32 results on '"Celia A. Schiffer"'
Search Results
2. Discovery of Quinoxaline-Based P1–P3 Macrocyclic NS3/4A Protease Inhibitors with Potent Activity against Drug-Resistant Hepatitis C Virus Variants
- Author
-
Nese Kurt Yilmaz, Desaboini Nageswara Rao, Hasahn L. Conway, Mohsan Saeed, Alicia Newton, Adam K. Hedger, Jacqueto Zephyr, Ashley N. Matthew, Akbar Ali, Mina Henes, Elise T Chan, Wei Huang, Christos J. Petropoulos, and Celia A. Schiffer
- Subjects
Male ,Macrocyclic Compounds ,Hepatitis C virus ,medicine.medical_treatment ,Hepacivirus ,Drug resistance ,Viral Nonstructural Proteins ,Crystallography, X-Ray ,medicine.disease_cause ,Antiviral Agents ,Cocrystal ,Article ,Rats, Sprague-Dawley ,Structure-Activity Relationship ,chemistry.chemical_compound ,Quinoxaline ,Quinoxalines ,Drug Resistance, Viral ,Drug Discovery ,Genotype ,medicine ,Animals ,Protease Inhibitors ,Protease ,Molecular Structure ,Chemistry ,Glecaprevir ,Hepatitis C ,Biochemistry ,Molecular Medicine ,Serine Proteases ,Ns3 4a protease ,Protein Binding - Abstract
The three pan-genotypic HCV NS3/4A protease inhibitors (PIs) currently in clinical use-grazoprevir, glecaprevir, and voxilaprevir-are quinoxaline-based P2-P4 macrocycles and thus exhibit similar resistance profiles. Using our quinoxaline-based P1-P3 macrocyclic lead compounds as an alternative chemical scaffold, we explored structure-activity relationships (SARs) at the P2 and P4 positions to develop pan-genotypic PIs that avoid drug resistance. A structure-guided strategy was used to design and synthesize two series of compounds with different P2 quinoxalines in combination with diverse P4 groups of varying sizes and shapes, with and without fluorine substitutions. Our SAR data and cocrystal structures revealed the interplay between the P2 and P4 groups, which influenced inhibitor binding and the overall resistance profile. Optimizing inhibitor interactions in the S4 pocket led to PIs with excellent antiviral activity against clinically relevant PI-resistant HCV variants and genotype 3, providing potential pan-genotypic inhibitors with improved resistance profiles.
- Published
- 2021
3. Deciphering Antifungal Drug Resistance in Pneumocystis jirovecii DHFR with Molecular Dynamics and Machine Learning
- Author
-
Florian Leidner, Celia A. Schiffer, and Nese Kurt Yilmaz
- Subjects
General Chemical Engineering ,Antifungal drug ,Drug resistance ,Library and Information Sciences ,Machine learning ,computer.software_genre ,01 natural sciences ,0103 physical sciences ,Dihydrofolate reductase ,medicine ,Pneumocystis jirovecii ,Homology modeling ,010304 chemical physics ,Resistance (ecology) ,biology ,business.industry ,Active site ,General Chemistry ,biology.organism_classification ,Trimethoprim ,0104 chemical sciences ,Computer Science Applications ,010404 medicinal & biomolecular chemistry ,biology.protein ,Artificial intelligence ,business ,computer ,medicine.drug - Abstract
Drug resistance impacts the effectiveness of many new therapeutics. Mutations in the therapeutic target confer resistance; however, deciphering which mutations, often remote from the enzyme active site, drive resistance is challenging. In a series of Pneumocystis jirovecii dihydrofolate reductase variants, we elucidate which interactions are key bellwethers to confer resistance to trimethoprim using homology modeling, molecular dynamics, and machine learning. Six molecular features involving mainly residues that did not vary were the best indicators of resistance.
- Published
- 2021
4. Drug Design Strategies to Avoid Resistance in Direct-Acting Antivirals and Beyond
- Author
-
Florian Leidner, Ashley N. Matthew, Linah N. Rusere, Gordon J. Lockbaum, Mina Henes, Debra A. Ragland, Desaboini Nageswara Rao, Jacqueto Zephyr, Djade I. Soumana, Celia A. Schiffer, Janet L. Paulsen, Nese Kurt Yilmaz, Kristina L. Prachanronarong, Ellen A. Nalivaika, Shurong Hou, Jennifer Timm, and Akbar Ali
- Subjects
Drug ,media_common.quotation_subject ,Human immunodeficiency virus (HIV) ,Drug design ,Resistance (psychoanalysis) ,Hepacivirus ,Drug resistance ,010402 general chemistry ,medicine.disease_cause ,DIRECT ACTING ANTIVIRALS ,Antiviral Agents ,01 natural sciences ,Article ,Machine Learning ,Structure-Activity Relationship ,Viral Proteins ,Drug Resistance, Viral ,medicine ,Humans ,Enzyme Inhibitors ,media_common ,Health consequences ,010405 organic chemistry ,Chemistry ,Computational Biology ,General Chemistry ,Orthomyxoviridae ,0104 chemical sciences ,Pharmaceutical Preparations ,Drug development ,Risk analysis (engineering) ,Virus Diseases ,Drug Design ,Mutation ,HIV-1 ,Protein Binding ,Signal Transduction - Abstract
Drug resistance is prevalent across many diseases, rendering therapies ineffective with severe financial and health consequences. Rather than accepting resistance after the fact, proactive strategies need to be incorporated into the drug design and development process to minimize the impact of drug resistance. These strategies can be derived from our experience with viral disease targets where multiple generations of drugs had to be developed to combat resistance and avoid antiviral failure. Significant efforts including experimental and computational structural biology, medicinal chemistry, and machine learning have focused on understanding the mechanisms and structural basis of resistance against direct-acting antiviral (DAA) drugs. Integrated methods show promise for being predictive of resistance and potency. In this review, we give an overview of this research for human immunodeficiency virus type 1, hepatitis C virus, and influenza virus and the lessons learned from resistance mechanisms of DAAs. These lessons translate into rational strategies to avoid resistance in drug design, which can be generalized and applied beyond viral targets. While resistance may not be completely avoidable, rational drug design can and should incorporate strategies at the outset of drug development to decrease the prevalence of drug resistance.
- Published
- 2021
5. HIV-1 Protease Inhibitors Incorporating Stereochemically Defined P2′ Ligands To Optimize Hydrogen Bonding in the Substrate Envelope
- Author
-
Gordon J. Lockbaum, Celia A. Schiffer, Ellen A. Nalivaika, Nese Kurt Yilmaz, Linah N. Rusere, Mina Henes, Sook-Kyung Lee, Klajdi Kosovrasti, Ronald Swanstrom, Akbar Ali, and Ean Spielvogel
- Subjects
Models, Molecular ,Anti-HIV Agents ,Stereochemistry ,medicine.medical_treatment ,Stereoisomerism ,Microbial Sensitivity Tests ,Crystallography, X-Ray ,Ligands ,01 natural sciences ,Article ,Cell Line ,Substrate Specificity ,Structure-Activity Relationship ,03 medical and health sciences ,HIV Protease ,HIV-1 protease ,Drug Discovery ,medicine ,Humans ,Moiety ,Structure–activity relationship ,Molecule ,030304 developmental biology ,0303 health sciences ,Protease ,Dose-Response Relationship, Drug ,Molecular Structure ,biology ,Chemistry ,Hydrogen bond ,Diastereomer ,Hydrogen Bonding ,HIV Protease Inhibitors ,0104 chemical sciences ,010404 medicinal & biomolecular chemistry ,HEK293 Cells ,HIV-1 ,biology.protein ,Molecular Medicine - Abstract
A structure-guided design strategy was used to improve the resistance profile of HIV-1 protease inhibitors by optimizing hydrogen bonding and van der Waals interactions with the protease while staying within the substrate envelope. Stereoisomers of 4-(1-hydroxyethyl)benzene and 4-(1,2-dihydroxyethyl)benzene moieties were explored as P2' ligands providing pairs of diastereoisomers epimeric at P2', which exhibited distinct potency profiles depending on the configuration of the hydroxyl group and size of the P1' group. While compounds with the 4-(1-hydroxyethyl)benzene P2' moiety maintained excellent antiviral potency against a panel of multidrug-resistant HIV-1 strains, analogues with the polar 4-(1,2-dihydroxyethyl)benzene moiety were less potent, and only the (R)-epimer incorporating a larger 2-ethylbutyl P1' group showed improved potency. Crystal structures of protease-inhibitor complexes revealed strong hydrogen bonding interactions of both (R)- and (S)-stereoisomers of the hydroxyethyl group with Asp30'. Notably, the (R)-dihydroxyethyl group was involved in a unique pattern of direct hydrogen bonding interactions with the backbone amides of Asp29' and Asp30'. The SAR data and analysis of crystal structures provide insights for optimizing these promising HIV-1 protease inhibitors.
- Published
- 2019
6. Target-Specific Prediction of Ligand Affinity with Structure-Based Interaction Fingerprints
- Author
-
Celia A. Schiffer, Nese Kurt Yilmaz, and Florian Leidner
- Subjects
Computer science ,General Chemical Engineering ,HIV Infections ,Computational biology ,Library and Information Sciences ,Ligands ,01 natural sciences ,Article ,Machine Learning ,HIV Protease ,Drug Discovery ,0103 physical sciences ,Feature (machine learning) ,Humans ,Potency ,Interpretability ,010304 chemical physics ,Basis (linear algebra) ,HIV Protease Inhibitors ,General Chemistry ,Ligand (biochemistry) ,Small molecule ,0104 chemical sciences ,Computer Science Applications ,Hierarchical clustering ,Molecular Docking Simulation ,010404 medicinal & biomolecular chemistry ,Drug Design ,HIV-1 ,Gradient boosting ,Protein Binding - Abstract
Discovery and optimization of small molecule inhibitors as therapeutic drugs have immensely benefited from rational structure-based drug design. With recent advances in high-resolution structure determination, computational power, and machine learning methodology, it is becoming more tractable to elucidate the structural basis of drug potency. However, the applicability of machine learning models to drug design is limited by the interpretability of the resulting models in terms of feature importance. Here, we take advantage of the large number of available inhibitor-bound HIV-1 protease structures and associated potencies to evaluate inhibitor diversity and machine learning models to predict ligand affinity. First, using a hierarchical clustering approach, we grouped HIV-1 protease inhibitors and identified distinct core structures. Explicit features including protein-ligand interactions were extracted from high-resolution cocrystal structures as 3D-based fingerprints. We found that a gradient boosting machine learning model with this explicit feature attribution can predict binding affinity with high accuracy. Finally, Shapley values were derived to explain local feature importance. We found specific van der Waals (vdW) interactions of key protein residues are pivotal for the predicted potency. Protein-specific and interpretable prediction models can guide the optimization of many small molecule drugs for improved potency.
- Published
- 2019
7. Hydration Structure and Dynamics of Inhibitor-Bound HIV-1 Protease
- Author
-
Nese Kurt Yilmaz, Yves A. Muller, Celia A. Schiffer, Janet L. Paulsen, and Florian Leidner
- Subjects
0301 basic medicine ,Surface Properties ,medicine.medical_treatment ,Molecular Dynamics Simulation ,010402 general chemistry ,01 natural sciences ,Article ,03 medical and health sciences ,Molecular dynamics ,HIV Protease ,HIV-1 protease ,Catalytic Domain ,Drug Resistance, Viral ,medicine ,Molecule ,Physical and Theoretical Chemistry ,Topology (chemistry) ,Protease ,biology ,Hydrogen bond ,Chemistry ,Water ,Hydrogen Bonding ,HIV Protease Inhibitors ,0104 chemical sciences ,Computer Science Applications ,030104 developmental biology ,Solvation shell ,Chemical physics ,HIV-1 ,biology.protein ,Protein folding - Abstract
Water is an essential in many biological processes, and the hydration structure plays a critical role in facilitating protein folding, dynamics and ligand binding. A variety of biophysical spectroscopic techniques have been used to probe the water solvating proteins, often complemented with molecular dynamics (MD) simulations to resolve the spatial and dynamic features of the hydration shell, but comparing relative water structure is challenging. In this study 1 microsecond MD simulations were performed to identify and characterize hydration sites around HIV-1 protease bound to an inhibitor, darunavir (DRV). The water density, hydration site occupancy, extent and anisotropy of fluctuations, coordinated water molecules, and hydrogen bonds were characterized and compared to the properties of bulk water. The water density of the principal hydration shell was found to be higher than bulk, dependent on the topology and physio-chemical identity of the biomolecular surface. The dynamics of water molecules occupying principal hydration sites was highly dependent on the number of water-water interactions, and inversely correlated with hydrogen bonds to the protein–inhibitor complex. While many waters were conserved following the symmetry of homodimeric HIV protease, the asymmetry induced by DRV resulted in asymmetric lower-occupancy hydration sites at the concave surface of the active site. Key interactions between water molecules and the protease, that stabilize the protein in the inhibited form, were altered in a drug resistant variant of the protease indicating that modulation of solvent–solute interactions might play a key role in conveying drug resistance. Our analysis provides insights into the interplay between an enzyme inhibitor complex and the hydration shell and has implications in elucidating water structure in a variety of biological processes and applications including ligand binding, inhibitor design and resistance.
- Published
- 2018
8. Probing Structural Changes among Analogous Inhibitor-Bound Forms of HIV-1 Protease and a Drug-Resistant Mutant in Solution by Nuclear Magnetic Resonance
- Author
-
Celia A. Schiffer, Shahid N. Khan, John D. Persons, Janet L. Paulsen, Rieko Ishima, N. KurtYilmaz, and Michel Guerrero
- Subjects
0301 basic medicine ,Protein Conformation ,medicine.medical_treatment ,Mutant ,Molecular Dynamics Simulation ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Article ,03 medical and health sciences ,Molecular dynamics ,chemistry.chemical_compound ,Nuclear magnetic resonance ,HIV Protease ,HIV-1 protease ,Amide ,Drug Resistance, Viral ,medicine ,Amino Acid Sequence ,Nuclear Magnetic Resonance, Biomolecular ,Darunavir ,Protease ,biology ,Hydrogen bond ,Chemistry ,Chemical shift ,HIV Protease Inhibitors ,0104 chemical sciences ,030104 developmental biology ,Mutation ,biology.protein ,medicine.drug - Abstract
In the era of state-of-the-art inhibitor design and high-resolution structural studies, detection of significant but small protein structural differences in the inhibitor-bound forms is critical to further developing the inhibitor. Here, we probed differences in HIV-1 protease (PR) conformation among darunavir and four analogous inhibitor-bound forms and compared them with a drug-resistant mutant using nuclear magnetic resonance chemical shifts. Changes in amide chemical shifts of wild-type (WT) PR among these inhibitor-bound forms, ΔCSP, were subtle but detectable and extended10 Å from the inhibitor-binding site, asymmetrically between the two subunits of PR. Molecular dynamics simulations revealed differential local hydrogen bonding as the molecular basis of this remote asymmetric change. Inhibitor-bound forms of the drug-resistant mutant also showed a similar long-range ΔCSP pattern. Differences in ΔCSP values of the WT and the mutant (ΔΔCSPs) were observed at the inhibitor-binding site and in the surrounding region. Comparing chemical shift changes among highly analogous inhibitors and ΔΔCSPs effectively eliminated local environmental effects stemming from different chemical groups and enabled exploitation of these sensitive parameters to detect subtle protein conformational changes and to elucidate asymmetric and remote conformational effects upon inhibitor interaction.
- Published
- 2018
9. Elucidating the Interdependence of Drug Resistance from Combinations of Mutations
- Author
-
N. KurtYilmaz, Troy W. Whitfield, Sook Kyung Lee, Debra A. Ragland, Ronald Swanstrom, Celia A. Schiffer, and Konstantin B. Zeldovich
- Subjects
0301 basic medicine ,Polyproteins ,medicine.medical_treatment ,Drug Resistance ,Drug resistance ,Cleavage (embryo) ,01 natural sciences ,Genome ,Article ,Virus ,03 medical and health sciences ,HIV Protease ,Catalytic Domain ,0103 physical sciences ,medicine ,Humans ,Physical and Theoretical Chemistry ,Darunavir ,Genetics ,Protease ,010304 chemical physics ,biology ,Active site ,HIV Protease Inhibitors ,Computer Science Applications ,030104 developmental biology ,Amino Acid Substitution ,Mutation ,biology.protein ,medicine.drug - Abstract
HIV-1 protease is responsible for the cleavage of 12 non-homologous sites within the Gag and Gag-Pro-Pol polyproteins in the viral genome. Under the selective pressure of protease inhibition, the virus evolves mutations within (primary) and outside of (secondary) the active site allowing the protease to process substrates while simultaneously countering inhibition. The primary protease mutations impede inhibitor binding directly, while the secondary mutations are considered accessory mutations that compensate for a loss in fitness. However, the role of secondary mutations in conferring drug resistance remains a largely unresolved topic. We have shown previously that mutations distal to the active site are able to perturb binding of darunavir (DRV) via the protein’s internal hydrogen-bonding network. In this study we show that mutations distal to the active site, regardless of context, can play an interdependent role in drug resistance. Applying eigenvalue decomposition to collections of hydrogen bonding and van der Waals interactions from a series of molecular dynamics simulations of 15 diverse HIV-1 protease variants, we identify sites in the protease where amino acid substitutions lead to perturbations in non-bonded interactions with DRV and/or the hydrogen-bonding network of the protease itself. While primary mutations are known to drive resistance in HIV-1 protease, these findings delineate the significant contributions of accessory mutations to resistance. Identifying the variable positions in the protease that have the greatest impact on drug resistance may aid in future structure-based design of inhibitors.
- Published
- 2017
10. Hepatitis C Virus NS3/4A Protease Inhibitors Incorporating Flexible P2 Quinoxalines Target Drug Resistant Viral Variants
- Author
-
N. KurtYilmaz, Jacqueto Zephyr, Celia A. Schiffer, Christos J. Petropoulos, Ashley N. Matthew, Caitlin. J. Hill, Akbar Ali, Muhammad Jahangir, Wei Huang, and Alicia Newton
- Subjects
0301 basic medicine ,medicine.medical_treatment ,Hepacivirus ,Drug resistance ,Molecular Dynamics Simulation ,Viral Nonstructural Proteins ,Crystallography, X-Ray ,Antiviral Agents ,01 natural sciences ,Article ,Structure-Activity Relationship ,03 medical and health sciences ,chemistry.chemical_compound ,Quinoxaline ,Quinoxalines ,Drug Resistance, Viral ,Drug Discovery ,Catalytic triad ,medicine ,Humans ,Structure–activity relationship ,Potency ,Protease Inhibitors ,EC50 ,Protease ,010405 organic chemistry ,0104 chemical sciences ,Molecular Docking Simulation ,030104 developmental biology ,Biochemistry ,chemistry ,Target drug ,Molecular Medicine - Abstract
A substrate envelope-guided design strategy is reported for improving the resistance profile of HCV NS3/4A protease inhibitors. Analogues of 5172-mcP1P3 were designed by incorporating diverse quinoxalines at the P2 position that predominantly interact with the invariant catalytic triad of the protease. Exploration of structure-activity relationships showed that inhibitors with small hydrophobic substituents at the 3-position of P2 quinoxaline maintain better potency against drug resistant variants, likely due to reduced interactions with residues in the S2 subsite. In contrast, inhibitors with larger groups at this position were highly susceptible to mutations at Arg155, Ala156 and Asp168. Excitingly, several inhibitors exhibited exceptional potency profiles with EC50 values ≤ 5 nM against major drug resistant HCV variants. These findings support that inhibitors designed to interact with evolutionarily constrained regions of the protease, while avoiding interactions with residues not essential for substrate recognition, are less likely to be susceptible to drug resistance.
- Published
- 2017
11. Molecular Basis for Differential Patterns of Drug Resistance in Influenza N1 and N2 Neuraminidase
- Author
-
Kelly M. Thayer, Jennifer P. Wang, Konstantin B. Zeldovich, Jeffrey D. Jensen, N. KurtYilmaz, Kristina L. Prachanronarong, L. Safak Yilmaz, Robert W. Finberg, Ayşegül Özen, Celia A. Schiffer, Daniel N. Bolon, and Timothy F. Kowalik
- Subjects
0301 basic medicine ,Oseltamivir ,Static Electricity ,030106 microbiology ,Neuraminidase ,Drug resistance ,Plasma protein binding ,Molecular Dynamics Simulation ,medicine.disease_cause ,Antiviral Agents ,Substrate Specificity ,03 medical and health sciences ,chemistry.chemical_compound ,Zanamivir ,Protein structure ,Drug Resistance, Viral ,Influenza, Human ,medicine ,Influenza A virus ,Humans ,Enzyme Inhibitors ,Physical and Theoretical Chemistry ,Protein Structure, Quaternary ,Genetics ,Binding Sites ,biology ,Active site ,Hydrogen Bonding ,Virology ,Computer Science Applications ,030104 developmental biology ,chemistry ,Mutagenesis, Site-Directed ,biology.protein ,Thermodynamics ,Protein Binding ,medicine.drug - Abstract
Neuraminidase (NA) inhibitors are used for the prevention and treatment of influenza A virus infections. Two subtypes of NA, N1 and N2, predominate in viruses that infect humans, but differential patterns of drug resistance have emerged in each subtype despite highly homologous active sites. To understand the molecular basis for the selection of these drug resistance mutations, structural and dynamic analyses on complexes of N1 and N2 NA with substrates and inhibitors were performed. Comparison of dynamic substrate and inhibitor envelopes and interactions at the active site revealed how differential patterns of drug resistance have emerged for specific drug resistance mutations, at residues I222, S246, and H274 in N1 and E119 in N2. Our results show that the differences in intermolecular interactions, especially van der Waals contacts, of the inhibitors versus substrates at the NA active site effectively explain the selection of resistance mutations in the two subtypes. Avoiding such contacts that render inhibitors vulnerable to resistance by better mimicking the dynamics and intermolecular interactions of substrates can lead to the development of novel inhibitors that avoid drug resistance in both subtypes.
- Published
- 2016
12. Dengue Protease Substrate Recognition: Binding of the Prime Side
- Author
-
Celia A. Schiffer, Kuan-Hung Lin, Nese Kurt Yilmaz, Kristina L. Prachanronarong, and Ellen A. Nalivaika
- Subjects
0301 basic medicine ,viruses ,medicine.medical_treatment ,Aedes aegypti ,Viral Nonstructural Proteins ,Biology ,Dengue virus ,medicine.disease_cause ,Article ,Substrate Specificity ,Dengue fever ,Dengue ,03 medical and health sciences ,0302 clinical medicine ,medicine ,Humans ,Peptide sequence ,Polyproteins ,Serine protease ,NS3 ,Binding Sites ,Protease ,Serine Endopeptidases ,Dengue Virus ,biochemical phenomena, metabolism, and nutrition ,medicine.disease ,biology.organism_classification ,Virology ,NS2-3 protease ,030104 developmental biology ,Infectious Diseases ,030220 oncology & carcinogenesis ,biology.protein - Abstract
Dengue virus (DENV), transmitted predominantly in tropical and subtropical regions by the mosquito Aedes aegypti, infects millions of people and leads to dengue fever and thousands of deaths each year. There are no direct-acting antivirals to combat DENV, and molecular and structural knowledge is required to develop such compounds. The dengue NS2B/NS3 protease is a promising target for direct-acting antivirals, as viral polyprotein cleavage during replication is required for the maturation of the viral particle. The NS2B/NS3 protease processes 8 of the 13 viral polyprotein cleavage sites to allow viral maturation. Although these sites share little sequence homology beyond the P1 and P2 positions, most are well conserved among the serotypes. How the other substrate residues, especially at the P′ side, affect substrate recognition remains unclear. We exploited the tight-binding general serine protease inhibitor aprotinin to investigate protease–substrate interactions at the molecular level. We engineered aprotinin’s binding loop with sequences mimicking the P′ side of DENV substrates. P′ residues significantly modulate substrate affinity to protease, with inhibition constants varying from nanomolar to sub-millimolar. Structural and dynamic analysis revealed the molecular basis of this modulation and allowed identifying optimal residues for each of the P′ positions. In addition, isothermal titration calorimetry showed binding to be solely entropy driven for all constructs. Potential flaviviral P′ side inhibitors could benefit from mimicking the optimal residues at P′ positions and incorporate hydrophobicity and rigidity to maintain entropic advantage for potency.
- Published
- 2016
13. Drug Resistance Conferred by Mutations Outside the Active Site through Alterations in the Dynamic and Structural Ensemble of HIV-1 Protease
- Author
-
Celia A. Schiffer, Hong Cao, Ellen A. Nalivaika, Debra A. Ragland, Yufeng Cai, N. KurtYilmaz, Rajintha M. Bandaranayake, Kristina L. Prachanronarong, and Madhavi N. L. Nalam
- Subjects
Models, Molecular ,Protein Conformation ,medicine.medical_treatment ,Drug resistance ,Plasma protein binding ,medicine.disease_cause ,01 natural sciences ,Biochemistry ,Article ,Catalysis ,03 medical and health sciences ,Colloid and Surface Chemistry ,HIV Protease ,HIV-1 protease ,Drug Resistance, Viral ,0103 physical sciences ,medicine ,Binding site ,Darunavir ,030304 developmental biology ,Sulfonamides ,0303 health sciences ,Mutation ,Binding Sites ,Protease ,010304 chemical physics ,biology ,Chemistry ,Active site ,HIV Protease Inhibitors ,General Chemistry ,Virology ,Molecular biology ,3. Good health ,HIV-1 ,biology.protein ,Protein Binding ,medicine.drug - Abstract
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 and dynamic ensemble of HIV-1 protease active site. These alterations correlate with the observed inhibitor binding affinities for the mutants, and suggest a network hypothesis on how the effect of distal mutations are propagated to pivotal residues at the active site and may contribute to conferring drug resistance.
- Published
- 2014
14. Correction to Structural Adaptation of Darunavir Analogues against Primary Mutations in HIV-1 Protease
- Author
-
Linah N. Rusere, Celia A. Schiffer, Gordon J. Lockbaum, Mina Henes, Akbar Ali, Gily S. Nachum, Florian Leidner, Daniel N. Bolon, Klajdi Kosovrasti, Nese Kurt Yilmaz, and Ellen A. Nalivaika
- Subjects
Infectious Diseases ,HIV-1 protease ,biology ,business.industry ,biology.protein ,medicine ,Adaptation ,business ,Virology ,Darunavir ,medicine.drug - Published
- 2019
15. Mavyret: A Pan-Genotypic Combination Therapy for the Treatment of Hepatitis C Infection
- Author
-
Nese Kurt Yilmaz, Celia A. Schiffer, and Ashley N. Matthew
- Subjects
0301 basic medicine ,medicine.medical_specialty ,Combination therapy ,Extramural ,business.industry ,030106 microbiology ,MEDLINE ,Hepatitis C ,medicine.disease ,Biochemistry ,03 medical and health sciences ,0302 clinical medicine ,Internal medicine ,Genotype ,medicine ,business ,030217 neurology & neurosurgery - Published
- 2017
16. Efficient Computation of Small-Molecule Configurational Binding Entropy and Free Energy Changes by Ensemble Enumeration
- Author
-
Hong Cao, Bruce Tidor, Madhavi N. L. Nalam, Bracken M. King, Tariq M. Rana, Nathaniel W. Silver, G. S. Kiran Kumar Reddy, Celia A. Schiffer, and Akbar Ali
- Subjects
Quantitative Biology::Biomolecules ,0303 health sciences ,010304 chemical physics ,Chemistry ,business.industry ,Computation ,Conditional mutual information ,Configuration entropy ,Enthalpy ,Degrees of freedom (statistics) ,Thermodynamics ,Dihedral angle ,01 natural sciences ,Small molecule ,Article ,3. Good health ,Computer Science Applications ,03 medical and health sciences ,0103 physical sciences ,Artificial intelligence ,Physical and Theoretical Chemistry ,Entropy (energy dispersal) ,business ,030304 developmental biology - Abstract
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 fraction of the full configurational space, and we see significant improvement in relative accuracy when using an ensemble versus single-conformer approach to ligand ranking. We also find that using approximate metrics based on the single-conformation enthalpy differences between the global minimum energy configuration in the bound as well as unbound states also correlates well with experiment. Using a novel, additive entropy expansion based on conditional mutual information, we also analyze the source of ligand configurational entropy loss upon binding in terms of both uncoupled per degree of freedom losses as well as changes in coupling between inhibitor degrees of freedom. We estimate entropic free energy losses of approximately +24 kcal/mol, 12 kcal/mol of which stems from loss of translational and rotational entropy. Coupling effects contribute only a small fraction to the overall entropy change (1−2 kcal/mol) but suggest differences in how inhibitor dihedral angles couple to each other in the bound versus unbound states. The importance of accounting for flexibility in drug optimization and design is also discussed.
- Published
- 2013
17. A Sensitive Assay Using a Native Protein Substrate for Screening HIV-1 Maturation Inhibitors Targeting the Protease Cleavage Site between the Matrix and Capsid
- Author
-
Emily A. Hull-Ryde, Nancy Cheng, Marc Potempa, Sook-Kyung Lee, William P. Janzen, Ronald Swanstrom, and Celia A. Schiffer
- Subjects
Infectivity ,Protease ,Virus Assembly ,medicine.medical_treatment ,Biology ,Virus Replication ,Biochemistry ,Molecular biology ,Fusion protein ,Article ,Cell Line ,Substrate Specificity ,chemistry.chemical_compound ,Capsid ,HIV Protease ,chemistry ,Viral replication ,Cell culture ,HIV-1 ,medicine ,Humans ,Fluorescein ,Fluorescence anisotropy - Abstract
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. By limiting the size of CA and increasing the size of MA (with an N-terminal GST fusion), we were able to measure significant differences in polarization values as a function of HIV-1 protease cleavage. The sensitivity of the assay was tested in the presence of increasing amounts of an HIV-1 protease inhibitor, which resulted in a gradual decrease in the fluorescence polarization values demonstrating that the assay is sensitive in discerning changes in protease processing. The high-throughput screening assay validation in 384-well plates showed that the assay is reproducible and robust with an average Z' value of 0.79 and average coefficient of variation values of3%. The robustness and reproducibility of the assay were further validated using the LOPAC(1280) compound library, demonstrating that the assay provides a sensitive high-throughput screening platform that can be used with large compound libraries for identifying novel maturation inhibitors targeting the MA/CA site of the HIV-1 Gag polyprotein.
- Published
- 2013
18. Evaluating the Role of Macrocycles in the Susceptibility of Hepatitis C Virus NS3/4A Protease Inhibitors to Drug Resistance
- Author
-
Ayşegül Özen, Cihan Aydin, Hong Cao, Celia A. Schiffer, Keith P. Romano, Akbar Ali, Djade I. Soumana, Wei Huang, Reinhold Gildemeister, Christos J. Petropoulos, and Alicia Newton
- Subjects
Macrocyclic Compounds ,medicine.medical_treatment ,Vaniprevir ,Hepacivirus ,Drug resistance ,Viral Nonstructural Proteins ,Biology ,Biochemistry ,Article ,Telaprevir ,chemistry.chemical_compound ,Boceprevir ,Drug Resistance, Viral ,medicine ,Humans ,NS3 ,Protease ,Molecular Structure ,Danoprevir ,Intracellular Signaling Peptides and Proteins ,General Medicine ,Virology ,chemistry ,Molecular Medicine ,Asunaprevir ,Carrier Proteins ,medicine.drug - Abstract
The hepatitis C virus (HCV) infects an estimated 150 million people worldwide and is the major cause of viral hepatitis, cirrhosis, and liver cancer. The available antiviral therapies, which include PEGylated interferon, ribavirin, and one of the HCV NS3/4A protease inhibitors telaprevir or boceprevir, are ineffective for some patients and cause severe side effects. More potent NS3/4A protease inhibitors are in clinical development, but the long-term effectiveness of these drugs is challenged by the development of drug resistance. Here, we investigated the role of macrocycles in the susceptibility of NS3/4A protease inhibitors to drug resistance in asunaprevir, danoprevir, vaniprevir, and MK-5172, with similar core structures but varied P2 moieties and macrocyclizations. Linear and macrocyclic analogues of these drugs were designed, synthesized, and tested against wild-type and drug-resistant variants R155K, V36M/R155K, A156T, and D168A in enzymatic and antiviral assays. Macrocyclic inhibitors were generally more potent, but the location of the macrocycle was critical for retaining activity against drug-resistant variants: the P1-P3 macrocyclic inhibitors were less susceptible to drug resistance than the linear and P2-P4 macrocyclic analogues. In addition, the heterocyclic moiety at P2 largely determined the inhibitor resistance profile, susceptibility to drug resistance, and the extent of modulation by the helicase domain. Our findings suggest that to design robust inhibitors that retain potency to drug-resistant NS3/4A protease variants, inhibitors should combine P1-P3 macrocycles with flexible P2 moieties that optimally contact with the invariable catalytic triad of this enzyme.
- Published
- 2013
19. Cooperative Effects of Drug-Resistance Mutations in the Flap Region of HIV-1 Protease
- Author
-
Robert W. Shafer, Jennifer E. Foulkes-Murzycki, Christina Rosi, Nese Kurt Yilmaz, and Celia A. Schiffer
- Subjects
Models, Molecular ,chemistry.chemical_classification ,Genetics ,Mutation ,Protease ,medicine.medical_treatment ,General Medicine ,Drug resistance ,Calorimetry ,Biology ,medicine.disease_cause ,Biochemistry ,Article ,Enzyme ,HIV Protease ,chemistry ,HIV-1 protease ,Cleave ,Drug Resistance, Viral ,medicine ,biology.protein ,Thermodynamics ,Molecular Medicine - Abstract
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 in the flaps (residues 48 and 54) of the enzyme reveal that the thermodynamic effects are not additive. Rather, the thermodynamic profile of the variants is interdependent on the cooperative effects exerted by a particular combination of mutations simultaneously present.
- Published
- 2012
20. Hydrophobic Core Flexibility Modulates Enzyme Activity in HIV-1 Protease
- Author
-
Celia A. Schiffer, Daniel N. Bolon, Seema Mittal, Madhavi N. L. Nalam, and Yufeng Cai
- Subjects
Models, Molecular ,Protein Conformation ,medicine.medical_treatment ,Molecular Dynamics Simulation ,Crystallography, X-Ray ,Biochemistry ,Article ,Catalysis ,Enzyme activator ,Colloid and Surface Chemistry ,Protein structure ,HIV Protease ,HIV-1 protease ,medicine ,chemistry.chemical_classification ,Protease ,biology ,Chemistry ,Active site ,Substrate (chemistry) ,General Chemistry ,Enzyme assay ,Enzyme Activation ,Enzyme ,Mutation ,biology.protein ,Hydrophobic and Hydrophilic Interactions - Abstract
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 overall structure of the protease is retained. However, cross-linking the cysteines led to drastic loss in enzyme activity, which was regained upon reducing the disulfide cross-links. Molecular dynamics simulations showed that altered dynamics propagated throughout the enzyme from the engineered disulfide. Thus, altered flexibility within the hydrophobic core can modulate HIV-1 protease activity, supporting the hypothesis that drug resistant mutations distal from the active site can alter the balance between substrate turnover and inhibitor binding by modulating enzyme activity.
- Published
- 2012
21. First-In-Class Small Molecule Inhibitors of the Single-Strand DNA Cytosine Deaminase APOBEC3G
- Author
-
Mohan Somasundaran, Shivender M.D. Shandilya, Ming Li, Anurag Rathore, Reuben S. Harris, Celia A. Schiffer, Daniel A. Harki, Akbar Ali, Michael A. Parniak, Krishan K. Pandey, Angela L. Perkins, Jonathan Solberg, Nevan J. Krogan, Michael A. Carpenter, Jeffrey R. Johnson, William L. Brown, and Derek J. Hook
- Subjects
Models, Molecular ,Ribonuclease H ,APOBEC-3G Deaminase ,HIV Integrase ,Crystallography, X-Ray ,Biochemistry ,Article ,Small Molecule Libraries ,Structure-Activity Relationship ,chemistry.chemical_compound ,Cytidine Deaminase ,Humans ,Enzyme Inhibitors ,APOBEC3A ,Binding site ,Cells, Cultured ,Dose-Response Relationship, Drug ,Molecular Structure ,biology ,Cytosine deaminase ,Active site ,General Medicine ,Cytidine deaminase ,Enzyme Activation ,HEK293 Cells ,chemistry ,Uracil-DNA glycosylase ,biology.protein ,Molecular Medicine ,DNA - Abstract
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 C321-to-L, F, Y, or W mimicked chemical inhibition. A strong specificity for APOBEC3G was evident, as most compounds failed to inhibit the related APOBEC3A enzyme or the unrelated enzymes E. coli uracil DNA glycosylase, HIV-1 RNase H, or HIV-1 integrase. Partial, but not complete, sensitivity could be conferred to APOBEC3A by introducing the entire C321 loop from APOBEC3G. Thus, a structural model is presented in which the mechanism of inhibition is both specific and competitive, by binding a pocket adjacent to the APOBEC3G active site, reacting with C321, and blocking access to substrate DNA cytosines.
- Published
- 2012
22. Structure-Based Design, Synthesis, and Structure−Activity Relationship Studies of HIV-1 Protease Inhibitors Incorporating Phenyloxazolidinones
- Author
-
G. S. Kiran Kumar Reddy, Akbar Ali, Celia A. Schiffer, Hong Cao, Madhavi N. L. Nalam, Saima Ghafoor Anjum, and Tariq M. Rana
- Subjects
Models, Molecular ,Anti-HIV Agents ,Stereochemistry ,medicine.medical_treatment ,Molecular Sequence Data ,Stereoisomerism ,Crystallography, X-Ray ,medicine.disease_cause ,Article ,Structure-Activity Relationship ,Drug Resistance, Multiple, Viral ,HIV-1 protease ,Drug Discovery ,medicine ,Humans ,Potency ,Structure–activity relationship ,HIV Protease Inhibitor ,Binding site ,Oxazolidinones ,Mutation ,Binding Sites ,Protease ,Molecular Structure ,biology ,Chemistry ,HIV Protease Inhibitors ,Drug Design ,HIV-1 ,biology.protein ,Molecular Medicine - Abstract
A series of new HIV-1 protease inhibitors with the hydroxyethylamine core and different phenyloxazolidinone P2 ligands were designed and synthesized. Variation of phenyl substitutions at the P2 and P2' moieties significantly affected the binding affinity and antiviral potency of the inhibitors. In general, compounds with 2- and 4-substituted phenyloxazolidinones at P2 exhibited lower binding affinities than 3-substituted analogues. Crystal structure analyses of ligand-enzyme complexes revealed different binding modes for 2- and 3-substituted P2 moieties in the protease S2 binding pocket, which may explain their different binding affinities. Several compounds with 3-substituted P2 moieties demonstrated picomolar binding affinity and low nanomolar antiviral potency against patient-derived viruses from HIV-1 clades A, B, and C, and most retained potency against drug-resistant viruses. Further optimization of these compounds using structure-based design may lead to the development of novel protease inhibitors with improved activity against drug-resistant strains of HIV-1.
- Published
- 2010
23. Additivity in the Analysis and Design of HIV Protease Inhibitors
- Author
-
Bruce Tidor, Tariq M. Rana, Akbar Ali, G. S. Kiran Kumar Reddy, Michael D. Altman, Celia A. Schiffer, Saima Ghafoor Anjum, Michael K. Gilson, Robert N. Jorissen, and Sripriya Chellappan
- Subjects
Models, Molecular ,Quantitative structure–activity relationship ,Binding Sites ,Crystallography ,Molecular model ,Chemistry ,Stereochemistry ,Substituent ,Quantitative Structure-Activity Relationship ,HIV Protease Inhibitors ,Affinities ,Article ,Kinetics ,chemistry.chemical_compound ,HIV Protease ,Drug Design ,Additive function ,Molecular descriptor ,Drug Discovery ,Fluorescence Resonance Energy Transfer ,Molecular Medicine ,HIV Protease Inhibitor ,Carbamates ,Additive model - Abstract
We explore the applicability of an additive treatment of substituent effects to the analysis and design of HIV protease inhibitors. Affinity data for a set of inhibitors with a common chemical framework were analyzed to provide estimates of the free energy contribution of each chemical substituent. These estimates were then used to design new inhibitors, whose high affinities were confirmed by synthesis and experimental testing. Derivations of additive models by least-squares and ridge-regression methods were found to yield statistically similar results. The additivity approach was also compared with standard molecular descriptor-based QSAR; the latter was not found to provide superior predictions. Crystallographic studies of HIV protease-inhibitor complexes help explain the perhaps surprisingly high degree of substituent additivity in this system, and allow some of the additivity coefficients to be rationalized on a structural basis.
- Published
- 2009
24. HIV-1 Protease Inhibitors from Inverse Design in the Substrate Envelope Exhibit Subnanomolar Binding to Drug-Resistant Variants
- Author
-
Bruce Tidor, Michael D. Altman, Celia A. Schiffer, Hong Cao, Visvaldas Kairys, Michael K. Gilson, Akbar Ali, G. S. Kiran Kumar Reddy, Tariq M. Rana, Saima Ghafoor Anjum, Madhavi N. L. Nalam, Sripriya Chellappan, and Miguel X. Fernandes
- Subjects
Models, Molecular ,medicine.medical_treatment ,Crystallography, X-Ray ,Biochemistry ,Article ,Catalysis ,Structure-Activity Relationship ,Colloid and Surface Chemistry ,HIV Protease ,HIV-1 protease ,Drug Resistance, Viral ,medicine ,Structure–activity relationship ,HIV Protease Inhibitor ,Binding site ,Furans ,Darunavir ,Sulfonamides ,Protease ,biology ,Chemistry ,Wild type ,Substrate (chemistry) ,HIV Protease Inhibitors ,General Chemistry ,Kinetics ,Drug Design ,HIV-1 ,biology.protein ,Carbamates ,Algorithms ,medicine.drug - Abstract
The acquisition of drug-resistance mutations by infectious pathogens remains a pressing health concern, and the development of strategies to combat this threat is a priority. Here we have applied a general strategy, inverse design using the substrate envelope, to develop inhibitors of HIV-1 protease. Structure-based computation was used to design inhibitors predicted to stay within a consensus substrate volume in the binding site. Two rounds of design, synthesis, experimental testing, and structural analysis were carried out, resulting in a total of 51 compounds. Improvements in design methodology led to a roughly 1000-fold affinity enhancement to a wild-type protease for the best binders, from Ki of 30–50 nM in round one to below 100 pM in round two. Crystal structures of a subset of complexes revealed a binding mode similar to each design that respected the substrate envelope in nearly all cases. All four best binders from round one exhibited broad specificity against a clinically relevant panel of drug-resistant HIV-1 protease variants, losing no more than 6–13 fold affinity relative to wild type. Testing a subset of second-round compounds against the panel of resistant variants revealed three classes of inhibitors — robust binders (maximum affinity loss of 14–16 fold), moderate binders (35–80 fold), and susceptible binders (greater than 100 fold). Although for especially high-affinity inhibitors additional factors may also be important, overall, these results suggest that designing inhibitors using the substrate envelope may be a useful strategy in the development of therapeutics with low susceptibility to resistance.
- Published
- 2008
25. Design and Synthesis of HIV-1 Protease Inhibitors Incorporating Oxazolidinones as P2/P2‘ Ligands in Pseudosymmetric Dipeptide Isosteres
- Author
-
G. S. Kiran Kumar Reddy, Robin S. Nathans, Saima Ghafoor Anjum, Celia A. Schiffer, Madhavi N. L. Nalam, Akbar Ali, Tariq M. Rana, and Hong Cao
- Subjects
Models, Molecular ,Stereochemistry ,medicine.medical_treatment ,Pyrimidinones ,Ligands ,Article ,Lopinavir ,Cell Line ,Structure-Activity Relationship ,chemistry.chemical_compound ,Drug Resistance, Multiple, Viral ,HIV Protease ,HIV-1 protease ,Drug Discovery ,Hydrolase ,medicine ,Humans ,HIV Protease Inhibitor ,Structure–activity relationship ,Protease inhibitor (pharmacology) ,Oxazolidinones ,Protease ,Dipeptide ,Molecular Structure ,biology ,Stereoisomerism ,Dipeptides ,HIV Protease Inhibitors ,chemistry ,Enzyme inhibitor ,Drug Design ,HIV-1 ,biology.protein ,Molecular Medicine - Abstract
A series of novel HIV-1 protease inhibitors based on two pseudosymmetric dipeptide isosteres have been synthesized and evaluated. The inhibitors were designed by incorporating N-phenyloxazolidinone-5-carboxamides into the hydroxyethylene and (hydroxyethyl)hydrazine dipeptide isosteres as P2 and P2' ligands. Compounds with (S)-phenyloxazolidinones attached at a position proximal to the central hydroxyl group showed low nM inhibitory activities against wild-type HIV-1 protease. Selected compounds were further evaluated for their inhibitory activities against a panel of multidrug-resistant protease variants and for their antiviral potencies in MT-4 cells. The crystal structures of lopinavir (LPV) and two new inhibitors containing phenyloxazolidinone-based ligands in complex with wild-type HIV-1 protease have been determined. A comparison of the inhibitor-protease structures with the LPV-protease structure provides valuable insight into the binding mode of the new inhibitors to the protease enzyme. Based on the crystal structures and knowledge of structure-activity relationships, new inhibitors can be designed with enhanced enzyme inhibitory and antiviral potencies.
- Published
- 2007
26. Discovery of HIV-1 Protease Inhibitors with Picomolar Affinities Incorporating N-Aryl-oxazolidinone-5-carboxamides as Novel P2 Ligands
- Author
-
G. S. Kiran Kumar Reddy, Hong Cao, Madhavi N. L. Nalam, Akbar Ali, Tariq M. Rana, Saima Ghafoor Anjum, and Celia A. Schiffer
- Subjects
Stereochemistry ,medicine.drug_class ,medicine.medical_treatment ,Carboxamide ,Crystallography, X-Ray ,Ligands ,Structure-Activity Relationship ,Drug Resistance, Multiple, Viral ,HIV Protease ,HIV-1 protease ,Drug Discovery ,Hydrolase ,Fluorescence Resonance Energy Transfer ,medicine ,Structure–activity relationship ,Oxazoles ,chemistry.chemical_classification ,Sulfonamides ,Protease ,Molecular Structure ,biology ,Stereoisomerism ,HIV Protease Inhibitors ,Amides ,Protease inhibitor (biology) ,Enzyme ,chemistry ,Enzyme inhibitor ,Mutation ,biology.protein ,Molecular Medicine ,medicine.drug - Abstract
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, as compounds with other substitutions show lower binding affinities. Attempts to replace the isobutyl group at P1' with small cyclic moieties caused significant loss of affinities in the resulting compounds. Crystal structure analysis of the two most potent inhibitors in complex with the HIV-1 protease provided valuable information on the interactions between the inhibitor and the protease enzyme. In both inhibitor - enzyme complexes, the carbonyl group of the oxazolidinone ring makes hydrogenbond interactions with relatively conserved Asp29 residue of the protease. Potent inhibitors from each series incorporating various phenyloxazolidinone based P2 ligands were selected and their activities against a panel of multidrug-resistant (MDR) protease variants were determined. Interestingly, the most potent protease inhibitor starts out with extremely tight affinity for the wild-type enzyme (K(i) = 0.8 pM), and even against the MDR variants it retains picomolar to low nanomolar K(i), which is highly comparable with the best FDA-approved protease inhibitors.
- Published
- 2006
27. RecA Dimers Serve as a Functional Unit for Assembly of Active Nucleoprotein Filaments
- Author
-
Kendall L. Knight, Melissa A. Calmann, Celia A. Schiffer, Dharia A. McGrew, Anthony L. Forget, and Michelle M. Kudron
- Subjects
Adenosine Triphosphatases ,DNA repair ,Recombinant Fusion Proteins ,RAD51 ,macromolecular substances ,biochemical phenomena, metabolism, and nutrition ,Biology ,Biochemistry ,Molecular biology ,Article ,Nucleoprotein ,Protein filament ,Rec A Recombinases ,chemistry.chemical_compound ,Nucleoproteins ,chemistry ,Recombinase ,Biophysics ,bacteria ,Repressor lexA ,Homologous recombination ,Dimerization ,DNA - Abstract
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 as well as their coordinated activity during the catalysis of homologous recombination.
- Published
- 2006
28. Design of HIV-1 Protease Inhibitors Active on Multidrug-Resistant Virus
- Author
-
Dominique Louis Nestor Ghislain Surleraux, Anik Peeters, Sandra De Meyer, Rudi Wilfried Jan Pauwels, Moses Prabu-Jeyabalan, Piet Wigerinck, Celia A. Schiffer, Geert M. E. Pille, Hilde Azijn, Louis J. R. Maes, Herman Augustinus De Kock, Sandrine Marie Helene Vendeville, Wim Gaston Verschueren, Marie-Pierre de Béthune, and Nancy M. King
- Subjects
Models, Molecular ,Molecular model ,medicine.medical_treatment ,Calorimetry ,In Vitro Techniques ,Crystallography, X-Ray ,Virus ,Cell Line ,Dogs ,Drug Resistance, Multiple, Viral ,Drug Stability ,HIV-1 protease ,Drug Discovery ,medicine ,Animals ,Humans ,Protease inhibitor (pharmacology) ,Rats, Wistar ,chemistry.chemical_classification ,Benzoxazoles ,Sulfonamides ,Binding Sites ,Protease ,biology ,HIV Protease Inhibitors ,Rats ,Multiple drug resistance ,Thiazoles ,Enzyme ,Biochemistry ,chemistry ,Enzyme inhibitor ,HIV-1 ,Microsomes, Liver ,biology.protein ,Thermodynamics ,Molecular Medicine - Abstract
On the basis of structural data gathered during our ongoing HIV-1 protease inhibitors program, from which our clinical candidate TMC114 9 was selected, we have discovered new series of fused heteroaromatic sulfonamides. The further extension into the P2' region was aimed at identifying new classes of compounds with an improved broad spectrum activity and acceptable pharmacokinetic properties. Several of these compounds display an exceptional broad spectrum activity against a panel of highly cross-resistant mutants. Certain members of these series exhibit favorable pharmacokinetic profiles in rat and dog. Crystal structures and molecular modeling were used to rationalize the broad spectrum profile resulting from the extension into the P2' pocket of the HIV-1 protease.
- Published
- 2005
29. Nitric Oxide-Mediated Inhibition of Hdm2−p53 Binding
- Author
-
Alonzo H. Ross, Stephen N. Jones, Marie-Claire Daou, Christopher M. Schonhoff, and Celia A. Schiffer
- Subjects
Protein Conformation ,Recombinant Fusion Proteins ,Molecular Sequence Data ,Enzyme-Linked Immunosorbent Assay ,Biochemistry ,Dithiothreitol ,Nitric oxide ,chemistry.chemical_compound ,Residue (chemistry) ,Proto-Oncogene Proteins ,Humans ,Amino Acid Sequence ,Cysteine ,Glutathione Transferase ,Alanine ,Binding Sites ,Sequence Homology, Amino Acid ,Chemistry ,Nitrosylation ,Nuclear Proteins ,Proto-Oncogene Proteins c-mdm2 ,Glutathione ,In vitro ,Neoplasm Proteins ,Mutation ,Mutagenesis, Site-Directed ,Triazenes ,Tumor Suppressor Protein p53 ,Protein Binding ,P53 binding - Abstract
It has become increasingly evident that nitric oxide exerts its effects, in part, by S-nitrosylation of cysteine residues. We tested in vitro whether nitric oxide may indirectly control p53 by S-nitrosylation and inactivation of the p53 negative regulator, Hdm2. Treatment of Hdm2 with a nitric oxide donor inhibits Hdm2-p53 binding, a critical step in Hdm2 regulation of p53. The presence of excess amounts of cysteine or dithiothreitol blocks this inhibition of binding. Moreover, nitric oxide inhibition of Hdm2-p53 binding was found to be reversible. Sulfhydryl sensitivity and reversibility are consistent with nitrosylation. Finally, we have identified a critical cysteine residue that nitric oxide modifies to disrupt Hdm2-p53 binding. This cysteine is proximal to the Hdm2-p53 binding interface and is conserved across species from zebrafish to humans. Mutation of this residue from a cysteine to an alanine does not interfere with binding but rather eliminates the sensitivity of Hdm2 to nitric oxide inactivation.
- Published
- 2002
30. Crystal structure of human thymidylate synthase: a structural mechanism for guiding substrates into the active site
- Author
-
Daniel V. Santi, Robert M. Stroud, V. Jo Davisson, Ian J. Clifton, and Celia A. Schiffer
- Subjects
Models, Molecular ,Stereochemistry ,Protein subunit ,Molecular Sequence Data ,Crystal structure ,Biochemistry ,Structure-Activity Relationship ,Thymidine Monophosphate ,Humans ,Amino Acid Sequence ,Enzyme kinetics ,Conserved Sequence ,chemistry.chemical_classification ,Binding Sites ,Crystallography ,biology ,Active site ,Hydrogen Bonding ,Thymidylate Synthase ,biology.organism_classification ,Protein Structure, Tertiary ,Eukaryotic Cells ,Enzyme ,chemistry ,Docking (molecular) ,DNA Transposable Elements ,biology.protein ,Eukaryote ,Deoxyuracil Nucleotides ,Sequence Alignment ,Synchrotrons ,Cysteine - Abstract
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 residues between 156 and 157 (h146-h153) are known to be alpha-helical in other eukaryotes, and lie close together on the outside of the protein in regions of disordered electron density in this crystal form. Two cysteines [cys 202 (h199) and 213 (h210)] are close enough to form a disulfide bond within each subunit, and a third cysteine [cys 183 (h180)] is positioned to form a disulfide bond with the active site cysteine [cys 198 (h195)] in its unliganded conformation. The amino terminal 27 residues, unique to human TS, contains 8 proline residues, is also in a region of disordered electron density, and is likely to be flexible prior to substrate binding. The drug resistance mutation, Y6H, confers a 4-fold reduction in FdUMP affinity and 8-fold reduction in kcat for the dUMP reaction. Though indirectly connected to the active site, the structure suggests a mechanism of resistance that possibly involves a change in structure. This structure offers a unique opportunity for structure-based drug design aimed at the unliganded form of the human enzyme.
- Published
- 1995
31. Exploring the Role of the Solvent in the Denaturation of a Protein: A Molecular Dynamics Study of the DNA Binding Domain of the 434 Repressor
- Author
-
Volker Dötsch, Kurt Wüthrich, Celia A. Schiffer, and Wilfred F. van Gunsteren
- Subjects
Models, Molecular ,Protein Denaturation ,Protein Folding ,Magnetic Resonance Spectroscopy ,Chemical Phenomena ,HMG-box ,Molecular Sequence Data ,Biochemistry ,Viral Proteins ,Molecular dynamics ,Computer Simulation ,Denaturation (biochemistry) ,Amino Acid Sequence ,Binding Sites ,Aqueous solution ,Chemistry, Physical ,Chemistry ,Hydrogen Bonding ,DNA ,DNA-binding domain ,Repressor Proteins ,Solvent ,Crystallography ,Solvent models ,Solvents ,Biophysics ,Binding domain - Abstract
Molecular dynamics simulations of the DNA binding domain of 434 repressor are presented which aim at unraveling the role of solvent in protein denaturation. Four altered solvent models, each mimicking various possible aspects of the addition of a denaturant to the aqueous solvent, were used in the simulations to analyze their effects on the stability of the protein. The solvent was altered by selectively changing the Coulombic interaction between water and protein atoms and between different water molecules. The use of a modified solvent model has the advantage of mimicking the presence of denaturant without having denaturant molecules present in the simulation, which would require much longer simulations. In these simulations, only an increase in the solvent-protein Coulombic interaction causes initiation of protein unfolding in a manner consistent with NMR data. The altered solvent thus provides a model of a denaturing environment for studying protein unfolding.
- Published
- 1995
32. Stabilization of Type-I .beta.-Turn Conformations in Peptides Containing the NPNA-Repeat Motif of the Plasmodium falciparum Circumsporozoite Protein by Substituting Proline for (S)-.alpha.-Methylproline
- Author
-
Christian Bisang, Christoph K. Weber, Janice Inglis, Ilian Jelesarov, Celia A. Schiffer, Wilfred F. van Gunsteren, Hans Rudolf Bosshard, and John A. Robinson
- Subjects
Circumsporozoite protein ,Colloid and Surface Chemistry ,Alpha-methylproline ,biology ,Biochemistry ,Stereochemistry ,Chemistry ,Plasmodium falciparum ,General Chemistry ,Proline ,biology.organism_classification ,Catalysis - Published
- 1995
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.