Robert Stroud
580 California St., Suite 400
San Francisco, CA, 94104
HIV integrase: from structure to drug design
2002, Acta Crystallographica Section A Foundations of Crystallography
https://doi.org/10.1107/S0108767302085604visibility
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Abstract
HIV-1 integrase catalyzes the insertion of viral DNA into the human chromosome, and as such, it is a target for the development of new anti-HIV drugs. Structural studies of HIV-1 integrase have been limited due to its insolubility. We have engineered a soluble, functional integrase by introducing five point mutations, and have solved the structure of the viral DNA binding core and C-terminal domains to 2.8 Å resolution. The Y-shaped, dimeric molecule reveals a putative DNA binding region consisting of residues contributed by both monomers of the dimer. This implies that a dimer is the minimal DNA binding unit. A kink at T210 occurs at a proteolytic cleavage site, suggesting a functional flexibility in the molecule that may be crucial for integration. The C-terminal domain is an SH3-like fold, and provides the majority of the crystal contacts, consistent with the role of the domain in oligomerization of integrase. Based on this structure, we are outlining new strategies to discover novel drug leads targeting integrase.
Related papers
Discovery of a small-molecule HIV-1 integrase inhibitor-binding site
Proceedings of the National Academy of Sciences, 2006
Herein, we report the identification of a unique HIV-1 integrase (IN) inhibitor-binding site using photoaffinity labeling and mass spectrometric analysis. We chemically incorporated a photo-activatable benzophenone moiety into a series of coumarin-containing IN inhibitors. A representative of this series was covalently photocrosslinked with the IN core domain and subjected to HPLC purification. Fractions were subsequently analyzed by using MALDI-MS and electrospray ionization (ESI)-MS to identify photo-crosslinked products. In this fashion, a single binding site for an inhibitor located within the tryptic peptide 128 AACWWAGIK 136 was identified. Site-directed mutagenesis followed by in vitro inhibition assays resulted in the identification of two specific amino acid residues, C130 and W132, in which substitutions resulted in a marked resistance to the IN inhibitors. Docking studies suggested a specific disruption in functional oligomeric IN complex formation. The combined approach of photo-affinity labeling͞MS analysis with site-directed mutagenesis͞molecular modeling is a powerful approach for elucidating inhibitor-binding sites of proteins at the atomic level. This approach is especially important for the study of proteins that are not amenable to traditional x-ray crystallography and NMR techniques. This type of structural information can help illuminate processes of inhibitor resistance and thereby facilitate the design of more potent second-generation inhibitors.
Structural basis for a new mechanism of inhibition of Human Immunodeficiency Virus Type 1 Integrase identified by fragment screening and structure based design
2011
Background: HIV-1 integrase is a clinically validated therapeutic target for the treatment of HIV-1 infection, with one approved therapeutic currently on the market. This enzyme represents an attractive target for the development of new inhibitors to HIV-1 that are effective against the current resistance mutations. Methods: A fragment-based screening method employing surface plasmon resonance and NMR was initially used to detect interactions between integrase and fragments. The binding sites of the fragments were elucidated by crystallography and the structural information used to design and synthesize improved ligands.
Fragment-Based Design of Ligands Targeting a Novel Site on the Integrase Enzyme of Human Immunodeficiency Virus 1
ChemMedChem, 2011
HIV-1 integrase inhibitors that compete with the target DNA substrate define a unique strand transfer conformation for integrase
Proceedings of the National Academy of Sciences, 2000
Diketo acids such as L-731,988 are potent inhibitors of HIV-1 integrase that inhibit integration and viral replication in cells. These compounds exhibit the unique ability to inhibit the strand transfer activity of integrase in the absence of an effect on 3 end processing. To understand the reasons for this distinct inhibitory profile, we developed a scintillation proximity assay that permits analysis of radiolabeled inhibitor binding and integrase function. High-affinity binding of L-731,988 is shown to require the assembly of a specific complex on the HIV-1 long terminal repeat. The interaction of L-731,988 with the complex and the efficacy of L-731,988 in strand transfer can be abrogated by the interaction with target substrates, suggesting competition between the inhibitor and the target DNA. The L-731,988 binding site and that of the target substrate are thus distinct from that of the donor substrate and are defined by a conformation of integrase that is only adopted after assembly with the viral end. These results elucidate the basis for diketo acid inhibition of strand transfer and have implications for integrase-directed HIV-1 drug discovery efforts.
Structural basis for a new mechanism of inhibition of HIV-1 integrase identified by fragment screening and structure-based design
Antiviral Chemistry and Chemotherapy, 2011
Background: HIV-1 integrase is a clinically validated therapeutic target for the treatment of HIV-1 infection, with one approved therapeutic currently on the market. This enzyme represents an attractive target for the development of new inhibitors to HIV-1 that are effective against the current resistance mutations. Methods: A fragment-based screening method employing surface plasmon resonance and NMR was initially used to detect interactions between integrase and fragments. The binding sites of the fragments were elucidated by crystallography and the structural information used to design and synthesize improved ligands. Results: The location of binding of fragments to the catalytic core of integrase was found to be in a previously undescribed binding site, adjacent to the mobile loop. Enzyme assays confirmed that formation of enzyme-fragment complexes inhibits the catalytic activity of integrase and the structural data was utilized to further develop these fragments into more potent novel enzyme inhibitors. Conclusions: We have defined a new site in integrase as a valid region for the structure-based design of allosteric integrase inhibitors. Using a structure-based design process we have improved the activity of the initial fragments 45-fold.
Inhibiting HIV-1 integrase by shifting its oligomerization equilibrium
Proceedings of the National Academy of Sciences, 2007
Proteins are involved in various equilibria that play a major role in their activity or regulation. The design of molecules that shift such equilibria is of great therapeutic potential. This fact was demonstrated in the cases of allosteric inhibitors, which shift the equilibrium between active and inactive (R and T) states, and chemical chaperones, which shift folding equilibrium of proteins. Here, we expand these concepts and propose the shifting of oligomerization equilibrium of proteins as a general methodology for drug design. We present a strategy for inhibiting proteins by ''shiftides'': ligands that specifically bind to an inactive oligomeric state of a disease-related protein and modulate its activity by shifting the oligomerization equilibrium of the protein toward it. We demonstrate the feasibility of our approach for the inhibition of the HIV-1 integrase (IN) protein by using peptides derived from its cellularbinding protein, LEDGF/p75, which specifically inhibit IN activity by a noncompetitive mechanism. The peptides inhibit the DNA-binding of IN by shifting the IN oligomerization equilibrium from the active dimer toward the inactive tetramer, which is unable to catalyze the first integration step of 3 end processing. The LEDGF/ p75-derived peptides inhibit the enzymatic activity of IN in vitro and consequently block HIV-1 replication in cells because of the lack of integration. These peptides are promising anti-HIV lead compounds that modulate oligomerization of IN via a previously uncharacterized mechanism, which bears advantages over the conventional interface dimerization inhibitors.
A Symmetric Region of the HIV-1 Integrase Dimerization Interface Is Essential for Viral Replication
PLoS ONE
HIV-1 integrase (IN) is an important target for contemporary antiretroviral drug design research. Historically, efforts at inactivating the enzyme have focused upon blocking its active site. However, it has become apparent that new classes of allosteric inhibitors will be necessary to advance the antiretroviral field in light of the emergence of viral strains resistant to contemporary clinically used IN drugs. In this study we have characterized the importance of a close network of IN residues, distant from the active site, as important for the obligatory multimerization of the enzyme and viral replication as a whole. Specifically, we have determined that the configuration of six residues within a highly symmetrical region at the IN dimerization interface, composed of a four-tiered aromatic interaction flanked by two salt bridges, significantly contributes to proper HIV-1 replication. Additionally, we have utilized a quantitative luminescence assay to examine IN oligomerization and ...
A targeted DNA substrate mechanism for the inhibition of HIV-1 integrase by inhibitors with antiretroviral activity
We recently reported that viral DNA could be the primary target of raltegravir (RAL), an efficient anti-HIV-1 drug, which acts by inhibiting integrase. To elucidate this mechanism, we conducted a comparative analysis of RAL and TB11, a diketoacid abandoned as an anti-HIV-1 drug for its weak efficiency and marked toxicity, and tested the effects of the catalytic cofactor Mg 2+ (5 mM) on drug-binding properties. We used circular dichroism and fluorescence to determine drug affinities for viral DNA long terminal repeats (LTRs) and peptides derived from the integrase active site and DNA retardation assays to assess drug intercalation into DNA base pairs. We found that RAL bound more tightly to LTR ends than did TB11 (a diketo acid bearing an azido group) and that Mg 2+ significantly increased the affinity of both RAL and TB11. We also observed a good relationship between drug binding with processed LTR and strand transfer inhibition. This unusual type of inhibition was caused by Mg 2+ -assisted binding of drugs to DNA substrate, rather than to enzyme. Notably, while RAL bound exclusively to the cleavable/cleaved site, TB11 further intercalated into DNA base pairs and interacted with the integrase-derived peptides. These unwanted binding sites explain the weaker bioavailability and higher toxicity of TB11 compared with the more effective RAL.
Acta Cryst. (2002). A58 (Supplement), C13 Acta Cryst. (2002). A58 (Supplement), C13
HIV INTEGRASE: FROM STRUCTURE TO DRUG DESIGN ENZYMES: EVOLUTION OF FUNCTION FROM A STRUCTURAL
J.C.H. Chen1 L.J.W. Micercke1 J. Krucinski1 A.H. Tang2 J.S. Finer-Moore1 PERSPECTIVE
A.D. Leavitt2 R.M. Stroud1 J. Thornton1 R.A. Laskowski1 G. Bartlett1 C. Porter1 A. Todd2 C,
1 2
UCSF Department of Biochemistry And Biophysics Box 0448 513 Parnassus Orengo
Avenue San Francisco CA 94143 USA 2Department of Laboratory Medicine 1
European Bioinformatics Institute The Wellcome Trust Genome Campus
Hinxton Cambridge CB10 1SD UNITED KINGDOM 2University College,
HIV-1 integrase catalyzes the insertion of viral DNA into the human London
chromosome, and as such, it is a target for the development of new anti-HIV
drugs. Structural studies of HIV-1 integrase have been limited due to its As more gene sequences are determined, the demand for better methods to
insolubility. We have engineered a soluble, functional integrase by introducing predict protein function from sequence grows. Currently the only reliable
five point mutations, and have solved the structure of the viral DNA binding approach is to predict function by recognising sequence and/or structural
core and C-terminal domains to 2.8 Å resolution. The Y-shaped, dimeric homology to a related protein, whose function is known. An overview will
molecule reveals a putative DNA binding region consisting of residues be presented of our current knowledge of how sequences, structures and
contributed by both monomers of the dimer. This implies that a dimer is the functions evolve. In particular, we will consider the evolution of enzyme
minimal DNA binding unit. A kink at T210 occurs at a proteolytic cleavage function, the evolution of enzyme function within homologous families(1), and
site, suggesting a functional flexibility in the molecule that may be crucial for the evolution of biochemical pathways(2,3). Approaches to capture the
integration. The C-terminal domain is an SH3-like fold, and provides the structures data relating to enzyme active sites as 3D templates will be described
majority of the crystal contacts, consistent with the role of the domain in (4). An analysis of our results so far will be presented.
oligomerization of integrase. Based on this structure, we are outlining new (1) Todd, A.E., Orengo, C.A. & Thornton, J.M. (2001). Evolution of Function
strategies to discover novel drug leads targeting integrase. in Protein Superfamilies, from a Structural Perspective. Journal of Molecular
Biology, 307, 113-1143 (2) Teichmann, S.A., Rison S.C.G., Thornton,
Keywords: HIV INTEGRASE DNA BINDING J.M., Riley, M., Gough, J., & Chothia, C. (2001). The Evolution and Structural
Anatomy of the Small Metabolic Pathways in Escherichia coli. Journal of
Molecular Biology, 311, 693-708.
(3) Rison, S. Teichmann, S. & Thornton, J.M. (2002). Homology, pathway
distance and chromosomal localisation of the small molecule metabolism
enzymes in escherichia coli. Journal of molecular biology. 318, 911-932.
(4) Bartlett, G., Porter, C., Borkakoti, N. & Thornton, J.M. Computational
Analysis of Catalytic Residues in Enzyme Active Sites (Submitted)
Keywords: ENZYMES FUNCTION EVOLUTION
Acta Cryst. (2002). A58 (Supplement), C13 Acta Cryst. (2002). A58 (Supplement), C13
EVOLUTIONARY FOOTPRINTS FROM ANCIENT TIMES: THE MECHANISM OF SUCCINYL-CoA SYNTHETASE
NOVEL USE OF COMMON FOLDS IN THE BIOSYNTHETIC M.E. Fraser1 W.T. Wolodko2
1
PATHWAY FOR COBALAMIN Department of Biochemistry, University of Western Ontario, London, Ontario
I. Rayment N6A 5C1 Canada 2Department of Biochemistry, University of Alberta,
University of Wisconsin Biochemistry 433 Babcock Drive MADISON WI Edmonton, Alberta T6G 2H7 Canada
53711 USA
Succinyl-CoA synthetase (SCS) catalyzes the reversible reaction succinyl-CoA
The evolution of a new enzymatic activity from an existing functional enzyme + NDP + Pi = succinate + CoA + NTP where N is adenosine or guanosine.
is universally accepted as a way that nature adapts to new opportunities. SCS consists of two different subunits, α and β. During the reaction, a
Although there are clear cases where this has occurred in recent years, such as histidine residue of the α-subunit is transiently phosphorylated. The crystal
the development of pathways to utilize chemical compounds that have never structure of SCS showed that CoA binds to the α-subunit with the
existed in the biosphere, there is also good reason to believe that this has been a phosphorylated histidine residue approximately 7 Å from the thiol group of
common strategy since the beginning of life. In most cases the footprint of CoA. The structure of the complex of SCS with ADP-Mg2+ proved that the
sequence similarity has long been lost through the accumulation of point nucleotide binds to the predicted site in the 'ATP-grasp' fold of the β-subunit,
mutations and genetic rearrangements so that the only vestige of similarity however 35 Å away from where the active site histidine residue is seen in the
resides in the structural fold. The latter remains since every evolutionary step structure. We postulated that the loop that includes the active site histidine
must proceed through a stable folded protein. Thus the structural study of residue flips to shuttle the phosphoryl group between site I, where CoA and,
ancient biosynthetic pathways can provide insight into the folds that were presumably, succinate bind, and site II where nucleotide binds. Our most
present at the time of their inception. Recent structural studies of the enzymes interesting mutation to date is the change of a glutamate residue in site II to
in the cobalamin biosynthetic pathway reveal that several common protein alanine, which was predicted to destabilize only the binding at site II. The
folds have been adapted to meet the novel enzymatic problems presented by mutant protein showed very low enzymatic activity, and, surprisingly, in our
the synthesis of nature's most complex cofactor. The structures of assay it could not be phosphorylated by succinyl-CoA and inorganic phosphate
adenosylcobinamide kinase/adenosylcobinamide phosphate or by nucleotide triphosphate. The structure showed that two residues in site-I,
guanylyltransferase, ATP:corrinoid adenosyl transferase and L-threonine-O-3- Cys 123α-Pro 124α, were in a very different conformation than in the wild-
phosphate decarboxylase will be discussed. type enzyme. We postulate, when in this conformation the protein cannot bind
succinyl-CoA for phosphorylation by succinyl-CoA and inorganic phosphate.
Keywords: ENZYME EVOLUTION COBALAMIN BIOSYNTHETIC
PATHWAYS Keywords: MECHANISM, ENZYME, PHOSPHORYLATED
HISTIDINE