Jerry Alexandratos

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.

The catalytic domain of avian sarcoma virus integrase: conformation of the active-site residues in the presence of divalent cations

BACKGROUND Members of the structurally-related superfamily of enzymes that includes RNase H, RuvC resolvase, MuA transposase, and retroviral integrase require divalent cations for enzymatic activity. So far, cation positions are reported in the X-ray crystal structures of only two of these proteins, E. coli and human immunodeficiency virus 1 (HIV-1) RNase H. Details of the placement of metal ions in the active site of retroviral integrases are necessary for the understanding of the catalytic mechanism of these enzymes. RESULTS The structure of the enzymatically active catalytic domain (residues 52-207) of avian sarcoma virus integrase (ASV IN) has been solved in the presence of divalent cations (Mn2+ or Mg2+), at 1.7-2.2 A resolution. A single ion of either type interacts with the carboxylate groups of the active site aspartates and uses four water molecules to complete its octahedral coordination. The placement of the aspartate side chains and metal ions is very similar to that observed in the RNase H members of this superfamily; however, the conformation of the catalytic aspartates in the active site of ASV IN differs significantly from that reported for the analogous residues in HIV-1 IN. CONCLUSIONS Binding of the required metal ions does not lead to significant structural modifications in the active site of the catalytic domain of ASV IN. This indicates that at least one metal-binding site is preformed in the structure, and suggests that the observed constellation of the acidic residues represents a catalytically competent active site. Only a single divalent cation was observed even at extremely high concentrations of the metals. We conclude that either only one metal ion is needed for catalysis, or that a second metal-binding site can only exist in the presence of substrate and/or other domains of the protein. The unexpected differences between the active sites of ASV IN and HIV-1 IN remain unexplained; they may reflect the effects of crystal contacts on the active site of HIV-1 IN, or a tendency for structural polymorphism.

Binding of different divalent cations to the active site of avian sarcoma virus integrase and their effects on enzymatic activity.

Retroviral integrases (INs) contain two known metal binding domains. The N-terminal domain includes a zinc finger motif and has been shown to bind Zn2+, whereas the central catalytic core domain includes a triad of acidic amino acids that bind Mn2+ or Mg2+, the metal cofactors required for enzymatic activity. The integration reaction occurs in two distinct steps; the first is a specific endonucleolytic cleavage step called “processing,” and the second is a polynucleotide transfer or “joining” step. Our previous results showed that the metal preference for in vitro activity of avian sarcoma virus IN is Mn2+ > Mg2+ and that a single cation of either metal is coordinated by two of the three critical active site residues (Asp-64 and Asp-121) in crystals of the isolated catalytic domain. Here, we report that Ca2+, Zn2+, and Cd2+ can also bind in the active site of the catalytic domain. Furthermore, two zinc and cadmium cations are bound at the active site, with all three residues of the active site triad (Asp-64, Asp-121, and Glu-157) contributing to their coordination. These results are consistent with a two-metal mechanism for catalysis by retroviral integrases. We also show that Zn2+ can serve as a cofactor for the endonucleolytic reactions catalyzed by either the full-length protein, a derivative lacking the N-terminal domain, or the isolated catalytic domain of avian sarcoma virus IN. However, polynucleotidyl transferase activities are severely impaired or undetectable in the presence of Zn2+. Thus, although the processing and joining steps of integrase employ a similar mechanism and the same active site triad, they can be clearly distinguished by their metal preferences.

The catalytic domain of human immunodeficiency virus integrase: ordered active site in the F185H mutant

We solved the structure and traced the complete active site of the catalytic domain of the human immunodeficiency virus type 1 integrase (HIV‐1 IN) with the F185H mutation. The only previously available crystal structure, the F185K mutant of this domain, lacks one of the catalytically important residues, E152, located in a stretch of 12 disordered residues [Dyda et al. (1994) Science 266, 1981–1986]. It is clear, however, that the active site of HIV‐1 IN observed in either structure cannot correspond to that of the functional enzyme, since the cluster of three conserved carboxylic acids does not create a proper metal‐binding site. The conformation of the loop was compared with two different conformations found in the catalytic domain of the related avian sarcoma virus integrase [Bujacz et al. (1995) J. Mol. Biol. 253, 333‐246]. Flexibility of the active site region of integrases may be required in order for the enzyme to assume a functional conformation in the presence of substrate and/or cofactors.

Crystal Structure of a Dimerized Cockroach Allergen Bla g 2 Complexed with a Monoclonal Antibody

The crystal structure of a 1:1 complex between the German cockroach allergen Bla g 2 and the Fab′ fragment of a monoclonal antibody 7C11 was solved at 2.8-Å resolution. Bla g 2 binds to the antibody through four loops that include residues 60-70, 83-86, 98-100, and 129-132. Cation-π interactions exist between Lys-65, Arg-83, and Lys-132 in Bla g 2 and several tyrosines in 7C11. In the complex with Fab′, Bla g 2 forms a dimer, which is stabilized by a quasi-four-helix bundle comprised of an α-helix and a helical turn from each allergen monomer, exhibiting a novel dimerization mode for an aspartic protease. A disulfide bridge between C51a and C113, unique to the aspartic protease family, connects the two helical elements within each Bla g 2 monomer, thus facilitating formation of the bundle. Mutation of these cysteines, as well as the residues Asn-52, Gln-110, and Ile-114, involved in hydrophobic interactions within the bundle, resulted in a protein that did not dimerize. The mutant proteins induced less β-hexosaminidase release from mast cells than the wild-type Bla g 2, suggesting a functional role of dimerization in allergenicity. Because 7C11 shares a binding epitope with IgE, the information gained by analysis of the crystal structure of its complex provided guidance for site-directed mutagenesis of the allergen epitope. We have now identified key residues involved in IgE antibody binding; this information will be useful for the design of vaccines for immunotherapy.

Piecing together the structure of retroviral integrase, an important target in AIDS therapy.

Integrase (IN) is one of only three enzymes encoded in the genomes of all retroviruses, and is the one least characterized in structural terms. IN catalyzes processing of the ends of a DNA copy of the retroviral genome and its concerted insertion into the chromosome of the host cell. The protein consists of three domains, the central catalytic core domain flanked by the N‐terminal and C‐terminal domains, the latter being involved in DNA binding. Although the Protein Data Bank contains a number of NMR structures of the N‐terminal and C‐terminal domains of HIV‐1 and HIV‐2, simian immunodeficiency virus and avian sarcoma virus IN, as well as X‐ray structures of the core domain of HIV‐1, avian sarcoma virus and foamy virus IN, plus several models of two‐domain constructs, no structure of the complete molecule of retroviral IN has been solved to date. Although no experimental structures of IN complexed with the DNA substrates are at hand, the catalytic mechanism of IN is well understood by analogy with other nucleotidyl transferases, and a variety of models of the oligomeric integration complexes have been proposed. In this review, we present the current state of knowledge resulting from structural studies of IN from several retroviruses. We also attempt to reconcile the differences between the reported structures, and discuss the relationship between the structure and function of this enzyme, which is an important, although so far rather poorly exploited, target for designing drugs against HIV‐1 infection.

Atomic resolution structures of the core domain of avian sarcoma virus integrase and its D64N mutant.

Six crystal structures of the core domain of integrase (IN) from avian sarcoma virus (ASV) and its active-site derivative containing an Asp64 --> Asn substitution have been solved at atomic resolution ranging 1.02-1.42 A. The high-quality data provide new structural information about the active site of the enzyme and clarify previous inconsistencies in the description of this fragment. The very high resolution of the data and excellent quality of the refined models explain the dynamic properties of IN and the multiple conformations of its disordered residues. They also allow an accurate description of the solvent structure and help to locate other molecules bound to the enzyme. A detailed analysis of the flexible active-site region, in particular the loop formed by residues 144-154, suggests conformational changes which may be associated with substrate binding and enzymatic activity. The pH-dependent conformational changes of the active-site loop correlates with the pH vs activity profile observed for ASV IN.

Integrase of Mason-Pfizer monkey virus

The gene encoding an integrase of Mason–Pfizer monkey virus (M‐PMV) is located at the 3′‐end of the pol open reading frame. The M‐PMV integrase has not been previously isolated and characterized. We have now cloned, expressed, isolated, and characterized M‐PMV integrase and compared its activities and primary structure with those of HIV‐1 and other retroviral integrases. M‐PMV integrase prefers untranslated 3′‐region‐derived long‐terminal repeat sequences in both the 3′‐processing and the strand transfer activity assays. While the 3′‐processing reaction catalyzed by M‐PMV integrase was significantly increased in the presence of Mn2+ and Co2+ and was readily detectable in the presence of Mg2+ and Ni2+ cations, the strand transfer activity was strictly dependent only on Mn2+. M‐PMV integrase displays more relaxed substrate specificity than HIV‐1 integrase, catalyzing the cleavage and the strand transfer of M‐PMV and HIV‐1 long‐terminal repeat‐derived substrates with similar efficiency. The structure‐based sequence alignment of M‐PMV, HIV‐1, SIV, and ASV integrases predicted critical amino acids and motifs of M‐PMV integrase for metal binding, interaction with nucleic acids, dimerization, protein structure maintenance and function, as well as for binding of human immunodeficiency virus type 1 and Rous avian sarcoma virus integrase inhibitors 5‐CI‐TEP, DHPTPB and Y‐3.

Crystal structure of avian sarcoma virus integrase with bound essential cations

Publisher Summary Over the time, study has resulted in a general understanding of the enzymatic mechanism of retroviral integrase (IN), but analyses that are more detailed have been hampered by the lack of precise structural information. It is a virus-encoded enzyme that catalyzes nonspecific insertion of viral DNA into multiple sites on host DNA. As DNA integration is an essential step in the retroviral replication cycle, this enzyme is an attractive target for inhibition of human immunodeficiency virus (HIV). Research in this field took a subsequent leap when the crystal structures of the catalytic domains of both HIV-1 IN and avian sarcoma virus (ASV) IN became available. Precise data on the interaction of these enzymes and the essential ligands are necessary for understanding the structural basis of the reaction mechanism and for guiding rational drug design. Details of the location of metal ions, required for their enzymatic activity, in the active site of retroviral integrases can enhance the understanding of the catalytic mechanism of these enzymes and their relationship to that of other members of the superfamily. This chapter presents the structure of ASV IN catalytic domain with the essential cations Mg 2+ or Mn 2+ bound in the active site. In addition, the chapter presents the structure of an inactive complex of the catalytic domain of ASV IN with Zn 2+ . The structure of the catalytic domain of ASV IN complexed with three different divalent cations is presented. These results clearly show that the active site of this enzyme is preformed, in that only relatively small movements of side chains and no shifts of the main chain are needed to provide an environment suitable for cation binding. This is in contrast with the related core HIV-1 IN. However, antibody-binding experiments have shown that HIV-1 IN undergoes a conformational change when incubated with divalent cation cofactors.

Crystal structure of the catalytic domain of avian sarcoma virus integrase

Publisher Summary This chapter presents the structure of the catalytic domain of avian sarcoma virus (ASV) integrase (IN). Retroviruses contain an RNA genome that, upon infection of the host cell, is first reverse transcribed into DNA and then integrated into the host genome. IN and reverse transcriptase (RT) are encoded in the retroviral pol gene. The current model for IN function states that (1) IN acts as a multimer synchronizing the insertion of the two viral ends at the host target DNA site, (2) IN recognizes specific sequence and structure characteristics in the viral DNA ends and nearly random target sequences in host DNA, and (3) IN contains a single active site that carries out both the processing and joining reactions. The organization of the active site observed in ASV IN is not an artifact as it was confirmed in several well-refined high resolution structures.