Ying-Chuan Lin

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.

A copper(I)-catalyzed 1,2,3-triazole azide-alkyne click compound, is a potent inhibitor of a multidrug-resistant HIV-1 protease variant

Treatment with HIV-1 protease inhibitors, a component of highly active antiretroviral therapy (HAART), often results in viral resistance. Structural and biochemical characterization of a 6X protease mutant arising from in vitro selection with compound 1, a C 2-symmetric diol protease inhibitor, has been previously described. We now show that compound 2, a copper(I)-catalyzed 1,2,3-triazole derived compound previously shown to be potently effective against wild-type protease (IC 50 = 6.0 nM), has low nM activity (IC 50 = 15.7 nM) against the multidrug-resistant 6X protease mutant. Compound 2 displays similar efficacy against wild-type and 6X HIV-1 in viral replication assays. While structural studies of compound 1 bound to wild type and mutant proteases revealed a progressive change in binding mode in the mutants, the 1.3 A resolution 6X protease-compound 2 crystal structure reveals nearly identical interactions for 2 as in the wild-type protease complex with very little change in compound 2 or protease conformation.

Rapid Discovery and Structure−Activity Profiling of Novel Inhibitors of Human Immunodeficiency Virus Type 1 Protease Enabled by the Copper(I)-Catalyzed Synthesis of 1,2,3-Triazoles and Their Further Functionalization

Building from the results of a computational screen of a range of triazole-containing compounds for binding efficiency to human immunodeficiency virus type 1 protease (HIV-1-Pr), a novel series of potent inhibitors has been developed. The copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), which provides ready access to 1,4-disubstituted-1,2,3-triazoles, was used to unite a focused library of azide-containing fragments with a diverse array of functionalized alkyne-containing building blocks. In combination with direct screening of the crude reaction products, this method led to the rapid identification of a lead structure and readily enabled optimization of both azide and alkyne fragments. Replacement of the triazole with a range of alternative linkers led to greatly reduced protease inhibition; however, further functionalization of the triazoles at the 5-position gave a series of compounds with increased activity, exhibiting Ki values as low as 8 nM.

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

Feline immunodeficiency virus (FIV) as a model for study of lentivirus infections: parallels with HIV.

FIV is a significant pathogen in the cat and is, in addition, the smallest available natural model for the study of lentivirus infections. Although divergent at the amino acid level, the cat lentivirus has an abundance of structural and pathophysiological commonalities with HIV and thus serves well as a model for development of intervention strategies relevant to infection in both cats and man. The following review highlights both the strengths and shortcomings of the FIV/cat model, particular as regards development of antiviral drugs.

Molecular mechanisms of FIV infection.

Feline immunodeficiency virus (FIV) is an important viral pathogen worldwide in the domestic cat, which is the smallest animal model for the study of natural lentivirus infection. Thus, understanding the molecular mechanisms by which FIV carries out its life cycle and causes an acquired immune deficiency syndrome (AIDS) in the cat is of high priority. FIV has an overall genome size similar to HIV, the causative agent of AIDS in man, and shares with the human virus genomic features that may serve as common targets for development of broad-based intervention strategies. Specific targets include enzymes encoded by the two lentiviruses, such as protease (PR), reverse transcriptase (RT), RNAse H, and integrase (IN). In addition, both FIV and HIV encode Vif and Rev elements essential for virus replication and also share the use of the chemokine receptor CXCR4 for entry into the host cell. The following review is a brief overview of the current state of characterization of the feline/FIV model and development of its use for generation and testing of anti-viral agents.

Alteration of Substrate and Inhibitor Specificity of Feline Immunodeficiency Virus Protease

ABSTRACT Feline immunodeficiency virus (FIV) protease is structurally very similar to human immunodeficiency virus (HIV) protease but exhibits distinct substrate and inhibitor specificities. We performed mutagenesis of subsite residues of FIV protease in order to define interactions that dictate this specificity. The I37V, N55M, M56I, V59I, and Q99V mutants yielded full activity. The I37V, N55M, V59I, and Q99V mutants showed a significant increase in activity against the HIV-1 reverse transcriptase/integrase and P2/nucleocapsid junction peptides compared with wild-type (wt) FIV protease. The I37V, V59I, and Q99V mutants also showed an increase in activity against two rapidly cleaved peptides selected by cleavage of a phage display library with HIV-1 protease. Mutations at Q54K, I98P, and L101I dramatically reduced activity. Mutants containing a I35D or I57G substitution showed no activity against either FIV or HIV substrates. FIV proteases all failed to cut HIV-1 matrix/capsid, P1/P6, P6/protease, and protease/reverse transcriptase junctions, indicating that none of the substitutions were sufficient to change the specificity completely. The I37V, N55M, M56I, V59I, and Q99V mutants, compared with wt FIV protease, all showed inhibitor specificity more similar to that of HIV-1 protease. The data also suggest that FIV protease prefers a hydrophobic P2/P2′ residue like Val over Asn or Glu, which are utilized by HIV-1 protease, and that S2/S2′ might play a critical role in distinguishing FIV and HIV-1 protease by specificity. The findings extend our observations regarding the interactions involved in substrate binding and aid in the development of broad-based inhibitors.

Viral Evolution in Response to the Broad-Based Retroviral Protease Inhibitor TL-3

ABSTRACT TL-3 is a protease inhibitor developed using the feline immunodeficiency virus protease as a model. It has been shown to efficiently inhibit replication of human, simian, and feline immunodeficiency viruses and therefore has broad-based activity. We now demonstrate that TL-3 efficiently inhibits the replication of 6 of 12 isolates with confirmed resistance mutations to known protease inhibitors. To dissect the spectrum of molecular changes in protease and viral properties associated with resistance to TL-3, a panel of chronological in vitro escape variants was generated. We have virologically and biochemically characterized mutants with one (V82A), three (M46I/F53L/V82A), or six (L24I/M46I/F53L/L63P/V77I/V82A) changes in the protease and structurally modeled the protease mutant containing six changes. Virus containing six changes was found to be 17-fold more resistant to TL-3 in cell culture than was wild-type virus but maintained similar in vitro replication kinetics compared to the wild-type virus. Analyses of enzyme activity of protease variants with one, three, and six changes indicated that these enzymes, compared to wild-type protease, retained 40, 47, and 61% activity, respectively. These results suggest that deficient protease enzymatic activity is sufficient for function, and the observed protease restoration might imply a selective advantage, at least in vitro, for increased protease activity.

Crystal structure of an FIV/HIV chimeric protease complexed with the broad-based inhibitor, TL-3.

We have obtained the 1.7 Å crystal structure of FIV protease (PR) in which 12 critical residues around the active site have been substituted with the structurally equivalent residues of HIV PR (12X FIV PR). The chimeric PR was crystallized in complex with the broad-based inhibitor TL-3, which inhibits wild type FIV and HIV PRs, as well as 12X FIV PR and several drug-resistant HIV mutants [1–4]. Biochemical analyses have demonstrated that TL-3 inhibits these PRs in the order HIV PR > 12X FIV PR > FIV PR, with Ki values of 1.5 nM, 10 nM, and 41 nM, respectively [2–4]. Comparison of the crystal structures of the TL-3 complexes of 12X FIV and wild-typeFIV PR revealed theformation of additinal van der Waals interactions between the enzyme inhibitor in the mutant PR. The 12X FIV PR retained the hydrogen bonding interactions between residues in the flap regions and active site involving the enzyme and the TL-3 inhibitor in comparison to both FIV PR and HIV PR. However, the flap regions of the 12X FIV PR more closely resemble those of HIV PR, having gained several stabilizing intra-flap interactions not present in wild type FIV PR. These findings offer a structural explanation for the observed inhibitor/substrate binding properties of the chimeric PR.

Molecular Basis for the Relative Substrate Specificity of Human Immunodeficiency Virus Type 1 and Feline Immunodeficiency Virus Proteases

ABSTRACT We have used a random hexamer phage library to delineate similarities and differences between the substrate specificities of human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus (FIV) proteases (PRs). Peptide sequences were identified that were specifically cleaved by each protease, as well as sequences cleaved equally well by both enzymes. Based on amino acid distinctions within the P3-P3′ region of substrates that appeared to correlate with these cleavage specificities, we prepared a series of synthetic peptides within the framework of a peptide sequence cleaved with essentially the same efficiency by both HIV-1 and FIV PRs, Ac-KSGVF↓VVNGLVK-NH2 (arrow denotes cleavage site). We used the resultant peptide set to assess the influence of specific amino acid substitutions on the cleavage characteristics of the two proteases. The findings show that when Asn is substituted for Val at the P2 position, HIV-1 PR cleaves the substrate at a much greater rate than does FIV PR. Likewise, Glu or Gln substituted for Val at the P2′ position also yields peptides specifically susceptible to HIV-1 PR. In contrast, when Ser is substituted for Val at P1′, FIV PR cleaves the substrate at a much higher rate than does HIV-1 PR. In addition, Asn or Gln at the P1 position, in combination with an appropriate P3 amino acid, Arg, also strongly favors cleavage by FIV PR over HIV PR. Structural analysis identified several protease residues likely to dictate the observed specificity differences. Interestingly, HIV PR Asp30 (Ile-35 in FIV PR), which influences specificity at the S2 and S2′ subsites, and HIV-1 PR Pro-81 and Val-82 (Ile-98 and Gln-99 in FIV PR), which influence specificity at the S1 and S1′ subsites, are residues which are often involved in development of drug resistance in HIV-1 protease. The peptide substrate KSGVF↓VVNGK, cleaved by both PRs, was used as a template for the design of a reduced amide inhibitor, Ac-GSGVFΨ(CH2NH)VVNGL-NH2. This compound inhibited both FIV and HIV-1 PRs with approximately equal efficiency. These findings establish a molecular basis for distinctions in substrate specificity between human and feline lentivirus PRs and offer a framework for development of efficient broad-based inhibitors.