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Articles 1 - 30 of 127
Full-Text Articles in Life Sciences
Crystal Structure Of Apobec3a Bound To Single-Stranded Dna Reveals Structural Basis For Cytidine Deamination And Specificity, Takahide Kouno, Tania V. Silvas, Brendan J. Hilbert, Shivender Shandilya, Markus-Frederik Bohn, Brian A. Kelch, William E. Royer, Mohan Somasundaran, Nese Kurt Yilmaz, Hiroshi Matsuo, Celia A. Schiffer
Crystal Structure Of Apobec3a Bound To Single-Stranded Dna Reveals Structural Basis For Cytidine Deamination And Specificity, Takahide Kouno, Tania V. Silvas, Brendan J. Hilbert, Shivender Shandilya, Markus-Frederik Bohn, Brian A. Kelch, William E. Royer, Mohan Somasundaran, Nese Kurt Yilmaz, Hiroshi Matsuo, Celia A. Schiffer
Celia A. Schiffer
Nucleic acid editing enzymes are essential components of the immune system that lethally mutate viral pathogens and somatically mutate immunoglobulins, and contribute to the diversification and lethality of cancers. Among these enzymes are the seven human APOBEC3 deoxycytidine deaminases, each with unique target sequence specificity and subcellular localization. While the enzymology and biological consequences have been extensively studied, the mechanism by which APOBEC3s recognize and edit DNA remains elusive. Here we present the crystal structure of a complex of a cytidine deaminase with ssDNA bound in the active site at 2.2 A. This structure not only visualizes the active site …
Dengue Virus Ns2b/Ns3 Protease Inhibitors Exploiting The Prime Side, Kuan-Hung Lin, Akbar Ali, Linah Rusere, Djade I. Soumana, Nese Kurt Yilmaz, Celia A. Schiffer
Dengue Virus Ns2b/Ns3 Protease Inhibitors Exploiting The Prime Side, Kuan-Hung Lin, Akbar Ali, Linah Rusere, Djade I. Soumana, Nese Kurt Yilmaz, Celia A. Schiffer
Celia A. Schiffer
The mosquito-transmitted dengue virus (DENV) infects millions of people in tropical and subtropical regions. Maturation of DENV particles requires proper cleavage of the viral polyprotein, including processing of 8 of the 13 substrate cleavage sites by dengue virus NS2B/NS3 protease. With no available direct-acting antiviral targeting DENV, NS2/NS3 protease is a promising target for inhibitor design. Current design efforts focus on the nonprime side of the DENV protease active site, resulting in highly hydrophilic and nonspecific scaffolds. However, the prime side also significantly modulates DENV protease binding affinity, as revealed by engineering the binding loop of aprotinin, a small protein …
Interdependence Of Inhibitor Recognition In Hiv-1 Protease, Janet L. Paulsen, Florian Leidner, Debra A. Ragland, Nese Kurt Yilmaz, Celia A. Schiffer
Interdependence Of Inhibitor Recognition In Hiv-1 Protease, Janet L. Paulsen, Florian Leidner, Debra A. Ragland, Nese Kurt Yilmaz, Celia A. Schiffer
Celia A. Schiffer
Molecular recognition is a highly interdependent process. Subsite couplings within the active site of proteases are most often revealed through conditional amino acid preferences in substrate recognition. However, the potential effect of these couplings on inhibition and thus inhibitor design is largely unexplored. The present study examines the interdependency of subsites in HIV-1 protease using a focused library of protease inhibitors, to aid in future inhibitor design. Previously a series of darunavir (DRV) analogs was designed to systematically probe the S1' and S2' subsites. Co-crystal structures of these analogs with HIV-1 protease provide the ideal opportunity to probe subsite interdependency. …
Structural And Molecular Analysis Of A Protective Epitope Of Lyme Disease Antigen Ospa And Antibody Interactions, Shivender Shandilya, Nese Kurt Yilmaz, Ejemel Monir, Andrew Sadowski, William D. Thomas, Mark S. Klempner, Celia A. Schiffer, Yan Wang
Structural And Molecular Analysis Of A Protective Epitope Of Lyme Disease Antigen Ospa And Antibody Interactions, Shivender Shandilya, Nese Kurt Yilmaz, Ejemel Monir, Andrew Sadowski, William D. Thomas, Mark S. Klempner, Celia A. Schiffer, Yan Wang
Celia A. Schiffer
The murine monoclonal antibody LA-2 recognizes a clinically protective epitope on outer surface protein (OspA) of Borrelia burgdorferi, the causative agent of Lyme disease in North America. Human antibody equivalence to LA-2 is the best serologic correlate of protective antibody responses following OspA vaccination. Understanding the structural and functional basis of the LA-2 protective epitope is important for developing OspA-based vaccines and discovering prophylactic antibodies against Lyme disease. Here, we present a detailed structure-based analysis of the LA-2/OspA interaction interface and identification of residues mediating antibody recognition. Mutations were introduced into both OspA and LA-2 based on computational predictions on …
The Ssdna Mutator Apobec3a Is Regulated By Cooperative Dimerization, Markus-Frederik Bohn, Shivender Shandilya, Tania Silvas, Ellen Nalivaika, Takahide Kouno, Brian Kelch, Sean Ryder, Nese Yilmaz, Mohan Somasundaran, Celia Schiffer
The Ssdna Mutator Apobec3a Is Regulated By Cooperative Dimerization, Markus-Frederik Bohn, Shivender Shandilya, Tania Silvas, Ellen Nalivaika, Takahide Kouno, Brian Kelch, Sean Ryder, Nese Yilmaz, Mohan Somasundaran, Celia Schiffer
Celia A. Schiffer
Deaminase activity mediated by the human APOBEC3 family of proteins contributes to genomic instability and cancer. APOBEC3A is by far the most active in this family and can cause rapid cell death when overexpressed, but in general how the activity of APOBEC3s is regulated on a molecular level is unclear. In this study, the biochemical and structural basis of APOBEC3A substrate binding and specificity is elucidated. We find that specific binding of single-stranded DNA is regulated by the cooperative dimerization of APOBEC3A. The crystal structure elucidates this homodimer as a symmetric domain swap of the N-terminal residues. This dimer interface …
Structure Of The Vif-Binding Domain Of The Antiviral Enzyme Apobec3g, Takahide Kouno, Elizabeth Luengas, Megumi Shigematsu, Shivender Shandilya, Jingying Zhang, Luan Chen, Mayuko Hara, Celia Schiffer, Reuben Harris, Hiroshi Matsuo
Structure Of The Vif-Binding Domain Of The Antiviral Enzyme Apobec3g, Takahide Kouno, Elizabeth Luengas, Megumi Shigematsu, Shivender Shandilya, Jingying Zhang, Luan Chen, Mayuko Hara, Celia Schiffer, Reuben Harris, Hiroshi Matsuo
Celia A. Schiffer
The human APOBEC3G (A3G) DNA cytosine deaminase restricts and hypermutates DNA-based parasites including HIV-1. The viral infectivity factor (Vif) prevents restriction by triggering A3G degradation. Although the structure of the A3G catalytic domain is known, the structure of the N-terminal Vif-binding domain has proven more elusive. Here, we used evolution- and structure-guided mutagenesis to solubilize the Vif-binding domain of A3G, thus permitting structural determination by NMR spectroscopy. A smaller zinc-coordinating pocket and altered helical packing distinguish the structure from previous catalytic-domain structures and help to explain the reported inactivity of this domain. This soluble A3G N-terminal domain is bound by …
Simultaneously Targeting The Ns3 Protease And Helicase Activities For More Effective Hepatitis C Virus Therapy, Jean Ndjomou, M Corby, Noreena Sweeney, Alicia Hanson, Cihan Aydin, Akbar Ali, Celia Schiffer, Kelin Li, Kevin Frankowski, Frank Schoenen, David Frick
Simultaneously Targeting The Ns3 Protease And Helicase Activities For More Effective Hepatitis C Virus Therapy, Jean Ndjomou, M Corby, Noreena Sweeney, Alicia Hanson, Cihan Aydin, Akbar Ali, Celia Schiffer, Kelin Li, Kevin Frankowski, Frank Schoenen, David Frick
Celia A. Schiffer
This study examines the specificity and mechanism of action of a recently reported hepatitis C virus (HCV) nonstructural protein 3 (NS3) helicase-protease inhibitor (HPI), and the interaction of HPI with the NS3 protease inhibitors telaprevir, boceprevir, danoprevir, and grazoprevir. HPI most effectively reduced cellular levels of subgenomic genotype 4a replicons, followed by genotypes 3a and 1b replicons. HPI had no effect on HCV genotype 2a or dengue virus replicon levels. Resistance evolved more slowly to HPI than telaprevir, and HPI inhibited telaprevir-resistant replicons. Molecular modeling and analysis of the ability of HPI to inhibit peptide hydrolysis catalyzed by a variety …
Structural Basis For Mutation-Induced Destabilization Of Profilin 1 In Als, Sivakumar Boopathy, Tania Silvas, Maeve Tischbein, Silvia Jansen, Shivender Shandilya, Jill Zitzewitz, John Landers, Bruce Goode, Celia Schiffer, Daryl Bosco
Structural Basis For Mutation-Induced Destabilization Of Profilin 1 In Als, Sivakumar Boopathy, Tania Silvas, Maeve Tischbein, Silvia Jansen, Shivender Shandilya, Jill Zitzewitz, John Landers, Bruce Goode, Celia Schiffer, Daryl Bosco
Celia A. Schiffer
Mutations in profilin 1 (PFN1) are associated with amyotrophic lateral sclerosis (ALS); however, the pathological mechanism of PFN1 in this fatal disease is unknown. We demonstrate that ALS-linked mutations severely destabilize the native conformation of PFN1 in vitro and cause accelerated turnover of the PFN1 protein in cells. This mutation-induced destabilization can account for the high propensity of ALS-linked variants to aggregate and also provides rationale for their reported loss-of-function phenotypes in cell-based assays. The source of this destabilization is illuminated by the X-ray crystal structures of several PFN1 proteins, revealing an expanded cavity near the protein core of the …
Inhibition Of Apobec3g Activity Impedes Double-Stranded Dna Repair, Ponnandy Prabhu, Shivender Shandilya, Elena Britan-Rosich, Adi Nagler, Celia Schiffer, Moshe Kotler
Inhibition Of Apobec3g Activity Impedes Double-Stranded Dna Repair, Ponnandy Prabhu, Shivender Shandilya, Elena Britan-Rosich, Adi Nagler, Celia Schiffer, Moshe Kotler
Celia A. Schiffer
The cellular cytidine deaminase APOBEC3G (A3G) was first described as an anti-HIV-1 restriction factor, acting by directly deaminating reverse transcripts of the viral genome. HIV-1 Vif neutralizes the activity of A3G, primarily by mediating degradation of A3G to establish effective infection in host target cells. Lymphoma cells, which express high amounts of A3G, can restrict Vif-deficient HIV-1. Interestingly, these cells are more stable in the face of treatments that result in double-stranded DNA damage, such as ionizing radiation and chemotherapies. Previously, we showed that the Vif-derived peptide (Vif25-39) efficiently inhibits A3G deamination, and increases the sensitivity of lymphoma cells to …
Rediii: A Pipeline For Automated Structure Solution, Markus-Frederik Bohn, Celia Schiffer
Rediii: A Pipeline For Automated Structure Solution, Markus-Frederik Bohn, Celia Schiffer
Celia A. Schiffer
High-throughput crystallographic approaches require integrated software solutions to minimize the need for manual effort. REdiii is a system that allows fully automated crystallographic structure solution by integrating existing crystallographic software into an adaptive and partly autonomous workflow engine. The program can be initiated after collecting the first frame of diffraction data and is able to perform processing, molecular-replacement phasing, chain tracing, ligand fitting and refinement without further user intervention. Preset values for each software component allow efficient progress with high-quality data and known parameters. The adaptive workflow engine can determine whether some parameters require modifications and choose alternative software strategies …
Modulation Of Hiv Protease Flexibility By The T80n Mutation, Hao Zhou, Shangyang Li, John Badger, Ellen Nalivaika, Yufeng Cai, Jennifer Foulkes-Murzycki, Celia Schiffer, Lee Makowski
Modulation Of Hiv Protease Flexibility By The T80n Mutation, Hao Zhou, Shangyang Li, John Badger, Ellen Nalivaika, Yufeng Cai, Jennifer Foulkes-Murzycki, Celia Schiffer, Lee Makowski
Celia A. Schiffer
The flexibility of HIV protease (HIVp) plays a critical role in enabling enzymatic activity and is required for substrate access to the active site. While the importance of flexibility in the flaps that cover the active site is well known, flexibility in other parts of the enzyme is also critical for function. One key region is a loop containing Thr 80, which forms the walls of the active site. Although not situated within the active site, amino acid Thr80 is absolutely conserved. The mutation T80N preserves the structure of the enzyme but catalytic activity is completely lost. To investigate the …
A Direct Interaction With Rna Dramatically Enhances The Catalytic Activity Of The Hiv-1 Protease In Vitro, Marc Potempa, Ellen Nalivaika, Debra Ragland, Sook-Kyung Lee, Celia Schiffer, Ronald Swanstrom
A Direct Interaction With Rna Dramatically Enhances The Catalytic Activity Of The Hiv-1 Protease In Vitro, Marc Potempa, Ellen Nalivaika, Debra Ragland, Sook-Kyung Lee, Celia Schiffer, Ronald Swanstrom
Celia A. Schiffer
Though the steps of human immunodeficiency virus type 1 (HIV-1) virion maturation are well documented, the mechanisms regulating the proteolysis of the Gag and Gag-Pro-Pol polyproteins by the HIV-1 protease (PR) remain obscure. One proposed mechanism argues that the maturation intermediate p15NC must interact with RNA for efficient cleavage by the PR. We investigated this phenomenon and found that processing of multiple substrates by the HIV-1 PR was enhanced in the presence of RNA. The acceleration of proteolysis occurred independently from the substrate's ability to interact with nucleic acid, indicating that a direct interaction between substrate and RNA is not …
A Balance Between Inhibitor Binding And Substrate Processing Confers Influenza Drug Resistance, Li Jiang, Ping Liu, Claudia Bank, Nicholas Renzette, Kristina Prachanronarong, L. Yilmaz, Daniel Caffrey, Konstantin Zeldovich, Celia Schiffer, Timothy Kowalik, Jeffrey Jensen, Robert Finberg, Jennifer Wang, Daniel Bolon
A Balance Between Inhibitor Binding And Substrate Processing Confers Influenza Drug Resistance, Li Jiang, Ping Liu, Claudia Bank, Nicholas Renzette, Kristina Prachanronarong, L. Yilmaz, Daniel Caffrey, Konstantin Zeldovich, Celia Schiffer, Timothy Kowalik, Jeffrey Jensen, Robert Finberg, Jennifer Wang, Daniel Bolon
Celia A. Schiffer
The therapeutic benefits of the neuraminidase (NA) inhibitor oseltamivir are dampened by the emergence of drug resistance mutations in influenza A virus (IAV). To investigate the mechanistic features that underlie resistance, we developed an approach to quantify the effects of all possible single-nucleotide substitutions introduced into important regions of NA. We determined the experimental fitness effects of 450 nucleotide mutations encoding positions both surrounding the active site and at more distant sites in an N1 strain of IAV in the presence and absence of oseltamivir. NA mutations previously known to confer oseltamivir resistance in N1 strains, including H275Y and N295S, …
Structural And Thermodynamic Effects Of Macrocyclization In Hcv Ns3/4a Inhibitor Mk-5172, Djade Soumana, Nese Yilmaz, Kristina Prachanronarong, Cihan Aydin, Akbar Ali, Celia Schiffer
Structural And Thermodynamic Effects Of Macrocyclization In Hcv Ns3/4a Inhibitor Mk-5172, Djade Soumana, Nese Yilmaz, Kristina Prachanronarong, Cihan Aydin, Akbar Ali, Celia Schiffer
Celia A. Schiffer
Recent advances in direct-acting antivirals against Hepatitis C Virus (HCV) have led to the development of potent inhibitors, including MK-5172, that target the viral NS3/4A protease with relatively low susceptibility to resistance. MK-5172 has a P2-P4 macrocycle and a unique binding mode among current protease inhibitors where the P2 quinoxaline packs against the catalytic residues H57 and D81. However, the effect of macrocyclization on this binding mode is not clear, as is the relation between macrocyclization, thermodynamic stabilization, and susceptibility to the resistance mutation A156T. We have determined high-resolution crystal structures of linear and P1-P3 macrocyclic analogs of MK-5172 bound …
Positive Selection Drives Preferred Segment Combinations During Influenza Virus Reassortment, Konstantin Zeldovich, Ping Liu, Nicholas Renzette, Matthieu Foll, Serena Pham, Sergey Venev, Glen Gallagher, Daniel Bolon, Evelyn Kurt-Jones, Jeffrey Jensen, Daniel Caffrey, Celia Schiffer, Timothy Kowalik, Jennifer Wang, Robert Finberg
Positive Selection Drives Preferred Segment Combinations During Influenza Virus Reassortment, Konstantin Zeldovich, Ping Liu, Nicholas Renzette, Matthieu Foll, Serena Pham, Sergey Venev, Glen Gallagher, Daniel Bolon, Evelyn Kurt-Jones, Jeffrey Jensen, Daniel Caffrey, Celia Schiffer, Timothy Kowalik, Jennifer Wang, Robert Finberg
Celia A. Schiffer
Influenza A virus (IAV) has a segmented genome that allows for the exchange of genome segments between different strains. This reassortment accelerates evolution by breaking linkage, helping IAV cross species barriers to potentially create highly virulent strains. Challenges associated with monitoring the process of reassortment in molecular detail have limited our understanding of its evolutionary implications. We applied a novel deep sequencing approach with quantitative analysis to assess the in vitro temporal evolution of genomic reassortment in IAV. The combination of H1N1 and H3N2 strains reproducibly generated a new H1N2 strain with the hemagglutinin and nucleoprotein segments originating from H1N1 …
A Computational Analysis Of The Structural Determinants Of Apobec3'S Catalytic Activity And Vulnerability To Hiv-1 Vif, Shivender Shandilya, Markus-Frederik Bohn, Celia Schiffer
A Computational Analysis Of The Structural Determinants Of Apobec3'S Catalytic Activity And Vulnerability To Hiv-1 Vif, Shivender Shandilya, Markus-Frederik Bohn, Celia Schiffer
Celia A. Schiffer
APOBEC3s (A3) are Zn(2+) dependent cytidine deaminases with diverse biological functions and implications for cancer and immunity. Four of the seven human A3s restrict HIV by 'hypermutating' the reverse-transcribed viral genomic DNA. HIV Virion Infectivity Factor (Vif) counters this restriction by targeting A3s to proteasomal degradation. However, there is no apparent correlation between catalytic activity, Vif binding, and sequence similarity between A3 domains. Our comparative structural analysis reveals features required for binding Vif and features influencing polynucleotide deaminase activity in A3 proteins. All Vif-binding A3s share a negatively charged surface region that includes residues previously implicated in binding the highly-positively …
Modulation Of Hiv Protease Flexibility By The T80n Mutation, Hao Zhou, Shangyang Li, John Badger, Ellen Nalivaika, Yufeng Cai, Jennifer Foulkes-Murzycki, Celia Schiffer, Lee Makowski
Modulation Of Hiv Protease Flexibility By The T80n Mutation, Hao Zhou, Shangyang Li, John Badger, Ellen Nalivaika, Yufeng Cai, Jennifer Foulkes-Murzycki, Celia Schiffer, Lee Makowski
Celia A. Schiffer
The flexibility of HIV protease plays a critical role in enabling enzymatic activity and is required for substrate access to the active site. While the importance of flexibility in the flaps that cover the active site is well known, flexibility in other parts of the enzyme is also critical for function. One key region is a loop containing Thr 80 which forms the walls of the active site. Although not situated within the active site, amino acid Thr80 is absolutely conserved. The mutation T80N preserves the structure of the enzyme but catalytic activity is completely lost. To investigate the potential …
Structure-Guided Design Of A High Affinity Inhibitor To Human Ctbp, Brendan Hilbert, Benjamin Morris, Keith Ellis, Janet Paulsen, Celia Schiffer, Steven Grossman, William Royer
Structure-Guided Design Of A High Affinity Inhibitor To Human Ctbp, Brendan Hilbert, Benjamin Morris, Keith Ellis, Janet Paulsen, Celia Schiffer, Steven Grossman, William Royer
Celia A. Schiffer
Oncogenic transcriptional coregulators C-terminal Binding Protein (CtBP) 1 and 2 possess regulatory d-isomer specific 2-hydroxyacid dehydrogenase (D2-HDH) domains that provide an attractive target for small molecule intervention. Findings that the CtBP substrate 4-methylthio 2-oxobutyric acid (MTOB) can interfere with CtBP oncogenic activity in cell culture and in mice confirm that such inhibitors could have therapeutic benefit. Recent crystal structures of CtBP 1 and 2 revealed that MTOB binds in an active site containing a dominant tryptophan and a hydrophilic cavity, neither of which are present in other D2-HDH family members. Here, we demonstrate the effectiveness of exploiting these active site …
Drug-Resistant Hiv-1 Protease Regains Functional Dynamics Through Cleavage Site Coevolution, Nevra Ozer, Aysegul Ozen, Celia Schiffer, Turkan Haliloglu
Drug-Resistant Hiv-1 Protease Regains Functional Dynamics Through Cleavage Site Coevolution, Nevra Ozer, Aysegul Ozen, Celia Schiffer, Turkan Haliloglu
Celia A. Schiffer
Drug resistance is caused by mutations that change the balance of recognition favoring substrate cleavage over inhibitor binding. Here, a structural dynamics perspective of the regained wild-type functioning in mutant HIV-1 proteases with coevolution of the natural substrates is provided. The collective dynamics of mutant structures of the protease bound to p1-p6 and NC-p1 substrates are assessed using the Anisotropic Network Model (ANM). The drug-induced protease mutations perturb the mechanistically crucial hinge axes that involve key sites for substrate binding and dimerization and mainly coordinate the intrinsic dynamics. Yet with substrate coevolution, while the wild-type dynamic behavior is restored in …
Structural Basis And Distal Effects Of Gag Substrate Coevolution In Drug Resistance To Hiv-1 Protease, Aysegul Ozen, Kuan-Hung Lin, Nese Yilmaz, Celia Schiffer
Structural Basis And Distal Effects Of Gag Substrate Coevolution In Drug Resistance To Hiv-1 Protease, Aysegul Ozen, Kuan-Hung Lin, Nese Yilmaz, Celia Schiffer
Celia A. Schiffer
Drug resistance mutations in response to HIV-1 protease inhibitors are selected not only in the drug target but elsewhere in the viral genome, especially at the protease cleavage sites in the precursor protein Gag. To understand the molecular basis of this protease-substrate coevolution, we solved the crystal structures of drug resistant I50V/A71V HIV-1 protease with p1-p6 substrates bearing coevolved mutations. Analyses of the protease-substrate interactions reveal that compensatory coevolved mutations in the substrate do not restore interactions lost due to protease mutations, but instead establish other interactions that are not restricted to the site of mutation. Mutation of a substrate …
Evolution Of The Influenza A Virus Genome During Development Of Oseltamivir Resistance In Vitro, Nicholas Renzette, Daniel R. Caffrey, Konstantin B. Zeldovich, Ping Liu, Glen R. Gallagher, Daniel Aiello, Alyssa J. Porter, Evelyn A. Kurt-Jones, Daniel N. Bolon, Yu-Ping Poh, Jeffrey D. Jensen, Celia A. Schiffer, Timothy F. Kowalik, Robert W. Finberg, Jennifer P. Wang
Evolution Of The Influenza A Virus Genome During Development Of Oseltamivir Resistance In Vitro, Nicholas Renzette, Daniel R. Caffrey, Konstantin B. Zeldovich, Ping Liu, Glen R. Gallagher, Daniel Aiello, Alyssa J. Porter, Evelyn A. Kurt-Jones, Daniel N. Bolon, Yu-Ping Poh, Jeffrey D. Jensen, Celia A. Schiffer, Timothy F. Kowalik, Robert W. Finberg, Jennifer P. Wang
Celia A. Schiffer
Influenza A virus (IAV) is a major cause of morbidity and mortality throughout the world. Current antiviral therapies include oseltamivir, a neuraminidase inhibitor that prevents the release of nascent viral particles from infected cells. However, the IAV genome can evolve rapidly, and oseltamivir resistance mutations have been detected in numerous clinical samples. Using an in vitro evolution platform and whole-genome population sequencing, we investigated the population genomics of IAV during the development of oseltamivir resistance. Strain A/Brisbane/59/2007 (H1N1) was grown in Madin-Darby canine kidney cells with or without escalating concentrations of oseltamivir over serial passages. Following drug treatment, the H274Y …
Structural Analysis Of Asunaprevir Resistance In Hcv Ns3/4a Protease, Djade Soumana, Akbar Ali, Celia Schiffer
Structural Analysis Of Asunaprevir Resistance In Hcv Ns3/4a Protease, Djade Soumana, Akbar Ali, Celia Schiffer
Celia A. Schiffer
Asunaprevir (ASV), an isoquinoline-based competitive inhibitor targeting the hepatitis C virus (HCV) NS3/4A protease, is very potent in vivo. However, the potency is significantly compromised by the drug resistance mutations R155K and D168A. In this study three crystal structures of ASV and an analogue were determined to analyze the structural basis of drug resistance susceptibility. These structures revealed that ASV makes extensive contacts with Arg155 outside the substrate envelope. Arg155 in turn is stabilized by Asp168, and thus when either residue is mutated, the enzyme's interaction with ASV's P2* isoquinoline is disrupted. Adding a P1-P3 macrocycle to ASV enhances the …
Improving The Resistance Profile Of Hepatitis C Ns3/4a Inhibitors: Dynamic Substrate Envelope Guided Design, Aysegul Ozen, Woody Sherman, Celia Schiffer
Improving The Resistance Profile Of Hepatitis C Ns3/4a Inhibitors: Dynamic Substrate Envelope Guided Design, Aysegul Ozen, Woody Sherman, Celia Schiffer
Celia A. Schiffer
Drug resistance is a principal concern in the treatment of quickly evolving diseases. The viral protease NS3/4A is a primary drug target for the hepatitis C virus (HCV) and is known to evolve resistance mutations in response to drug therapy. At the molecular level, drug resistance reflects a subtle change in the balance of molecular recognition by NS3/4A; the drug resistant protease variants are no longer effectively inhibited by the competitive active site inhibitors but can still process the natural substrates with enough efficiency for viral survival. In previous works we have developed the "substrate envelope" hypothesis, which posits that …
A Sensitive Assay Using A Native Protein Substrate For Screening Hiv-1 Maturation Inhibitors Targeting The Protease Cleavage Site Between The Matrix And Capsid, Sook-Kyung Lee, Nancy Cheng, Emily Hull-Ryde, Marc Potempa, Celia Schiffer, William Janzen, Ronald Swanstrom
A Sensitive Assay Using A Native Protein Substrate For Screening Hiv-1 Maturation Inhibitors Targeting The Protease Cleavage Site Between The Matrix And Capsid, Sook-Kyung Lee, Nancy Cheng, Emily Hull-Ryde, Marc Potempa, Celia Schiffer, William Janzen, Ronald Swanstrom
Celia A. Schiffer
The matrix/capsid processing site in the HIV-1 Gag precursor is likely the most sensitive target to inhibit HIV-1 replication. We have previously shown that modest incomplete processing at the site leads to a complete loss of virion infectivity. In the study presented here, a sensitive assay based on fluorescence polarization that can monitor cleavage at the MA/CA site in the context of the folded protein substrate is described. The substrate, an MA/CA fusion protein, was labeled with the fluorescein-based FlAsH (fluorescein arsenical hairpin) reagent that binds to a tetracysteine motif (CCGPCC) that was introduced within the N-terminal domain of CA. …
Efficient Computation Of Small-Molecule Configurational Binding Entropy And Free Energy Changes By Ensemble Enumeration, Nathaniel Silver, Bracken King, Madhavi Nalam, Hong Cao, Akbar Ali, G. S. Kiran Kumar Reddy, Tariq Rana, Celia Schiffer, Bruce Tidor
Efficient Computation Of Small-Molecule Configurational Binding Entropy And Free Energy Changes By Ensemble Enumeration, Nathaniel Silver, Bracken King, Madhavi Nalam, Hong Cao, Akbar Ali, G. S. Kiran Kumar Reddy, Tariq Rana, Celia Schiffer, Bruce Tidor
Celia A. Schiffer
Here we present a novel, end-point method using the dead-end-elimination and A* algorithms to efficiently and accurately calculate the change in free energy, enthalpy, and configurational entropy of binding for ligand-receptor association reactions. We apply the new approach to the binding of a series of human immunodeficiency virus (HIV-1) protease inhibitors to examine the effect ensemble reranking has on relative accuracy as well as to evaluate the role of the absolute and relative ligand configurational entropy losses upon binding in affinity differences for structurally related inhibitors. Our results suggest that most thermodynamic parameters can be estimated using only a small …
Drug Resistance Conferred By Mutations Outside The Active Site Through Alterations In The Dynamic And Structural Ensemble Of Hiv-1 Protease, Debra Ragland, Ellen Nalivaika, Madhavi Nalam, Kristina Prachanronarong, Hong Cao, Rajintha Bandaranayake, Yufeng Cai, Nese Yilmaz, Celia Schiffer
Drug Resistance Conferred By Mutations Outside The Active Site Through Alterations In The Dynamic And Structural Ensemble Of Hiv-1 Protease, Debra Ragland, Ellen Nalivaika, Madhavi Nalam, Kristina Prachanronarong, Hong Cao, Rajintha Bandaranayake, Yufeng Cai, Nese Yilmaz, Celia Schiffer
Celia A. Schiffer
HIV-1 protease inhibitors are part of the highly active antiretroviral therapy effectively used in the treatment of HIV infection and AIDS. Darunavir (DRV) is the most potent of these inhibitors, soliciting drug resistance only when a complex combination of mutations occur both inside and outside the protease active site. With few exceptions, the role of mutations outside the active site in conferring resistance remains largely elusive. Through a series of DRV-protease complex crystal structures, inhibition assays, and molecular dynamics simulations, we find that single and double site mutations outside the active site often associated with DRV resistance alter the structure …
Testing The Substrate-Envelope Hypothesis With Designed Pairs Of Compounds, Yang Shen, Michael Altman, Akbar Ali, Madhavi Nalam, Hong Cao, Tariq Rana, Celia Schiffer, Bruce Tidor
Testing The Substrate-Envelope Hypothesis With Designed Pairs Of Compounds, Yang Shen, Michael Altman, Akbar Ali, Madhavi Nalam, Hong Cao, Tariq Rana, Celia Schiffer, Bruce Tidor
Celia A. Schiffer
Acquired resistance to therapeutic agents is a significant barrier to the development of clinically effective treatments for diseases in which evolution occurs on clinical time scales, frequently arising from target mutations. We previously reported a general strategy to design effective inhibitors for rapidly mutating enzyme targets, which we demonstrated for HIV-1 protease inhibition [Altman et al. J. Am. Chem. Soc. 2008, 130, 6099-6113]. Specifically, we developed a computational inverse design procedure with the added constraint that designed inhibitors bind entirely inside the substrate envelope, a consensus volume occupied by natural substrates. The rationale for the substrate-envelope constraint is that it …
Influenza Virus Drug Resistance: A Time-Sampled Population Genetics Perspective, Matthieu Foll, Yu Poh, Nicholas Renzette, Anna Admetlla, Claudia Bank, Hyunjin Shim, Anna Malaspinas, Gregory Ewing, Ping Liu, Daniel Wegmann, Daniel Caffrey, Konstantin Zeldovich, Daniel Bolon, Jennifer Wang, Timothy Kowalik, Celia Schiffer, Robert Finberg, Jeffrey Jensen
Influenza Virus Drug Resistance: A Time-Sampled Population Genetics Perspective, Matthieu Foll, Yu Poh, Nicholas Renzette, Anna Admetlla, Claudia Bank, Hyunjin Shim, Anna Malaspinas, Gregory Ewing, Ping Liu, Daniel Wegmann, Daniel Caffrey, Konstantin Zeldovich, Daniel Bolon, Jennifer Wang, Timothy Kowalik, Celia Schiffer, Robert Finberg, Jeffrey Jensen
Celia A. Schiffer
The challenge of distinguishing genetic drift from selection remains a central focus of population genetics. Time-sampled data may provide a powerful tool for distinguishing these processes, and we here propose approximate Bayesian, maximum likelihood, and analytical methods for the inference of demography and selection from time course data. Utilizing these novel statistical and computational tools, we evaluate whole-genome datasets of an influenza A H1N1 strain in the presence and absence of oseltamivir (an inhibitor of neuraminidase) collected at thirteen time points. Results reveal a striking consistency amongst the three estimation procedures developed, showing strongly increased selection pressure in the presence …
Development Of A Novel Screening Strategy Designed To Discover A New Class Of Hiv Drugs, Nancy Cheng, Sook-Kyung Lee, P. Donover, Mel Reichman, Celia Schiffer, Emily Hull-Ryde, Ronald Swanstrom, William Janzen
Development Of A Novel Screening Strategy Designed To Discover A New Class Of Hiv Drugs, Nancy Cheng, Sook-Kyung Lee, P. Donover, Mel Reichman, Celia Schiffer, Emily Hull-Ryde, Ronald Swanstrom, William Janzen
Celia A. Schiffer
Current antiretroviral treatments target multiple pathways important for human immunodeficiency virus (HIV) multiplication, including viral entry, synthesis and integration of the DNA provirus, and the processing of viral polyprotein precursors. However, HIV is becoming increasingly resistant to these "combination therapies." Recent findings show that inhibition of HIV Gag protein cleavage into its two structural proteins, matrix (MA) and capsid (CA), has a devastating effect on viral production, revealing a potential new target class for HIV treatment. Unlike the widely used HIV protease inhibitors, this new class of inhibitor would target the substrate, not the protease enzyme itself. This approach offers …
Crystal Structures Of Human Ctbp In Complex With Substrate Mtob Reveal Active Site Features Useful For Inhibitor Design, Brendan Hilbert, Steven Grossman, Celia Schiffer, William Royer
Crystal Structures Of Human Ctbp In Complex With Substrate Mtob Reveal Active Site Features Useful For Inhibitor Design, Brendan Hilbert, Steven Grossman, Celia Schiffer, William Royer
Celia A. Schiffer
The oncogenic corepressors C-terminal Binding Protein (CtBP) 1 and 2 harbor regulatory d-isomer specific 2-hydroxyacid dehydrogenase (d2-HDH) domains. 4-Methylthio 2-oxobutyric acid (MTOB) exhibits substrate inhibition and can interfere with CtBP oncogenic activity in cell culture and mice. Crystal structures of human CtBP1 and CtBP2 in complex with MTOB and NAD(+) revealed two key features: a conserved tryptophan that likely contributes to substrate specificity and a hydrophilic cavity that links MTOB with an NAD(+) phosphate. Neither feature is present in other d2-HDH enzymes. These structures thus offer key opportunities for the development of highly selective anti-neoplastic CtBP inhibitors. Elsevier B.V. All …