Pharmacology, Toxicology and Environmental Health Commons^™

Pharmacology Of Legal And Illicit Drugs, Ashraf Mozayani

Ashraf Mozayani, Ph.D., PharmD

The pharmacology of any xenobiotic is a complex set of processes that is frequently divided into two categories: pharmacokinetics and pharmacodynamics. These two processes encompass the various steps that the body takes to transform the drug to facilitate its excretion (pharmacokinetics) and the interactions of the drug with the body which result in the observed effects (pharmacodynamics). The study of these processes includes the determination of kinetic parameters to describe the rate of elimination of the drug from the body, binding affinities to describe the interaction of the drug with endogenous receptors, and the determination of enzymes that are instrumental …

Pgf2Α-Associated Vascular Smooth Muscle Hypertrophy Is Ros Dependent And Involves The Activation Of Mtor, P70s6k, And Pten, Kevin Rice, Sreevani Uddemarri, Devashish Desai, Ryan Morrison, R. Harris, Eric Blough

Kevin M Rice

Prostaglandin F2α (PGF2α) increases reactive oxygen species (ROS) and induces vascular smooth muscle cell (VSMC) hypertrophy by largely unknown mechanism(s). To investigate the signaling events governing PGF2α –induced VSMC hypertrophy we examined the ability of the PGF2α analog, fluprostenol to elicit phosphorylation of Akt, the mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase (p70S6k), glycogen synthase kinase-3β (GSK-3β), phosphatase and tensin homolog (PTEN), extracellular signal-regulated kinase 1/2 (ERK1/2) and Jun N-terminal kinase (JNK) in growth arrested A7r5 VSMC. Fluprostenol-induced hypertrophy was associated with increased ROS, mTOR translocation from the nucleus to the cytoplasm, along with Akt, mTOR, GSK-3β, PTEN …

Pgf2Α-Associated Vascular Smooth Muscle Hypertrophy Is Ros Dependent And Involves The Activation Of Mtor, P70s6k, And Pten, Kevin Rice, Sreevani Uddemarri, Devashish Desai, Ryan Morrison, R. Harris, Eric Blough

Eric Blough

Prostaglandin F2α (PGF2α) increases reactive oxygen species (ROS) and induces vascular smooth muscle cell (VSMC) hypertrophy by largely unknown mechanism(s). To investigate the signaling events governing PGF2α –induced VSMC hypertrophy we examined the ability of the PGF2α analog, fluprostenol to elicit phosphorylation of Akt, the mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase (p70S6k), glycogen synthase kinase-3β (GSK-3β), phosphatase and tensin homolog (PTEN), extracellular signal-regulated kinase 1/2 (ERK1/2) and Jun N-terminal kinase (JNK) in growth arrested A7r5 VSMC. Fluprostenol-induced hypertrophy was associated with increased ROS, mTOR translocation from the nucleus to the cytoplasm, along with Akt, mTOR, GSK-3β, PTEN …

Prepubertal Organochlorine Pesticide Concentrations And Age Of Pubertal Onset Among Russian Boys, Thuy Lam, Paige Williams, Mary Lee, Susan Korrick, Linda Birnbaum, Jane Burns, Oleg Sergeyev, Boris Revich, Larisa Altshul, Donald Patterson, Wayman Turner, Russ Hauser

Mary M. Lee

BACKGROUND: In animal studies, organochlorine pesticide (OCP) exposure alters pubertal development; however, epidemiological data are limited and inconsistent. OBJECTIVE: To evaluate the associations of serum OCP concentrations [hexachlorobenzene (HCB), beta-hexachlorocyclohexane (beta-HCH), and p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE)] with male pubertal onset. METHODS: In Chapaevsk, Russia, a town environmentally contaminated with OCPs, 350 8-9 year old boys with measured OCPs were enrolled during 2003-2005 and were followed annually for eight years. We evaluated three measures of pubertal onset: testicular volume (TV) > 3 mL in either testis, or stage 2 or greater for genitalia (G2+), or pubic hair (P2+). We used multivariable interval-censored models to …

Association Between Chlorinated Pesticides In The Serum Of Prepubertal Russian Boys And Longitudinal Biomarkers Of Metabolic Function, Jane Burns, Paige Williams, Susan Korrick, Russ Hauser, Oleg Sergeyev, Boris Revich, Thuy Lam, Mary Lee

Mary M. Lee

Organochlorine pesticides (OCPs) have been linked to adult metabolic disorders; however, few studies have examined these associations in childhood. We prospectively evaluated the associations of baseline serum OCPs (hexachlorobenzene, beta-hexachlorocyclohexane, and p,p'-dichlorodiphenyldichloroethylene) in Russian boys with subsequent repeated measurements of serum glucose, insulin, lipids, leptin, and calculated homeostatic model assessment of insulin resistance (IR). During 2003-2005, we enrolled 499 boys aged 8-9 years in a prospective cohort; 318 had baseline serum OCPs and serum biomarkers measured at ages 10-13 years. Multivariable generalized estimating equation and mediation regression models were used to examine associations and direct and indirect (via body mass …

An Aryl Hydrocarbon Receptor From The Salamander Ambystoma Mexicanum Exhibits Low Sensitivity To 2,3,7,8-Tetrachlorodibenzo-P-Dioxin, Wade Powell

Wade Powell

Structural Basis And Distal Effects Of Gag Substrate Coevolution In Drug Resistance To Hiv-1 Protease, Aysegul Ozen, Kuan-Hung Lin, Nese Yilmaz, Celia Schiffer

Celia A. Schiffer

Drug resistance mutations in response to HIV-1 protease inhibitors are selected not only in the drug target but elsewhere in the viral genome, especially at the protease cleavage sites in the precursor protein Gag. To understand the molecular basis of this protease-substrate coevolution, we solved the crystal structures of drug resistant I50V/A71V HIV-1 protease with p1-p6 substrates bearing coevolved mutations. Analyses of the protease-substrate interactions reveal that compensatory coevolved mutations in the substrate do not restore interactions lost due to protease mutations, but instead establish other interactions that are not restricted to the site of mutation. Mutation of a substrate …

Structural Analysis Of Asunaprevir Resistance In Hcv Ns3/4a Protease, Djade Soumana, Akbar Ali, Celia Schiffer

Celia A. Schiffer

Asunaprevir (ASV), an isoquinoline-based competitive inhibitor targeting the hepatitis C virus (HCV) NS3/4A protease, is very potent in vivo. However, the potency is significantly compromised by the drug resistance mutations R155K and D168A. In this study three crystal structures of ASV and an analogue were determined to analyze the structural basis of drug resistance susceptibility. These structures revealed that ASV makes extensive contacts with Arg155 outside the substrate envelope. Arg155 in turn is stabilized by Asp168, and thus when either residue is mutated, the enzyme's interaction with ASV's P2* isoquinoline is disrupted. Adding a P1-P3 macrocycle to ASV enhances the …

A Sensitive Assay Using A Native Protein Substrate For Screening Hiv-1 Maturation Inhibitors Targeting The Protease Cleavage Site Between The Matrix And Capsid, Sook-Kyung Lee, Nancy Cheng, Emily Hull-Ryde, Marc Potempa, Celia Schiffer, William Janzen, Ronald Swanstrom

Celia A. Schiffer

The matrix/capsid processing site in the HIV-1 Gag precursor is likely the most sensitive target to inhibit HIV-1 replication. We have previously shown that modest incomplete processing at the site leads to a complete loss of virion infectivity. In the study presented here, a sensitive assay based on fluorescence polarization that can monitor cleavage at the MA/CA site in the context of the folded protein substrate is described. The substrate, an MA/CA fusion protein, was labeled with the fluorescein-based FlAsH (fluorescein arsenical hairpin) reagent that binds to a tetracysteine motif (CCGPCC) that was introduced within the N-terminal domain of CA. …

Drug Resistance Conferred By Mutations Outside The Active Site Through Alterations In The Dynamic And Structural Ensemble Of Hiv-1 Protease, Debra Ragland, Ellen Nalivaika, Madhavi Nalam, Kristina Prachanronarong, Hong Cao, Rajintha Bandaranayake, Yufeng Cai, Nese Yilmaz, Celia Schiffer

Celia A. Schiffer

HIV-1 protease inhibitors are part of the highly active antiretroviral therapy effectively used in the treatment of HIV infection and AIDS. Darunavir (DRV) is the most potent of these inhibitors, soliciting drug resistance only when a complex combination of mutations occur both inside and outside the protease active site. With few exceptions, the role of mutations outside the active site in conferring resistance remains largely elusive. Through a series of DRV-protease complex crystal structures, inhibition assays, and molecular dynamics simulations, we find that single and double site mutations outside the active site often associated with DRV resistance alter the structure …

Testing The Substrate-Envelope Hypothesis With Designed Pairs Of Compounds, Yang Shen, Michael Altman, Akbar Ali, Madhavi Nalam, Hong Cao, Tariq Rana, Celia Schiffer, Bruce Tidor

Celia A. Schiffer

Acquired resistance to therapeutic agents is a significant barrier to the development of clinically effective treatments for diseases in which evolution occurs on clinical time scales, frequently arising from target mutations. We previously reported a general strategy to design effective inhibitors for rapidly mutating enzyme targets, which we demonstrated for HIV-1 protease inhibition [Altman et al. J. Am. Chem. Soc. 2008, 130, 6099-6113]. Specifically, we developed a computational inverse design procedure with the added constraint that designed inhibitors bind entirely inside the substrate envelope, a consensus volume occupied by natural substrates. The rationale for the substrate-envelope constraint is that it …

Development Of A Novel Screening Strategy Designed To Discover A New Class Of Hiv Drugs, Nancy Cheng, Sook-Kyung Lee, P. Donover, Mel Reichman, Celia Schiffer, Emily Hull-Ryde, Ronald Swanstrom, William Janzen

Celia A. Schiffer

Current antiretroviral treatments target multiple pathways important for human immunodeficiency virus (HIV) multiplication, including viral entry, synthesis and integration of the DNA provirus, and the processing of viral polyprotein precursors. However, HIV is becoming increasingly resistant to these "combination therapies." Recent findings show that inhibition of HIV Gag protein cleavage into its two structural proteins, matrix (MA) and capsid (CA), has a devastating effect on viral production, revealing a potential new target class for HIV treatment. Unlike the widely used HIV protease inhibitors, this new class of inhibitor would target the substrate, not the protease enzyme itself. This approach offers …

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 …

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 …

Polychlorinated Biphenyl Exposure Alters Oxytocin Receptor Gene Expression And Maternal Behavior In Rat, Howard Cromwell

Howard Casey Cromwell

Polychlorinated biphenyl (PCB) is a persistent organic pollutant known to induce diverse molecular and behavioral alterations. Effects of PCB exposure could be transmitted to future generations via changes in behavior and gene expression. Previous work has shown that PCB-exposure can alter social behavior. The present study extends this work by examining a possible molecular mechanism for these changes. Pregnant rats (Sprague-Dawley) were exposed through diet to a combination of non-coplanar (PCB 47 - 2,20,4,40-tetrachlorobiphenyl) and coplanar (PCB 77 - 3,30,4,40- tetrachlorobiphenyl) congeners. Maternal care behaviors were examined by evaluating the rate and quality of nest building on the last 4 …

Connecting Through Consilience: Ecology, Society, Culture And Technology, Ruth Mirams, Alexander Hayes

Alexander Hayes Mr.

Amongst linguistic, cultural and geographic diversity, humanity is characterised by inquisitiveness, communication and a deep desire to connect with each other. Despite our advanced intelligence and technological capacity, we are creatures of nature - a species which occupies a habitat, depends on consumable resources and fragile in many ways. As a species, we currently face challenges including overpopulation, diminishing resources and habitat degradation. In essence, we are exhausting the resources we depend on. [1] Resource depletion, disruption, famine, growth and sustainability are all observable in other species and natural systems. Human societies and systems can be described through the same …

Research By Dr. Mozayani, Ashraf Mozayani

Ashraf Mozayani, Ph.D., PharmD

No abstract provided.

Books Written By Dr. Mozayani, Ashraf Mozayani

Ashraf Mozayani, Ph.D., PharmD

No abstract provided.

Books Published, Ashraf Mozayani

Ashraf Mozayani, Ph.D., PharmD

No abstract provided.

Prolactin And Fmri Response To Skf38393 In The Baboon, Brad D. Miller, Lauren A. Marks, Jonathan M. Koller, Blake J. Newman, G Larry Bretthorst, Kevin J. Black

Kevin J. Black, MD

Background: This study’s goal was to provide dose-response data for a dopamine agonist in the baboon using standard methods (replicate measurements at each dose, across a range of doses), as a standard against which to subsequently validate a novel pharmacological MRI (phMRI) method. Dependent variables were functional MRI (fMRI) data from brain regions selected a priori, and systemic prolactin release. Necessary first steps included estimating the magnitude and time course of prolactin response to anesthesia alone and to various doses of agonist. These first steps (“time course studies”) were performed with three agonists, and the results were used to select …

Acute Toxicity Of Copper Sulfate And Potassium Dichromate On Stygobiont Proasellus: General Aspects Of Groundwater Ecotoxicology And Future Perspectives, Ana Reboleira, Nelson Abrantes, Pedro Oromí, Fernando Gonçalves

Ana Sofia P.S. Reboleira

Karst systems harbor large groundwater resources for human consumption and represent an important habitat for rare and unprotected specialized animals, the so-called stygofauna. Due to the highly adapted features towards underground life, together with the geographic isolation provided by the subterranean aquifers, groundwater-dwelling animals may lose the ability to face sudden changes on their ecosystems, and therefore the risk of extinction is remarkably high. A little is known about their sensitiveness, especially linked to contamination pressure in urbanized karst areas. Understanding the impact of contaminants on stygofauna is important for setting groundwater environmental quality and management of karst systems. We …

Advanced Molecular Biologic Techniques In Toxicologic Disease, Jeanine Ward, Gyongyi Szabo, David Mcmanus, Edward Boyer

Gyongyi Szabo

The advancement of molecular biologic techniques and their capabilities to answer questions pertaining to mechanisms of pathophysiologic events have greatly expanded over the past few years. In particular, these opportunities have provided researchers and clinicians alike the framework from with which to answer clinical questions not amenable for elucidation using previous, more antiquated methods. Utilizing extremely small molecules, namely microRNA, DNA, protein, and nanoparticles, we discuss the background and utility of these approaches to the progressive, practicing physician. Finally, we consider the application of these tools employed as future bedside point of care tests, aiding in the ultimate goal of …

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 …

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, …

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.

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.

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 …

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 …

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 …

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 …