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Celia A. Schiffer

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Articles 31 - 60 of 115

Full-Text Articles in Biochemistry, Biophysics, and Structural Biology

Drug Resistance Mutations Alter Dynamics Of Inhibitor-Bound Hiv-1 Protease, Yufeng Cai, Wazo Myint, Janet Paulsen, Celia Schiffer, Rieko Ishima, Nese Yilmaz Jan 2015

Drug Resistance Mutations Alter Dynamics Of Inhibitor-Bound Hiv-1 Protease, Yufeng Cai, Wazo Myint, Janet Paulsen, Celia Schiffer, Rieko Ishima, Nese Yilmaz

Celia A. Schiffer

Under the selective pressure of therapy, HIV-1 protease mutants resistant to inhibitors evolve to confer drug resistance. Such mutations can impact both the dynamics and structures of the bound and unbound forms of the enzyme. Flap+ is a multidrug-resistant variant of HIV-1 protease with a combination of primary and secondary resistance mutations (L10I, G48V, I54V, V82A) and a strikingly altered thermodynamic profile for darunavir (DRV) binding relative to the wild-type protease. We elucidated the impact of these mutations on protein dynamics in the DRV-bound state using molecular dynamics simulations and NMR relaxation experiments. Both methods concur in that the conformational …

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Hiv-1 Protease-Substrate Coevolution In Nelfinavir Resistance, Madhavi Kolli, Aysegul Ozen, Nese Yilmaz, Celia Schiffer Jan 2015

Hiv-1 Protease-Substrate Coevolution In Nelfinavir Resistance, Madhavi Kolli, Aysegul Ozen, Nese Yilmaz, Celia Schiffer

Celia A. Schiffer

Resistance to various human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) challenges the effectiveness of therapies in treating HIV-1-infected individuals and AIDS patients. The virus accumulates mutations within the protease (PR) that render the PIs less potent. Occasionally, Gag sequences also coevolve with mutations at PR cleavage sites contributing to drug resistance. In this study, we investigated the structural basis of coevolution of the p1-p6 cleavage site with the nelfinavir (NFV) resistance D30N/N88D protease mutations by determining crystal structures of wild-type and NFV-resistant HIV-1 protease in complex with p1-p6 substrate peptide variants with L449F and/or S451N. Alterations of residue …

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Prototypical Recombinant Multi-Protease Inhibitor Resistant Infectious Molecular Clones Of Human Immunodeficiency Virus Type-1, Vici Varghese, Yumi Mitsuya, W. Jeffrey Fessel, Tommy F. Liu, George Melikian, David Katzenstein, Celia Schiffer, Susan Holmes, Robert Shafer Jan 2015

Prototypical Recombinant Multi-Protease Inhibitor Resistant Infectious Molecular Clones Of Human Immunodeficiency Virus Type-1, Vici Varghese, Yumi Mitsuya, W. Jeffrey Fessel, Tommy F. Liu, George Melikian, David Katzenstein, Celia Schiffer, Susan Holmes, Robert Shafer

Celia A. Schiffer

The many genetic manifestations of HIV-1 protease inhibitor (PI) resistance present challenges to research into the mechanisms of PI-resistance and the assessment of new PIs. To address these challenges, we created a panel of recombinant multi-PI resistant infectious molecular clones designed to represent the spectrum of clinically relevant multi-PI resistant viruses. To assess the representativeness of this panel, we examined the sequences of the panel's viruses in the context of a correlation network of PI-resistance amino acid substitutions in sequences from more than 10,000 patients. The panel of recombinant infectious molecular clones comprised 29 of 41 study-defined PI-resistance amino acid …

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Substrate Envelope-Designed Potent Hiv-1 Protease Inhibitors To Avoid Drug Resistance, Madhavi Nalam, Akbar Ali, G. S. Kiran Kumar Reddy, Hong Cao, Saima Anjum, Michael Altman, Nese Yilmaz, Bruce Tidor, Tariq Rana, Celia Schiffer Jan 2015

Substrate Envelope-Designed Potent Hiv-1 Protease Inhibitors To Avoid Drug Resistance, Madhavi Nalam, Akbar Ali, G. S. Kiran Kumar Reddy, Hong Cao, Saima Anjum, Michael Altman, Nese Yilmaz, Bruce Tidor, Tariq Rana, Celia Schiffer

Celia A. Schiffer

The rapid evolution of HIV under selective drug pressure has led to multidrug resistant (MDR) strains that evade standard therapies. We designed highly potent HIV-1 protease inhibitors (PIs) using the substrate envelope model, which confines inhibitors within the consensus volume of natural substrates, providing inhibitors less susceptible to resistance because a mutation affecting such inhibitors will simultaneously affect viral substrate processing. The designed PIs share a common chemical scaffold but utilize various moieties that optimally fill the substrate envelope, as confirmed by crystal structures. The designed PIs retain robust binding to MDR protease variants and display exceptional antiviral potencies against …

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The Interdomain Interface In Bifunctional Enzyme Protein 3/4a (Ns3/4a) Regulates Protease And Helicase Activities, Cihan Aydin, Sourav Mukherjee, Alicia Hanson, David Frick, Celia Schiffer Nov 2013

The Interdomain Interface In Bifunctional Enzyme Protein 3/4a (Ns3/4a) Regulates Protease And Helicase Activities, Cihan Aydin, Sourav Mukherjee, Alicia Hanson, David Frick, Celia Schiffer

Celia A. Schiffer

Hepatitis C (HCV) protein 3/4A (NS3/4A) is a bifunctional enzyme comprising two separate domains with protease and helicase activities, which are essential for viral propagation. Both domains are stable and have enzymatic activity separately, and the relevance and implications of having protease and helicase together as a single protein remains to be explored. Altered in vitro activities of isolated domains compared with the full-length NS3/4A protein suggest the existence of interdomain communication. The molecular mechanism and extent of this communication was investigated by probing the domain-domain interface observed in HCV NS3/4A crystal structures. We found in molecular dynamics simulations that …

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Cooperative Effects Of Drug-Resistance Mutations In The Flap Region Of Hiv-1 Protease, Jennifer Foulkes-Murzycki, Christina Rosi, Nese Yilmaz, Robert Shafer, Celia Schiffer Jul 2013

Cooperative Effects Of Drug-Resistance Mutations In The Flap Region Of Hiv-1 Protease, Jennifer Foulkes-Murzycki, Christina Rosi, Nese Yilmaz, Robert Shafer, Celia Schiffer

Celia A. Schiffer

Understanding the interdependence of multiple mutations in conferring drug resistance is crucial to the development of novel and robust inhibitors. As HIV-1 protease continues to adapt and evade inhibitors while still maintaining the ability to specifically recognize and efficiently cleave its substrates, the problem of drug resistance has become more complicated. Under the selective pressure of therapy, correlated mutations accumulate throughout the enzyme to compromise inhibitor binding, but characterizing their energetic interdependency is not straightforward. A particular drug resistant variant (L10I/G48V/I54V/V82A) displays extreme entropy-enthalpy compensation relative to wild-type enzyme but a similar variant (L10I/G48V/I54A/V82A) does not. Individual mutations of sites …

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Structural And Thermodynamic Basis Of Amprenavir/Darunavir And Atazanavir Resistance In Hiv-1 Protease With Mutations At Residue 50, Seema Mittal, Rajintha Bandaranayake, Nancy King, Moses Prabu-Jeyabalan, Madhavi Nalam, Ellen Nalivaika, Nese Yilmaz, Celia Schiffer Jul 2013

Structural And Thermodynamic Basis Of Amprenavir/Darunavir And Atazanavir Resistance In Hiv-1 Protease With Mutations At Residue 50, Seema Mittal, Rajintha Bandaranayake, Nancy King, Moses Prabu-Jeyabalan, Madhavi Nalam, Ellen Nalivaika, Nese Yilmaz, Celia Schiffer

Celia A. Schiffer

Drug resistance occurs through a series of subtle changes that maintain substrate recognition but no longer permit inhibitor binding. In HIV-1 protease, mutations at I50 are associated with such subtle changes that confer differential resistance to specific inhibitors. Residue I50 is located at the protease flap tips, closing the active site upon ligand binding. Under selective drug pressure, I50V/L substitutions emerge in patients, compromising drug susceptibility and leading to treatment failure. The I50V substitution is often associated with amprenavir (APV) and darunavir (DRV) resistance, while the I50L substitution is observed in patients failing atazanavir (ATV) therapy. To explain how APV, …

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Crystal Structure Of The Dna Cytosine Deaminase Apobec3f: The Catalytically Active And Hiv-1 Vif-Binding Domain, Markus-Frederik Bohn, Shivender Shandilya, John Albin, Takahide Kouno, Brett Anderson, Rebecca Mcdougle, Michael Carpenter, Anurag Rathore, Leah Evans, Ahkillah Davis, Jingying Zhang, Yongjian Lu, Mohan Somasundaran, Hiroshi Matsuo, Reuben Harris, Celia Schiffer Jun 2013

Crystal Structure Of The Dna Cytosine Deaminase Apobec3f: The Catalytically Active And Hiv-1 Vif-Binding Domain, Markus-Frederik Bohn, Shivender Shandilya, John Albin, Takahide Kouno, Brett Anderson, Rebecca Mcdougle, Michael Carpenter, Anurag Rathore, Leah Evans, Ahkillah Davis, Jingying Zhang, Yongjian Lu, Mohan Somasundaran, Hiroshi Matsuo, Reuben Harris, Celia Schiffer

Celia A. Schiffer

Human APOBEC3F is an antiretroviral single-strand DNA cytosine deaminase, susceptible to degradation by the HIV-1 protein Vif. In this study the crystal structure of the HIV Vif binding, catalytically active, C-terminal domain of APOBEC3F (A3F-CTD) was determined. The A3F-CTD shares structural motifs with portions of APOBEC3G-CTD, APOBEC3C, and APOBEC2. Residues identified to be critical for Vif-dependent degradation of APOBEC3F all fit within a predominantly negatively charged contiguous region on the surface of A3F-CTD. Specific sequence motifs, previously shown to play a role in Vif susceptibility and virion encapsidation, are conserved across APOBEC3s and between APOBEC3s and HIV-1 Vif. In this …

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Quantitative Comparison Of Errors In 15n Transverse Relaxation Rates Measured Using Various Cpmg Phasing Schemes, Wazo Myint, Yufeng Cai, Celia Schiffer, Rieko Ishima Oct 2012

Quantitative Comparison Of Errors In 15n Transverse Relaxation Rates Measured Using Various Cpmg Phasing Schemes, Wazo Myint, Yufeng Cai, Celia Schiffer, Rieko Ishima

Celia A. Schiffer

Nitrogen-15 Carr-Purcell-Meiboom-Gill (CPMG) transverse relaxation experiment are widely used to characterize protein backbone dynamics and chemical exchange parameters. Although an accurate value of the transverse relaxation rate, R(2), is needed for accurate characterization of dynamics, the uncertainty in the R(2) value depends on the experimental settings and the details of the data analysis itself. Here, we present an analysis of the impact of CPMG pulse phase alternation on the accuracy of the (15)N CPMG R(2). Our simulations show that R(2) can be obtained accurately for a relatively wide spectral width, either using the conventional phase cycle or using phase alternation …

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Extreme Entropy-Enthalpy Compensation In A Drug-Resistant Variant Of Hiv-1 Protease, Nancy King, Moses Prabu-Jeyabalan, Rajintha Bandaranayake, Madhavi Nalam, Ellen Nalivaika, Aysegul Ozen, Turkan Haliloglu, Nese Yilmaz, Celia Schiffer Oct 2012

Extreme Entropy-Enthalpy Compensation In A Drug-Resistant Variant Of Hiv-1 Protease, Nancy King, Moses Prabu-Jeyabalan, Rajintha Bandaranayake, Madhavi Nalam, Ellen Nalivaika, Aysegul Ozen, Turkan Haliloglu, Nese Yilmaz, Celia Schiffer

Celia A. Schiffer

The development of HIV-1 protease inhibitors has been the historic paradigm of rational structure-based drug design, where structural and thermodynamic analyses have assisted in the discovery of novel inhibitors. While the total enthalpy and entropy change upon binding determine the affinity, often the thermodynamics are considered in terms of inhibitor properties only. In the current study, profound changes are observed in the binding thermodynamics of a drug-resistant variant compared to wild-type HIV-1 protease, irrespective of the inhibitor bound. This variant (Flap+) has a combination of flap and active site mutations and exhibits extremely large entropy-enthalpy compensation compared to wild-type protease, …

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Decomposing The Energetic Impact Of Drug-Resistant Mutations: The Example Of Hiv-1 Protease-Drv Binding, Yufeng Cai, Celia Schiffer Oct 2012

Decomposing The Energetic Impact Of Drug-Resistant Mutations: The Example Of Hiv-1 Protease-Drv Binding, Yufeng Cai, Celia Schiffer

Celia A. Schiffer

HIV-1 protease is a major drug target for AIDS therapy. With the appearance of drug-resistant HIV-1 protease variants, understanding the mechanism of drug resistance becomes critical for rational drug design. Computational methods can provide more details about inhibitor-protease binding than crystallography and isothermal titration calorimetry. The latest FDA-approved HIV-1 protease inhibitor is Darunavir (DRV). Herein, each DRV atom is evaluated by free energy component analysis for its contribution to the binding affinity with wild-type protease and ACT, a drug-resistant variant. This information can contribute to the rational design of new HIV-1 protease inhibitors.

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Mass Spectrometry Tools For Analysis Of Intermolecular Interactions, Jared Auclair, Mohan Somasundaran, Karin Green, James Evans, Celia Schiffer, Dagmar Ringe, Gregory Petsko, Jeffrey Agar Oct 2012

Mass Spectrometry Tools For Analysis Of Intermolecular Interactions, Jared Auclair, Mohan Somasundaran, Karin Green, James Evans, Celia Schiffer, Dagmar Ringe, Gregory Petsko, Jeffrey Agar

Celia A. Schiffer

The small quantities of protein required for mass spectrometry (MS) make it a powerful tool to detect binding (protein-protein, protein-small molecule, etc.) of proteins that are difficult to express in large quantities, as is the case for many intrinsically disordered proteins. Chemical cross-linking, proteolysis, and MS analysis, combined, are a powerful tool for the identification of binding domains. Here, we present a traditional approach to determine protein-protein interaction binding sites using heavy water ((18)O) as a label. This technique is relatively inexpensive and can be performed on any mass spectrometer without specialized software.

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The Molecular Basis Of Drug Resistance Against Hepatitis C Virus Ns3/4a Protease Inhibitors, Keith Romano, Akbar Ali, Cihan Aydin, Djade Soumana, Aysegul Ozen, Laura Deveau, Casey Silver, Hong Cao, Alicia Newton, Christos Petropoulos, Wei Huang, Celia Schiffer Oct 2012

The Molecular Basis Of Drug Resistance Against Hepatitis C Virus Ns3/4a Protease Inhibitors, Keith Romano, Akbar Ali, Cihan Aydin, Djade Soumana, Aysegul Ozen, Laura Deveau, Casey Silver, Hong Cao, Alicia Newton, Christos Petropoulos, Wei Huang, Celia Schiffer

Celia A. Schiffer

Hepatitis C virus (HCV) infects over 170 million people worldwide and is the leading cause of chronic liver diseases, including cirrhosis, liver failure, and liver cancer. Available antiviral therapies cause severe side effects and are effective only for a subset of patients, though treatment outcomes have recently been improved by the combination therapy now including boceprevir and telaprevir, which inhibit the viral NS3/4A protease. Despite extensive efforts to develop more potent next-generation protease inhibitors, however, the long-term efficacy of this drug class is challenged by the rapid emergence of resistance. Single-site mutations at protease residues R155, A156 and D168 confer …

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Context Surrounding Processing Sites Is Crucial In Determining Cleavage Rate Of A Subset Of Processing Sites In Hiv-1 Gag And Gag-Pro-Pol Polyprotein Precursors By Viral Protease, Sook-Kyung Lee, Marc Potempa, Madhavi Kolli, Aysegul Ozen, Celia Schiffer, Ronald Swanstrom Oct 2012

Context Surrounding Processing Sites Is Crucial In Determining Cleavage Rate Of A Subset Of Processing Sites In Hiv-1 Gag And Gag-Pro-Pol Polyprotein Precursors By Viral Protease, Sook-Kyung Lee, Marc Potempa, Madhavi Kolli, Aysegul Ozen, Celia Schiffer, Ronald Swanstrom

Celia A. Schiffer

Processing of the human immunodeficiency virus type 1 (HIV-1) Gag and Gag-Pro-Pol polyproteins by the HIV-1 protease (PR) is essential for the production of infectious particles. However, the determinants governing the rates of processing of these substrates are not clearly understood. We studied the effect of substrate context on processing by utilizing a novel protease assay in which a substrate containing HIV-1 matrix (MA) and the N-terminal domain of capsid (CA) is labeled with a FlAsH (fluorescein arsenical hairpin) reagent. When the seven cleavage sites within the Gag and Gag-Pro-Pol polyproteins were placed at the MA/CA site, the rates of …

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Structural, Kinetic, And Thermodynamic Studies Of Specificity Designed Hiv-1 Protease, Oscar Alvizo, Seema Mittal, Stephen Mayo, Celia Schiffer Oct 2012

Structural, Kinetic, And Thermodynamic Studies Of Specificity Designed Hiv-1 Protease, Oscar Alvizo, Seema Mittal, Stephen Mayo, Celia Schiffer

Celia A. Schiffer

HIV-1 protease recognizes and cleaves more than 12 different substrates leading to viral maturation. While these substrates share no conserved motif, they are specifically selected for and cleaved by protease during viral life cycle. Drug resistant mutations evolve within the protease that compromise inhibitor binding but allow the continued recognition of all these substrates. While the substrate envelope defines a general shape for substrate recognition, successfully predicting the determinants of substrate binding specificity would provide additional insights into the mechanism of altered molecular recognition in resistant proteases. We designed a variant of HIV protease with altered specificity using positive computational …

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Methyl- And Normal-Cytosine Deamination By The Foreign Dna Restriction Enzyme Apobec3a, Michael Carpenter, Ming Li, Anurag Rathore, Lela Lackey, Emily Law, Allison Land, Brandon Leonard, Shivender Shandilya, Markus-Frederik Bohn, Celia Schiffer, William Brown, Reuben Harris Oct 2012

Methyl- And Normal-Cytosine Deamination By The Foreign Dna Restriction Enzyme Apobec3a, Michael Carpenter, Ming Li, Anurag Rathore, Lela Lackey, Emily Law, Allison Land, Brandon Leonard, Shivender Shandilya, Markus-Frederik Bohn, Celia Schiffer, William Brown, Reuben Harris

Celia A. Schiffer

Multiple studies have indicated that the TET oxidases and, more controversially, the AID/APOBEC deaminases have the capacity to convert genomic DNA 5-methylcytosine (MeC) into altered nucleobases that provoke excision repair and culminate in the replacement of the original MeC with a normal cytosine (C). We show that human APOBEC3A (A3A) efficiently deaminates both MeC to thymine (T) and normal C to uracil (U) in single-stranded DNA substrates. In comparison, the related enzyme APOBEC3G (A3G) has undetectable MeC-to-T activity and 10-fold less C-to-U activity. Upon 100-fold induction of endogenous A3A by interferon, the MeC status of bulk chromosomal DNA is unaltered …

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Design, Synthesis, And Biological And Structural Evaluations Of Novel Hiv-1 Protease Inhibitors To Combat Drug Resistance, Maloy Parai, David Huggins, Hong Cao, Madhavi Nalam, Akbar Ali, Celia Schiffer, Bruce Tidor, Tariq Rana Oct 2012

Design, Synthesis, And Biological And Structural Evaluations Of Novel Hiv-1 Protease Inhibitors To Combat Drug Resistance, Maloy Parai, David Huggins, Hong Cao, Madhavi Nalam, Akbar Ali, Celia Schiffer, Bruce Tidor, Tariq Rana

Celia A. Schiffer

A series of new HIV-1 protease inhibitors (PIs) were designed using a general strategy that combines computational structure-based design with substrate-envelope constraints. The PIs incorporate various alcohol-derived P2 carbamates with acyclic and cyclic heteroatomic functionalities into the (R)-hydroxyethylamine isostere. Most of the new PIs show potent binding affinities against wild-type HIV-1 protease and three multidrug resistant (MDR) variants. In particular, inhibitors containing the 2,2-dichloroacetamide, pyrrolidinone, imidazolidinone, and oxazolidinone moieties at P2 are the most potent with K(i) values in the picomolar range. Several new PIs exhibit nanomolar antiviral potencies against patient-derived wild-type viruses from HIV-1 clades A, B, and C …

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Hydrophobic Core Flexibility Modulates Enzyme Activity In Hiv-1 Protease, Seema Mittal, Yufeng Cai, Madhavi Nalam, Daniel Bolon, Celia Schiffer Oct 2012

Hydrophobic Core Flexibility Modulates Enzyme Activity In Hiv-1 Protease, Seema Mittal, Yufeng Cai, Madhavi Nalam, Daniel Bolon, Celia Schiffer

Celia A. Schiffer

Human immunodeficiency virus Type-1 (HIV-1) protease is crucial for viral maturation and infectivity. Studies of protease dynamics suggest that the rearrangement of the hydrophobic core is essential for enzyme activity. Many mutations in the hydrophobic core are also associated with drug resistance and may modulate the core flexibility. To test the role of flexibility in protease activity, pairs of cysteines were introduced at the interfaces of flexible regions remote from the active site. Disulfide bond formation was confirmed by crystal structures and by alkylation of free cysteines and mass spectrometry. Oxidized and reduced crystal structures of these variants show the …

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First-In-Class Small Molecule Inhibitors Of The Single-Strand Dna Cytosine Deaminase Apobec3g, Ming Li, Shivender Shandilya, Michael Carpenter, Anurag Rathore, William Brown, Angela Perkins, Daniel Harki, Jonathan Solberg, Derek Hook, Krishan Pandey, Michael Parniak, Jeffrey Johnson, Nevan Krogan, Mohan Somasundaran, Akbar Ali, Celia Schiffer, Reuben Harris Sep 2012

First-In-Class Small Molecule Inhibitors Of The Single-Strand Dna Cytosine Deaminase Apobec3g, Ming Li, Shivender Shandilya, Michael Carpenter, Anurag Rathore, William Brown, Angela Perkins, Daniel Harki, Jonathan Solberg, Derek Hook, Krishan Pandey, Michael Parniak, Jeffrey Johnson, Nevan Krogan, Mohan Somasundaran, Akbar Ali, Celia Schiffer, Reuben Harris

Celia A. Schiffer

APOBEC3G is a single-stranded DNA cytosine deaminase that comprises part of the innate immune response to viruses and transposons. Although APOBEC3G is the prototype for understanding the larger mammalian polynucleotide deaminase family, no specific chemical inhibitors exist to modulate its activity. High-throughput screening identified 34 compounds that inhibit APOBEC3G catalytic activity. Twenty of 34 small molecules contained catechol moieties, which are known to be sulfhydryl reactive following oxidation to the orthoquinone. Located proximal to the active site, C321 was identified as the binding site for the inhibitors by a combination of mutational screening, structural analysis, and mass spectrometry. Bulkier substitutions …

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Structural Insights Into Neuronal K+ Channel-Calmodulin Complexes, Karen Mruk, Shivender Shandilya, Robert Blaustein, Celia Schiffer, William Kobertz Sep 2012

Structural Insights Into Neuronal K+ Channel-Calmodulin Complexes, Karen Mruk, Shivender Shandilya, Robert Blaustein, Celia Schiffer, William Kobertz

Celia A. Schiffer

Calmodulin (CaM) is a ubiquitous intracellular calcium sensor that directly binds to and modulates a wide variety of ion channels. Despite the large repository of high-resolution structures of CaM bound to peptide fragments derived from ion channels, there is no structural information about CaM bound to a fully folded ion channel at the plasma membrane. To determine the location of CaM docked to a functioning KCNQ K(+) channel, we developed an intracellular tethered blocker approach to measure distances between CaM residues and the ion-conducting pathway. Combining these distance restraints with structural bioinformatics, we generated an archetypal quaternary structural model of …

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Crystal Structure Of Human Thymidylate Synthase: A Structural Mechanism For Guiding Substrates Into The Active Site, Celia Schiffer, Ian Clifton, V. Jo Davisson, Daniel Santi, Robert Stroud Nov 2011

Crystal Structure Of Human Thymidylate Synthase: A Structural Mechanism For Guiding Substrates Into The Active Site, Celia Schiffer, Ian Clifton, V. Jo Davisson, Daniel Santi, Robert Stroud

Celia A. Schiffer

The crystal structure of human thymidylate synthase, a target for anti-cancer drugs, is determined to 3.0 A resolution and refined to a crystallographic residual of 17.8%. The structure implicates the enzyme in a mechanism for facilitating the docking of substrates into the active site. This mechanism involves a twist of approximately 180 degrees of the active site loop, pivoted around the neighboring residues 184 and 204, and implicates ordering of external, eukaryote specific loops along with the well-characterized closure of the active site upon substrate binding. The highly conserved, but eukaryote-specific insertion of twelve residues 90-101 (h117-128), and of eight …

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Molecular Basis For Drug Resistance In Hiv-1 Protease, Akbar Ali, Rajintha M. Bandaranayake, Yufeng Cai, Nancy M. King, Madhavi Kolli, Seema Mittal, Jennifer E. Foulkes-Murzycki, Madhavi N. L. Nalam, Ellen A. Nalivaika, Aysegul Ozen, Moses Prabu-Jeyabalan, Kelly Thayer, Celia A. Schiffer Nov 2011

Molecular Basis For Drug Resistance In Hiv-1 Protease, Akbar Ali, Rajintha M. Bandaranayake, Yufeng Cai, Nancy M. King, Madhavi Kolli, Seema Mittal, Jennifer E. Foulkes-Murzycki, Madhavi N. L. Nalam, Ellen A. Nalivaika, Aysegul Ozen, Moses Prabu-Jeyabalan, Kelly Thayer, Celia A. Schiffer

Celia A. Schiffer

HIV-1 protease is one of the major antiviral targets in the treatment of patients infected with HIV-1. The nine FDA approved HIV-1 protease inhibitors were developed with extensive use of structure-based drug design, thus the atomic details of how the inhibitors bind are well characterized. From this structural understanding the molecular basis for drug resistance in HIV-1 protease can be elucidated. Selected mutations in response to therapy and diversity between clades in HIV-1 protease have altered the shape of the active site, potentially altered the dynamics and even altered the sequence of the cleavage sites in the Gag polyprotein. All …

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Evaluation Of The Substrate Envelope Hypothesis For Inhibitors Of Hiv-1 Protease, Sripriya Chellappan, Visvaldas Kairys, Miguel Fernandes, Celia Schiffer, Michael Gilson Nov 2011

Evaluation Of The Substrate Envelope Hypothesis For Inhibitors Of Hiv-1 Protease, Sripriya Chellappan, Visvaldas Kairys, Miguel Fernandes, Celia Schiffer, Michael Gilson

Celia A. Schiffer

Crystallographic data show that various substrates of HIV protease occupy a remarkably uniform region within the binding site; this region has been termed the substrate envelope. It has been suggested that an inhibitor that fits within the substrate envelope should tend to evade viral resistance because a protease mutation that reduces the affinity of the inhibitor will also tend to reduce the affinity of substrate, and will hence decrease the activity of the enzyme. Accordingly, inhibitors that fit the substrate envelope better should be less susceptible to clinically observed resistant mutations, since these must also allow substrates to bind. The …

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Expression, Purification, And Characterization Of Thymidylate Synthase From Lactococcus Lactis, Patricia Greene, Pak-Lam Yu, Jia Zhao, Celia Schiffer, Daniel Santi Nov 2011

Expression, Purification, And Characterization Of Thymidylate Synthase From Lactococcus Lactis, Patricia Greene, Pak-Lam Yu, Jia Zhao, Celia Schiffer, Daniel Santi

Celia A. Schiffer

The thymidylate synthase (TS) gene from Lactococcus lactis has been highly expressed in Escherichia coli. The TS protein was purified by sequential chromatography on Q-Sepharose and phenyl-Sepharose. Six grams of cell pellet yielded 140 mg of homogeneous TS. TS is a highly conserved enzyme, and several of the conserved amino acid residues that have been implicated in catalytic function are altered in L. lactis TS. By use of a 3-dimensional homology model, we have predicted covariant changes that might compensate for these differences. With the large amounts of L. lactis TS now available, studies can be pursued to understand the …

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Investigations Of Peptide Hydration Using Nmr And Molecular Dynamics Simulations: A Study Of Effects Of Water On The Conformation And Dynamics Of Antamanide, Jeffrey Peng, Celia Schiffer, Ping Xu, Wilfred Van Gunsteren, Richard Ernst Nov 2011

Investigations Of Peptide Hydration Using Nmr And Molecular Dynamics Simulations: A Study Of Effects Of Water On The Conformation And Dynamics Of Antamanide, Jeffrey Peng, Celia Schiffer, Ping Xu, Wilfred Van Gunsteren, Richard Ernst

Celia A. Schiffer

The influence of water binding on the conformational dynamics of the cyclic decapeptide antamanide dissolved in the model lipophilic environment chloroform is investigated by NMR relaxation measurements. The water-peptide complex has a lifetime of 35 mgrs at 250 K, which is longer than typical lifetimes of water-peptide complexes reported in aqueous solution. In addition, there is a rapid intracomplex mobility that probably involves librational motions of the bound water or water molecules hopping between different binding sites. Water binding restricts the flexibility of antamanide. The experimental findings are compared with GROMOS molecular dynamics simulations of antamanide with up to eight …

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Discovery Of Hiv-1 Protease Inhibitors With Picomolar Affinities Incorporating N-Aryl-Oxazolidinone-5-Carboxamides As Novel P2 Ligands, Akbar Ali, G. S. Kiran Kumar Reddy, Hong Cao, Saima Anjum, Madhavi Nalam, Celia Schiffer, Tariq Rana Nov 2011

Discovery Of Hiv-1 Protease Inhibitors With Picomolar Affinities Incorporating N-Aryl-Oxazolidinone-5-Carboxamides As Novel P2 Ligands, Akbar Ali, G. S. Kiran Kumar Reddy, Hong Cao, Saima Anjum, Madhavi Nalam, Celia Schiffer, Tariq Rana

Celia A. Schiffer

Here, we describe the design, synthesis, and biological evaluation of novel HIV-1 protease inhibitors incorporating N-phenyloxazolidinone-5-carboxamides into the (hydroxyethylamino)sulfonamide scaffold as P2 ligands. Series of inhibitors with variations at the P2 phenyloxazolidinone and the P2' phenylsulfonamide moieties were synthesized. Compounds with the (S)-enantiomer of substituted phenyloxazolidinones at P2 show highly potent inhibitory activities against HIV-1 protease. The inhibitors possessing 3-acetyl, 4-acetyl, and 3-trifluoromethyl groups at the phenyl ring of the oxazolidinone fragment are the most potent in each series, with K(i) values in the low picomolar (pM) range. The electron-donating groups 4-methoxy and 1,3-dioxolane are preferred at P2' phenyl ring, …

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The Role Of Protein-Solvent Interactions In Protein Unfolding, Celia Schiffer, Volker Dötsch Nov 2011

The Role Of Protein-Solvent Interactions In Protein Unfolding, Celia Schiffer, Volker Dötsch

Celia A. Schiffer

Protein unfolding occurs when the balance of forces between the protein's interaction with itself and the protein's interaction with its environment is disrupted. The disruption of this balance of forces may be as simple as a perturbance of the normal water structure around the protein. A decrease in the normal water-water interaction will result in an increase in the relative interaction of water with the protein. An increase in the number of interactions between water and the protein may initiate a protein's unfolding. This model for protein unfolding is supported by a range of recent experimental and computational data.

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Association Of A Novel Human Immunodeficiency Virus Type 1 Protease Substrate Cleft Mutation, L23i, With Protease Inhibitor Therapy And In Vitro Drug Resistance, Elizabeth Johnston, Mark Winters, Soo-Yon Rhee, Thomas Merigan, Celia Schiffer, Robert Shafer Nov 2011

Association Of A Novel Human Immunodeficiency Virus Type 1 Protease Substrate Cleft Mutation, L23i, With Protease Inhibitor Therapy And In Vitro Drug Resistance, Elizabeth Johnston, Mark Winters, Soo-Yon Rhee, Thomas Merigan, Celia Schiffer, Robert Shafer

Celia A. Schiffer

We observed a previously uncharacterized mutation in the protease substrate cleft, L23I, in 31 of 4,303 persons undergoing human immunodeficiency virus type 1 genotypic resistance testing. In combination with V82I, L23I was associated with a sevenfold reduction in nelfinavir susceptibility and a decrease in replication capacity. In combination with other drug resistance mutations, L23I was associated with multidrug resistance and a compensatory increase in replication capacity.

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Co-Evolution Of Nelfinavir-Resistant Hiv-1 Protease And The P1-P6 Substrate, Madhavi Kolli, Stephane Lastere, Celia Schiffer Nov 2011

Co-Evolution Of Nelfinavir-Resistant Hiv-1 Protease And The P1-P6 Substrate, Madhavi Kolli, Stephane Lastere, Celia Schiffer

Celia A. Schiffer

The selective pressure of the competitive protease inhibitors causes both HIV-1 protease and occasionally its substrates to evolve drug resistance. We hypothesize that this occurs particularly in substrates that protrude beyond the substrate envelope and contact residues that mutate in response to a particular protease inhibitor. To validate this hypothesis, we analyzed substrate and protease sequences for covariation. Using the chi2 test, we show a positive correlation between the nelfinavir-resistant D30N/N88D protease mutations and mutations at the p1-p6 cleavage site as compared to the other cleavage sites. Both nelfinavir and the substrate p1-p6 protrude beyond the substrate envelope and contact …

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Reca Dimers Serve As A Functional Unit For Assembly Of Active Nucleoprotein Filaments, Anthony Forget, Michelle Kudron, Dharia Mcgrew, Melissa Calmann, Celia Schiffer, Kendall Knight Nov 2011

Reca Dimers Serve As A Functional Unit For Assembly Of Active Nucleoprotein Filaments, Anthony Forget, Michelle Kudron, Dharia Mcgrew, Melissa Calmann, Celia Schiffer, Kendall Knight

Celia A. Schiffer

All RecA-like recombinase enzymes catalyze DNA strand exchange as elongated filaments on DNA. Despite numerous biochemical and structural studies of RecA and the related Rad51 and RadA proteins, the unit oligomer(s) responsible for nucleoprotein filament assembly and coordinated filament activity remains undefined. We have created a RecA fused dimer protein and show that it maintains in vivo DNA repair and LexA co-protease activities, as well as in vitro ATPase and DNA strand exchange activities. Our results support the idea that dimeric RecA is an important functional unit both for assembly of nucleoprotein filaments and for their coordinated activity during the …

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