautomerization reaction and the decarboxylation of oxaloacetate. Although the N‐terminal amine group of P1A is involved in both reactions, our results support a nucleophilic mechanism for the decarboxylase activity, in contrast to the general acid/base mechanism that has been previously established for the tautomerase activity. These findings demonstrate that a single catalytic group in a 4‐OT mutant can catalyze two reactions by two different mechanisms.

Cover Picture: ChemBioChem 9/2002

The cover picture shows a schematic representation of the mechanistic and catalytic diversity exhibited by mutants of 4-oxalocrotonate tautomerase (4-OT). A designed single amino acid substitution can alter the catalytic activity and mechanism of this enzyme. While the wild-type 4-OT catalyzes only the tautomerization of oxalocrotonate through a general acid/base machanism, the Pro1Ala mutant catalyzes two reactions—the original tautomerization reaction through an acid/base mechanism and the decarboxylation of oxaloacetate by a nucleophilic mechanism. This bifunctional mutant suggests that a new synthetic family of nucleophilic catalysts could be generated on the basis of the 4-OT scaffold through selection methods and rational protein engineering. For more details, see the article by Brik et al. on p. 845u2009ff.