The Olson laboratory brings to the Center two decades of research and development in computational docking and virtual screening, and the largest distributed-computing resource currently addressing HIV biology: FightAIDS@Home. The Olson laboratory and collaborators have a long history of creating innovative methods to address specific applications. As part of the Structural Biology of AIDS program, novel methods were developed to study:

1) coevolution of HIV resistance when faced by a panel of inhibitors, and use of this information to design inhibitors that are robust in the face of resistance;

2) methods for conformational clustering of known and predicted complexes, to identify spanning mutants and maximally-diverse mutants for analysis of mechanisms of drug resistance;

3) methods for identifying and characterizing novel inhibitor binding sites;

4) methods for docking, extension, and linking of fragments;

5) methods for combining molecular dynamics and docking, which have revealed the connection for the HIV PR active site and the exosite currently being targeted.

The role of the Olson group in the Center will be:

1) multi-scale modeling from ligand-protein binding to interactions within the intact viral capsid;

2) modeling evolutionary dynamics combining ex-vivo and patient drug resistance data with molecular structure information; and

3) fragment-based investigation of novel allosteric sites.

The Arnold laboratory has been studying HIV-1 RT structure and its implications for function, ligand binding, drug design, and drug resistance since 1987. In a long-term collaboration with the group of Stephen Hughes at NCI Frederick, the Arnold group has solved the structure of wild-type and mutant HIV-1 RT in many functional states, including complexes with DNA, RNA/DNA, and inhibitors. The Arnold group contributed to the discovery of two anti-AIDS drugs, TMC125/etravirine/Intelence and TMC278/rilpivirine/Edurant, in a multidisciplinary structure-based drug design effort. Progress in drug-like fragment screening includes identification of novel allosteric inhibitory sites in RT and initiation of HIV-1 IN studies (with Kvaratskhelia and Engelman), and this work will be extended in the Center effort.

Building on this experience in solving large macromolecular complexes (RT, bacterial RNA polymerase, and viruses) and in engineering HIV-1 RT to generate many useful crystal forms, the Arnold group will determine the structures of the HIV polyprotein precursors and the RT/tRNA/RNA initiation complex.

The Elder laboratory has been involved in molecular virology over the past 35 years, with emphasis on characterization of molecular mechanisms and life cycle of retroviruses. Of direct relevance to the HIVE center, the laboratory has expressed, purified and characterized a number of viral proteins employed in successful crystallization efforts, with specific focus on defining the molecular basis of drug resistance involving the viral protease (PR). In addition, Dr. Ying-Chuan Lin has developed an infectious FIV encoding PR with HIV-like drug sensitivity and has performed drug selections to document drug resistance mutations within PR and its polyprotein substrates for comparison to results with parental HIV.

The laboratory’s experience in studies of HIV and FIV structure and drug resistance/drug design will facilitate two major areas of the Center’s research effort. First, The laboratory will continue the preparation of Gag polyproteins, both full-length and partial constructs, along with both active and inactive forms of PR to provide the needed materials for crystallization trials by the Stout laboratory; for SAXS analysis in collaboration with the Tainer laboratory; and for HDX/MS with the Griffin laboratory. Constructs will include a series of partial Gag polyprotein constructs including MA-CA, MA-CA-SP1, SP1-NC, and NC-SP2-p6 as well as full-length Gag. Additional Gag-Pol constructs will be prepared to facilitate preparation of the full-length Gag-Pol polyprotein. Studies will be coordinated with the efforts of the Marcotrigiano laboratory, and will involve using both E. coli and mammalian expression systems. Studies will also continue to assess the role of viral RNA in facilitating PR cleavage of bottleneck sites within the Gag polyprotein, including the NC-SP2 and CA-SP1 cleavage sites. Parallel studies will utilize infectious HIV / FIV PR chimeric FIV to assess drug resistance evolution to HIV-relevant anti-PR drugs and identify relevant Gag-Pol mutations.

Alan Engelman’s research focuses on the integration step in the HIV-1 replication cycle. Dr. Engelman’s group has spearheaded HIV-1 preintegration complex (PIC) biochemistry methods that were critical to evaluate the role of the chromatin-associated lens epithelium-derived growth factor (LEDGF)/p75 in targeting HIV-1 PICs to active genes during integration. The Engelman group moreover solved the integrase-LEDGF/p75 co-crystal structure that formed the foundation for allosteric integrase inhibitor (ALLINI) discovery. Dr. Engelman’s group will focus on virological characterization of novel ALLINIs in this project. Dr. Engelman’s virology expertise furthermore extends to other HIVE Center projects, for example to test the functional relevance of novel macromolecular contacts gleaned from HIV-1 Pol and/or Gag-Pol polyprotein structures.

Kellie Ann Jurado is enrolled in the Graduate Program in Virology at Harvard Medical School. Kellie’s dissertation research utilizes small molecule pharmacophores as biological probes to discover novel aspects of integrase biology during HIV-1 replication.

The Finn and Fokin laboratories are synthetic chemists highly experienced in the development of drug candidates, chemical biology probe molecules, and bioconjugation techniques. They are the leading discoverers and developers of click chemistry methods and their applications to the synthesis of inhibitors/high affinity ligands and covalent modification of macromolecules. The copper-catalyzed azide-alkyne cycloaddition (CuAAC) has become one of the most widely used synthetic methods during the last decade.

The Finn-Fokin partnership provides the HIVE Center with synthetic chemistry expertise for the following purposes:

(a) optimizing molecules for the binding and inhibition of HIV and host proteins of interest;

(b) labeling molecules to aid studies of structure and dynamics;

(c) synthesizing molecules to test computational predictions of binding affinity or inhibition efficiency.

Particularly useful to the HIVE Center is the Finn-Fokin “in situ click” method for target-templated formation of selective and potent binding agents for biomolecules. The Finn group has also helped to develop backscattering interferometry (BSI) as a label-free technique for the quantitation of binding events for a wide variety of biological molecules. This capability will be used by many Center groups for independent validation of binding interactions identified by other techniques, and for the screening of candidate interactions among various HIV macromolecular components.

The Griffin laboratory has a broad background in drug discovery and development, as well as the study of protein structure with approaches to modulating protein function via synthetic small molecules that spans the last 20+ years. His research is focused on structure-function and chemical biology studies of nuclear receptors and GPCRs. His laboratory has built an automated platform to profile protein:protein and protein:ligand interactions by hydrogen/deuterium exchange (HDX) mass spectrometry.

In the HIVE Center, he serves as a HDX Core Director, providing HDX support to all the Center members. In addition, as Co-PI and consortium PI of the Scripps MLPCN Center “The Comprehensive Center for Chemical Probe Discovery and Optimization at Scripps,” he provides the HIVE Center with guidance on assay development and using the Fast Track mechanism to transfer assays into the MLPCN network.

The primary expertise of the Jones lab for many years has been the development of innovative methods for the chemical synthesis of modified nucleosides and nucleic acids. These efforts have included modifications of nucleosides that can be incorporated into strands of DNA and RNA to form cross-linkable tethers that will be important for the structure determinations in the HIVE Center. In addition to the cross-linkable rG that will be useful for studies of the RT/tRNA/RNA initiation complex we are planning to make cross-linkable pyrimidine phosphoramidites (dT/U or dC/C) to expand the scope of thiol cross-linking. We will coordinate with other members of the Center (Arnold, Parniak, Kvaratskelia, Engelman, and others) to produce large-scale amounts of strategically modified oligonucleotide reagents. Dr. Jones’s experience will complement the Finn/Fokin chemistry effort in the Center by creating nucleoside and nucleic acid probes of HIV interactions.

The Levy laboratory brings to this collaboration thirty years of experience and leadership in the development and application of molecular simulation methods to study the structure and dynamics of proteins and their complexes. He uses multi-scale modeling approaches (protein-protein docking, replica exchange molecular dynamics, kinetic networks), together with experimental information (e.g. SANS, SAXS, H/D exchange) obtained by HIVE Center investigators, to elucidate the structural ensembles which constitute the Gag and Pol polyproteins, and to construct the conformational pathways in the assembly process and model their kinetics. In addition the Levy group is developing information-theoretic statistical inference techniques to identify correlated sets of resistance mutations on HIV proteins and their partners. They will integrate these bioinformatic tools with biophysical data and structural models to assess how correlated mutations facilitate resistance through their effects on protein stability and activity.

The Marcotrigiano group has expertise in structural and functional characterization of viral polyprotein processing and large-scale recombinant protein production in mammalian cell lines. Similar to HIV, other positive-sense RNA viral genomes, including hepatitis C virus (HCV) and alphaviruses, encode a polyprotein that is proteolytically processed in a highly regulated manner into the final, mature forms. Recently, the Marcotrigiano group has determined the structure of a large portion of a precleavage form of Alphavirus polyprotein involved in genome replication. The structure demonstrated that prior to cleavage there is an extensive interface between the two viral proteins and access to the cleavage site is highly regulated. In addition, the Marcotrigaino group has developed methodologies that greatly diminish the cost and time of large-scale expression in mammalian cells. The technology combines the speed and high efficiency of viral infection with a novel adherent cell bioreactor that significantly reduces the cost, while improving the efficiency, quality, utility, and comprehensiveness for production of challenging proteins. The Marcotrigiano group will apply this mammalian expression system for the production of various polyprotein precursors.

Michael A. Parniak has over twenty years experience in HIV research, focusing on mechanisms of HIV resistance to antiretroviral drugs as well as drug discovery related to novel HIV targets. Parniak’s group was the first to identify pyrophosphorolysis as the underlying mechanism of HIV resistance to AZT, and discovered the first potent HIV ribonuclease H inhibitor (RNHI). In collaboration with the Arnold group and others, Parniak has identified novel allosteric binding sites in HIV RT that play roles in inhibition of RNH, inhibition of reverse transcription initiation, and that impact on Pol protein dimer stability. These studies will be extended in the HIVE Center effort to provide essential probes for the role of Pol polyprotein association in virion maturation and defining the RT/tRNA/RNA initiation complex as a novel drug target.

C. D. Stout has determined numerous crystal structures of HIV and FIV wild type and mutant PRs in complex with inhibitors and fragments, and of several membrane associated proteins, including cytochrome P450s (48 PDB entries) and cytochrome ba3 oxidase (9 PDB entries). Crystallization of the membrane proteins has entailed construct design and the use of detergents; the lipidic cubic phase (LCP) was used to obtain a structure of the oxidase at 1.8 Å resolution.

As part of the HIVE Center, the Stout group has several research goals:

Crystallization of Gag components and Gag-PR complexes

Crystal structures for Gag components with and without bound PR will be determined to define structural relationships during assembly and visualize the interaction of PR with flanking Gag domains during cleavage.  These structures are relevant to conformational states during viral assembly, rates of PR cleavage at specific junctions, the co-evolution of resistance in HIV, and development of new targets for antiviral therapies.

Constructs prepared in the Torbett and Elder laboratories include MA-CA, MA-CA-p2, p2-NC, and NC-p1-p6, as well as full-length Gag.  Protein expression will be coordinated with J. Marcotrigiano and will employ E. coli and mammalian systems.  Stable complexes of Gag components with PR bound will use D25 mutants of PR.  Biochemical analysis will establish monodispersity of the protein complexes, and crystallization screening will employ high-throughput robotics.  In collaboration with the Cherezov laboratory, MA will be engineered as a fusion protein with a trans-membrane domain, in order to crystallize MA in the membrane-like bilayer environment of the lipidic cubic phase.  Crystallographic experiments will be done using in-house and SSRL resources.

Development of allosteric inhibitors targeting HIV PR

Experiments will address the hypothesis that allosteric inhibitors of PR, acting in concert with active site inhibitors, can suppress the evolution of resistance.  It is proposed that the genetic barrier to the evolution of resistance is higher if multiple sites on the same HIV enzyme are targeted, as opposed to active sites on different enzymes, as in HAART.

Structural and molecular dynamics studies of PR reveal that clefts on the protein surface, distal from the active site, undergo local rearrangements correlated with motion of the flaps.  In collaboration with the Elder and Olson laboratories, two such have sites have been exploited using fragment screening and crystallography.  In collaboration with the Torbett, Olson, Finn, and Fokin laboratories, fragment hits are elaborated into larger, higher affinity inhibitors through combined use of biophysical assays, docking, chemical synthesis, and crystallography.  The compounds developed will be used to evaluate allosteric inhibition, interference with Gag-PR interactions, and ability to suppress the evolution of resistance in ex vivo viral selection experiments.

Bridget Carragher, Director, and Clint Potter, Co-Director, National Resource for Automated Molecular Microscopy, TSRI, will provide automated imaging techniques for solving the three-dimensional structures of Gag, Gag-Pol, and Pol complexes using cryoTEM, evaluating the monodispersity of HIV polyproteins in conjunction with crystallization, and visualizing microcrystals in the lipidic cubic phase.

Stephen H. Hughes, Director HIV Drug Resistance Program, NCI-Frederick, has worked with Arnold to study the structure and function of HIV-1 RT for 25 years. He will participate in the HIV polyprotein precursor studies, providing expression constructs, produce purified foamy virus polyproteins in large amounts for structural studies, study the impact of mutations on proteolytic susceptibility of Pol enzymes, and will test interesting compounds for anti-HIV activity in single-round replication systems. Hughes’ authoritative overview of HIV biology and research will help to guide the Center’s aim of connecting our studies and results throughout the HIV lifecycle.

David Looney MD, University of California San Diego School of Medicine, UCSD/VMRF CFAR Molecular Biology Core, has provided a clinical perspective in the TSRI HIV Program Project. He has been instrumental in compiling, annotating, and analyzing clinical data from Southern California VA Healthcare System and CFAR Network of Integrated Clinical Systems, which provides the primary support for work on pathways of resistance mutation. Within the Center, he will continue to provide new clinical data on resistance mutations to all inhibitors.

Jason Okulicz MD, MC, USAF, is on the Infectious Disease Service Staff at Lackland Air Force Base, Texas, will provide clinical data and sera/plasma/cell samples from participates in the U.S. Military HIV Natural History Study. Okulicz will participate within the Center on the antiretroviral-mediated viral co-evolution studies by providing viral samples from individuals for protease and gag sequencing. The sequence information from viral samples will be crucial for modeling the acquisition of resistance mutations.

Alan Rein, Director of the Retroviral Assembly Section, NCI-Frederick, and staff scientist Sid Datta will collaborate on studies of HIV Gag and Gag-Pol polyprotein precursors. Using a defined assembly system that he and his colleagues have developed, Rein and Datta will work with Center investigators to map specific Gag-Gag interactions by HDX and to explore Gag and Gag-Pol interactions in assembly. Their broad expertise in studying retroviral assembly will help to place the Center’s studies of HIV polyproteins in the broader context of HIV assembly and maturation.

Doug Richman MD, Director of UC San Diego Center for AIDS Research, will provide resources from the CFAR, including core services in flow cytometry, molecular biology, translation virology, bioinformatics and genomics, protein expression and proteomics, and as well as clinic investigations. Utilizing the available CFAR Cores will leverage valuable research and clinical services for all Center grant participants.

Robin Wilner, Vice President for Global Community Initiatives, IBM World Community Grid, will continue a long-standing collaboration on the FightAIDS@Home project, which provides a distributed network of over two million internet shared processors for use in docking and drug design.

Management of the Center builds upon over 25 years of experience in the Structural Biology of HIV program. Principal Investigator Arthur Olson led the TSRI program project for three funding cycles, building a highly collaborative program that effectively combined structure, biology, chemistry and computation into a full drug discovery cycle. Center Coordinator Margaret Graber brings 20 years of experience in administration of NIH and NSF funded program projects, including subawards, continuing with the three previous funding cycles of the Structural Biology of HIV program. In addition, she has organized national and international meetings, workshops, and conferences.

Co-principal Investigator Eddy Arnold administers the Collaborative Development Program, building on 10 years of experience in administration of program projects.  In/Outreach is coordinated by Richard Belew and David Goodsell. Dr. Belew has a long history of development of innovative methods for scientific data mining and communication. He applies this experience to the development of effective online tools for communication and archiving results within the Center and in the wider community, as well as methods for maximizing the effectiveness of face-to-face meeting through use of online preparation and sharing. For the past 9 years, David Goodsell has been centrally involved in outreach at the RCSB Protein Data Bank, creating materials for educators, students, and the general public. In collaboration with education researchers at the Milwaukee School of Engineering, he has helped to create innovative teaching materials that combine physical models, printed material and interactive online materials. In addition, he has provided materials for science museums and the news media, as well as coordinating outreach efforts to local high schools.