Discusses her research on the types of psychological stressors faced by the spouses of police officers and their methods of coping.
Discusses the legal precedent and research basis of evaluating a defendant's mental competency to stand trial, the variables that contribute to non-restorable incompetence to stand trial, and the differences between patients who are restorable to competency versus those who are not restorable to competency.
Prototypical Recombinant Multi-Protease Inhibitor Resistant Infectious Molecular Clones of Human Immunodeficiency Virus Type-1
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 substitutions and 23 of the 27 tightest amino acid substitution clusters. Based on their phenotypic properties, the clones were classified into four groups with increasing cross-resistance to the PIs most commonly used for salvage therapy: lopinavir (LPV), tipranavir (TPV), and darunavir (DRV). The panel of recombinant infectious molecular clones has been made available without restriction through the NIH AIDS Research and Reference Reagent Program. The public availability of the panel makes it possible to compare the inhibitory activity of different PIs with one another. The diversity of the panel and the high-level PI resistance of its clones suggest that investigational PIs active against the clones in this panel will retain antiviral activity against most, if not all clinically relevant PI-resistant viruses.
Celia Schiffer, a Professor in Biochemistry and Molecular Pharmacology; a former Director of UMass Center for AIDS Research; and a Founder and Co-Director for the Institute for Drug Resistance (University of Massachusetts Medical School, MA, USA). Schiffer has an undergraduate degree in physics from the University of Chicago, with a PhD in biophysics from University of California, San Francisco (CA, USA). She was a postdoctoral associate first at the ETH in Zurich and then at Genentech in San Francisco. Schiffer has published more than 100 peer reviewed journal articles. Her laboratory primarily uses structural biology, biophysical and chemistry techniques to study the molecular basis for drug resistance in anti-virals including: HIV, Hepatitis C, Influenza and Dengue. Her laboratory designs, synthesizes and tests new antiviral inhibitors that should be more robust to resistance. She has overseen more than US$30 million in research funding. She is also an outstanding mentor and educator, receiving a faculty mentoring award, a highly sought after PhD advisor.
Interview conducted by Hannah Coaker, Assistant Commissioning Editor.
A sensitive assay using a native protein substrate for screening HIV-1 maturation inhibitors targeting the protease cleavage site between the matrix and capsid
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 of <3%. 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.
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 different clades of HIV as well as a panel of 12 drug-resistant viral strains. The substrate envelope model proves to be a powerful strategy to develop potent and robust inhibitors that avoid drug resistance.
Efficient Computation of Small-Molecule Configurational Binding Entropy and Free Energy Changes by Ensemble Enumeration
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.
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 prevents designed inhibitors from making interactions beyond those required by substrates and thus limits the availability of mutations tolerated by substrates but not by designed inhibitors. The strategy resulted in subnanomolar inhibitors that bind robustly across a clinically derived panel of drug-resistant variants. To further test the substrate-envelope hypothesis, here we have designed, synthesized, and assayed derivatives of our original compounds that are larger and extend outside the substrate envelope. Our designs resulted in pairs of compounds that are very similar to one another, but one respects and one violates the substrate envelope. The envelope-respecting inhibitor demonstrates robust binding across a panel of drug-resistant protease variants, whereas the envelope-violating one binds tightly to wild type but loses affinity to at least one variant. This study provides strong support for the substrate-envelope hypothesis as a design strategy for inhibitors that reduce susceptibility to resistance mutations.
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 a distinct advantage in that inhibitors of MA/CA would only need to affect a subset of the Gag molecules to disable viral replication. To discover MA/CA-specific inhibitors, we constructed a modified MA/CA fusion peptide (MA/CADelta) that contains the HIV protease (PR) cleavage site as well as a tetracysteine motif for fluorescent labeling. The HIV PR cleavage of MA/CADelta can then be monitored via fluorescence polarization (FP). We have adapted this FP assay for high-throughput screening and validated it according to industry standards using a 384-well plate format. We have currently tested 24,000 compounds in this assay and here detail the screening methodology and the results of this screening campaign.
Improving the Resistance Profile of Hepatitis C NS3/4A Inhibitors: Dynamic Substrate Envelope Guided Design
Drug resistance is a principal concern in the treatment of quickly evolving diseases. The viral protease NS3/4A is a primary drug target for the hepatitis C virus (HCV) and is known to evolve resistance mutations in response to drug therapy. At the molecular level, drug resistance reflects a subtle change in the balance of molecular recognition by NS3/4A; the drug resistant protease variants are no longer effectively inhibited by the competitive active site inhibitors but can still process the natural substrates with enough efficiency for viral survival. In previous works we have developed the "substrate envelope" hypothesis, which posits that inhibitors should be less susceptible to drug resistance if they better mimic the natural substrate molecular recognition features. In this work, we perform molecular dynamics simulations on four native substrates bound to NS3/4A and discover a clearly conserved dynamic substrate envelope. We show that the most severe drug resistance mutations in NS3/4A occur at residues that are outside the substrate envelope. Comparative analysis of three NS3/4A inhibitors reveals structural and dynamic characteristics of inhibitors that could lead to resistance. We also suggest inhibitor modifications to improve resistance profiles based on the dynamic substrate envelope. This study provides a general framework for guiding the development of novel inhibitors that will be more robust against resistance by mimicking the static and dynamic binding characteristics of natural substrates.
Influenza A virus (IAV) is a major cause of morbidity and mortality throughout the world. Current antiviral therapies include oseltamivir, a neuraminidase inhibitor that prevents the release of nascent viral particles from infected cells. However, the IAV genome can evolve rapidly, and oseltamivir resistance mutations have been detected in numerous clinical samples. Using an in vitro evolution platform and whole-genome population sequencing, we investigated the population genomics of IAV during the development of oseltamivir resistance. Strain A/Brisbane/59/2007 (H1N1) was grown in Madin-Darby canine kidney cells with or without escalating concentrations of oseltamivir over serial passages. Following drug treatment, the H274Y resistance mutation fixed reproducibly within the population. The presence of the H274Y mutation in the viral population, at either a low or a high frequency, led to measurable changes in the neuraminidase inhibition assay. Surprisingly, fixation of the resistance mutation was not accompanied by alterations of viral population diversity or differentiation, and oseltamivir did not alter the selective environment. While the neighboring K248E mutation was also a target of positive selection prior to H274Y fixation, H274Y was the primary beneficial mutation in the population. In addition, once evolved, the H274Y mutation persisted after the withdrawal of the drug, even when not fixed in viral populations. We conclude that only selection of H274Y is required for oseltamivir resistance and that H274Y is not deleterious in the absence of the drug. These collective results could offer an explanation for the recent reproducible rise in oseltamivir resistance in seasonal H1N1 IAV strains in humans.
Crystal structures of human CtBP in complex with substrate MTOB reveal active site features useful for inhibitor design
The oncogenic corepressors C-terminal Binding Protein (CtBP) 1 and 2 harbor regulatory d-isomer specific 2-hydroxyacid dehydrogenase (d2-HDH) domains. 4-Methylthio 2-oxobutyric acid (MTOB) exhibits substrate inhibition and can interfere with CtBP oncogenic activity in cell culture and mice. Crystal structures of human CtBP1 and CtBP2 in complex with MTOB and NAD(+) revealed two key features: a conserved tryptophan that likely contributes to substrate specificity and a hydrophilic cavity that links MTOB with an NAD(+) phosphate. Neither feature is present in other d2-HDH enzymes. These structures thus offer key opportunities for the development of highly selective anti-neoplastic CtBP inhibitors. Elsevier B.V. All rights reserved.
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 30's interaction with the substrate are compensated by the coevolving L449F and S451N cleavage site mutations. This interdependency in the PR-p1-p6 interactions enhances intermolecular contacts and reinforces the overall fit of the substrate within the substrate envelope, likely enabling coevolution to sustain substrate recognition and cleavage in the presence of PR resistance mutations. IMPORTANCE: Resistance to human immunodeficiency virus type 1 (HIV-1) protease inhibitors challenges the effectiveness of therapies in treating HIV-1-infected individuals and AIDS patients. Mutations in HIV-1 protease selected under the pressure of protease inhibitors render the inhibitors less potent. Occasionally, Gag sequences also mutate and coevolve with protease, contributing to maintenance of viral fitness and to drug resistance. In this study, we investigated the structural basis of coevolution at the Gag p1-p6 cleavage site with the nelfinavir (NFV) resistance D30N/N88D protease mutations. Our structural analysis reveals the interdependency of protease-substrate interactions and how coevolution may restore substrate recognition and cleavage in the presence of protease drug resistance mutations.
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 ensemble and dynamics of protease are impacted by the drug resistance mutations in Flap+ variant. Surprisingly this change in ensemble dynamics is different from that observed in the unliganded form of the same variant (Cai, Y. et al. J. Chem. Theory Comput. 2012, 8, 3452-3462). Our comparative analysis of both inhibitor-free and bound states presents a comprehensive picture of the altered dynamics in drug-resistant mutant HIV-1 protease and underlies the importance of incorporating dynamic analysis of the whole system, including the unliganded state, into revealing drug resistance mechanisms.
Drug resistance conferred by mutations outside the active site through alterations in the dynamic and structural ensemble of HIV-1 protease
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.
Structural basis and distal effects of Gag substrate coevolution in drug resistance to HIV-1 protease
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 residue has distal effects on other residues' interactions as well, including through the induction of a conformational change in the protease. Additionally, molecular dynamics simulations suggest that restoration of active site dynamics is an additional constraint in the selection of coevolved mutations. Hence, protease-substrate coevolution permits mutational, structural, and dynamic changes via molecular mechanisms that involve distal effects contributing to drug resistance.
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 inhibitor's resistance barrier, likely due to poising the inhibitor to its bound conformation. Macrocyclic inhibitors with P2* extension moieties avoiding interaction with the protease S2 residues including Arg155 must be chosen for future design of more robust protease inhibitors.
Acute ischemic stroke imaging is one of the leading causes of death and disability worldwide. Neuroimaging plays a crucial role in early diagnosis and yields essential information regarding tissue integrity, a factor that remains a key therapeutic determinant. Given the widespread public health implications of stroke and central role of neuroimaging in overall management, acute stroke imaging remains a heavily debated, extensively researched, and rapidly evolving subject. There has been recent debate in the scientific community due to divided opinions on the use of CT perfusion and access-related limitations of MRI. In this paper we review and summarize recent updates relevant to acute stroke imaging and propose an imaging paradigm based on the recently available evidence.
The human intestine is a large and delicately balanced organ, responsible for efficiently absorbing nutrients and selectively eliminating disease-causing pathogens. The gut architecture consists of a single layer of epithelial cells that forms a barrier against the food antigens and resident microbiota within the lumen. This barrier is augmented by a thick layer of mucus on the luminal side and an underlying lamina propria containing a resident population of immune cells. Attempted breaches of the intestinal barrier by pathogenic bacteria result in the rapid induction of a coordinated innate immune response that includes release of antimicrobial peptides, activation of pattern recognition receptors, and recruitment of various immune cells. In recent years, the role of epithelial cells in initiating this immune response has been increasingly appreciated. In particular, epithelial cells are responsible for the release of a variety of factors that attract neutrophils, the body's trained bacterial killers. In this review we will highlight recent research that details a new understanding of how epithelial cells directionally secrete specific compounds at distinct stages of the inflammatory response in order to coordinate the immune response to intestinal microbes. In addition to their importance during the response to infection, evidence suggests that dysregulation of these pathways may contribute to pathologic inflammation during inflammatory bowel disease. Therefore, a continued understanding of the mechanisms by which epithelial cells control neutrophil migration into the intestine will have tremendous benefits in both the understanding of biological processes and the identification of potential therapeutic targets.
Neutropenia, defined as an absolute neutrophil count (ANC) < 1.5 x 10(9)/L, encompasses a wide range of diagnoses, from normal variants to life-threatening acquired and congenital disorders. This review addresses the diagnosis and management of isolated neutropenia, not multiple cytopenias due to splenomegaly, bone marrow replacement, or myelosuppression by chemotherapy or radiation. Laboratory evaluation generally includes repeat complete blood cell counts (CBCs) with differentials and bone marrow examination with cytogenetics. Neutrophil antibody testing may be useful but only in the context of clinical and bone marrow findings. The discovery of genes responsible for congenital neutropenias now permits genetic diagnosis in many cases. Management of severe chronic neutropenia includes commonsense precautions to avoid infection, aggressive treatment of bacterial or fungal infections, and administration of granulocyte colony-stimulating factor (G-CSF). Patients with severe chronic neutropenia, particularly those who respond poorly to G-CSF, have a risk of eventually developing myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML) and require monitoring for this complication, which also can occur without G-CSF therapy. Patients with cyclic, idiopathic, and autoimmune neutropenia have virtually no risk of evolving to MDS or AML. Hematopoietic stem cell transplantation is a curative therapy for congenital neutropenia with MDS/AML or with cytogenetic abnormalities indicating impending conversion.