David Sherman - US grants

Nonrheumatic atrial fibrillation (AF) occurs in 1.5 million Americans and carries a fivefold increased risk of stroke - some 75,000 strokes yearly. In November 1989, the first phase of the SPAF Study was terminated early due to unequivocal evidence that both aspirin and warfarin were superior to placebo for stroke prevention (p<.02, 49-81% reduction). Data were insufficient to determine the relative value of aspirin vs. warfarin. Major objectives of the multicenter, randomized, ongoing SPAF Study are to: 1. Compare the efficacy/safety of warfarin vs. aspirin for stroke prevention in AF pts. a. over a 2-4 yr follow-up period to assure sustained effects. b. assessing stroke severity/functional outcome vs. treatments. 2. Identify subgroups who might differentially respond to aspirin vs. warfarin, particularly age, sex and presence of carotid artery disease. 3. Organize a collaborative meta-analysis of recent randomized antithrombotic trials. Design: SPAF is a randomized treatment-efficacy trial ongoing at 15 clinical sites testing aspirin 325mg/day vs. warfarin (prothrombin time range 1.3-1.8 control) given nonblindedly. Primary events (ischemic stroke, systemic embolism) are assessed by a blinded Events Committee. The 1050 sample size allows independent determination of efficacy in pts under age 76 (n=675) and pts over age 75 (n=375). Power to detect clinically important differences in aspirin vs. warfarin is >.8 in each age group(alpha = .05, two-sided] By June 1990, 810 pts have been entered. Entry of pts over age 75 continues until June 1991 with follow-up of all pts until December 1992. Relevance: Recent randomized trials show that warfarin anticoagulation importantly reduces the stroke risk in AF pts. SPAF proved that aspirin is also effective, but that this effect is not uniform in all subpopulations. Determination of which pts should receive aspirin vs. warfarin is a critical clinical issue, affecting millions of people with AF. SPAF is the only clinical trial likely to yield an answer in the forseeable future.

chemoprevention; aspirin; carotid artery; cardiovascular disorder prevention; endarterectomy; stroke; preoperative state; postoperative state; human mortality; clinical research; human subject;

DESCRIPTION (Verbatim from the Applicant's Abstract): Combinatorial biology is a powerful approach for harnessing the tremendous biosynthetic capability of microorganisms, including primary and secondary pathways. A key aspect of this overall approach is the identification and characterization of genes and enzymes that prescribe the assembly of complex natural product molecules. Although access to rich genomic diversity is essential, tools for expression of genes in an appropriate microbial host is equally important to successful development of broad-based combinatorial biosynthetic systems. The aim of the proposed work is to develop a more thorough understanding of the functionality of individual polyketide synthase (PKS) modules (loading, processing, and termination), the mechanism of metabolic branching leading to construction of two distinct ring systems, the basis for flexible substrate specificity of two key tailoring enzymes, and overall pathway regulation in the pikromycin (pik) biosynthetic pathway from Streptomyces venezuelae. The pikromycin system includes a set of 18 genes residing on 60 kb of DNA with a locus containing two resistance genes, a polyketide synthase locus encoding six modules and a type II thioesterase, a desosamine biosynthetic locus comprised of 9 genes, a single cytochrome P450 hydroxylase and a putative gene involved in pathway regulation. In the first stage of this project, the role of a unique beta-ketosynthase domain will be investigated and a series of hybrid PKSs containing engineered loading domains will be constructed and analyzed for production of novel natural product modules. Second, the unusual ability of the pik PKS to generate 12- and 14-membered ring macrolactones will be studied to elucidate the genetic and biochemical basis for generating structural diversity. A series of engineered PKS systems will be assembled and based on contraction and expansion of the modular system in order to probe molecular recognition and metabolic flexibility. Finally, the mechanism of polyketide chain termination will be explored and hybrid PKSs constructed through manipulation of the thioesterase domain and thioesterase II of the pik PKS. Overall, we expect this work will provide key information on substrate specificity and function of the various pik-encoded catalytic domains and enzymes and provide insight into how PKS modules can be engineered to create novel biologically active molecules.

The overall aim of the proposed research is to understand the molecular mechanisms controlling the biosynthesis of and cellular resistance to the antitumor antibiotic, mitomycin C. This important chemotherapeutic agent is biosynthetically derived from a shikimate pathway metabolite (3-amino-5-hydroxybenzoic acid) and D-glucosamine. In this work, molecular genetic, biochemical and chemical approaches will be used to obtain information on the functional role of the set of genes and enzymes involved in constructing this important anticancer drug. Our initial work demonstrated that Streptomyces lavendulae (the mitomycin producer) had at least two genetic loci (mcr and mrd) that specify resistance to mitomycin. Identification of cosmid clones containing DNA adjacent to the resistance genes revealed that mitomycin biosynthetic genes are clustered around the mitomycin resistance determinant (mrd). Using probes for shikimate pathway genes, homologs to the dehydroquinase, dehydroquinate synthase, 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase and 3-amino-5-hydroxybenzoic acid synthase (AHBAS) were identified among a total of 47 genes within the 55 kb cluster. With this information in hand, gene disruption/replacement, mutant complementation, and biochemical experiments will be performed to probe the precise function of the individual mitomycin biosynthetic enzymes. This information will be used to identify and characterize the mechanism and specificity of the enzyme(s) responsible for coupling the AHBA precursor to the D-glucosamine sugar moiety. Subsequently, studies will be initiated to understand and manipulate enzymes involved in establishing the core mitosane structure and the specificity of tailoring enzymes that provide molecular diversity to this significant class of metabolites. Concurrently, our work will continue on the resistance mechanisms that provide cellular self-protection against mitomycins in the producing organism. Overall, this work will provide an important theoretical and experimental base for future combinatorial biology-based production of novel AHBA-derived natural products using molecular genetic technology.

The objective of the proposed research is to use in vitro metabolic engineering combined with combinatorial biology and microscale processing to evaluate the functional diversity of a complex metabolic network, namely, polyketide synthesis. The investigators will develop a microscale, microfluidic device for the in vitro combinatorial biosynthesis of complex polyketides. Also, because of the microscale component and the modularity of the polyketide synthesis, the investigators expect to use the principles of multi-step enzymatic networks (e.g., a metabolic pathway) to alter the progress of the pathway and generate unique compounds. The class of polyketides used in this research consists of a small number of polypeptides each containing multiple modules. Each module is responsible for one round of chain extension and post-condensation modifications. The modules will be immobilized into the channels of the microfabricated device, a mutant thioesterase domain will be engineered into each module (necessary for release and transport to the next module), and the products will be transported from one module to the next. This research could enable the synthesis of molecules with new and unusual functions. Also, this research could add to the fundamental understanding of metabolic pathways and microfluidics.

DESCRIPTION (provided by the applicant): Central to the pathogenic success of Mycobacterium tuberculosis (MTB) is its ability to persist within humans for long periods in a latent state, without causing any overt disease symptoms. Roughly one-third of the world population harbors latent MTB, greatly complicating efforts at tuberculosis control. A person with latent tuberculosis has about a 10 percent lifetime chance of developing active disease, and when such a person contracts HIV, the risk of developing reactivation TB increases to 8 - 10 percent per year. Hypoxic conditions within the human host are widely regarded as crucial for development of latent tuberculosis, but the MTB adaptive response to hypoxia is at present very poorly understood. The goal of this proposal is to define the MTB hypoxic response as it relates to latency and reactivation. The a-crystallin protein of MTB is powerfully induced by hypoxia and has been implicated in long-term survival. Alpha-crystallin will be used as a model system to determine the mechanisms by which oxygen tension control MTB gene expression. In addition, specific conditions in which Alpha-crystallin is necessary for achieving latency and reactivation will be determined. Finally, with Alpha-crystallin as an example, other proteins important for the adaptation to and from hypoxia will be identified and characterized. The result will be better tools to confront the threat to more than one billion persons with latent tuberculosis, millions of whom are now or will soon be co-infected with the human immunodeficiency virus, HIV.

DESCRIPTION (Provided by the applicant): Central to the pathogenic success of Mycobacterium tuberculosis (MTB) is its ability to persist within humans for long periods in a latent state, without causing any overt disease symptoms. Roughly one-third of the world population harbors latent MTB, greatly complicating efforts at tuberculosis control. A person with latent tuberculosis has about a 10 percent lifetime chance of developing active disease, and when such a person contracts HIV, the risk of developing reactivation TB increases to 8 - 10 percent per year. Hypoxic conditions within the human host are widely regarded as crucial for development of latent tuberculosis, but the MTB adaptive response to hypoxia is at present very poorly understood. The goal of this proposal is to define the MTB hypoxic response as it relates to latency and reactivation. We will mechanistically dissect this response and analyze the role of hypoxia in latent tuberculosis and reactivation. This proposal will define the genes whose response to reduced oxygen tension comprises the MTB hypoxia regulon. We will also focus on MTB alpha-crystallin (Acr), a component of the hypoxic response that is powerfully induced by microaerophilic conditions. We will determine the specific conditions in which expression of alpha-crystallin and its regulators is necessary for achieving latency or reactivation. Finally, we will dissect the alpha-crystallin regulatory machinery to determine the precise mechanisms by which oxygen tension controls MTB gene expression. The result will be better tools to confront the threat to more than one billion persons with latent tuberculosis, millions of whom are now or will soon be co-infected the the human immunodeficiency virus, HIV.

DESCRIPTION (provided by applicant): The aim of this competing continuation proposal is to develop a rational approach for metabolic engineering of secondary metabolite production using microbial genomic technologies. In this work both subset and genome-wide microarray methods will be used to analyze secondary metabolism in Streptomyces coelicolor. Quantitative physiological and modeling approaches will be combined to obtain information on temporal and conditional expression of global and pathway-specific regulatory factors for antibiotic biosynthetic pathways. In order to dissect further the control architecture in these multi-step biosynthetic systems, key regulatory elements will be investigated and their role in the circuitry of secondary metabolism defined. In addition, controlled expression of structural and regulatory genes in the actinorhodin and undecylprodigiosin biosynthesis will be analyzed to provide a genome-wide understanding of the intricate mechanisms affecting these secondary metabolic pathways. The specific objectives of this project are: I. Perform genome-wide microarray analysis to monitor expression of absA, eight absA-homologs, and the cutR/S and afsQ1/Q2 two-component regulatory genes involved in secondary metabolite biosynthesis in wild type S. coelicolor. II. Construction of the corresponding isogenic mutant strains for each of the two-component regulators noted in Aim I, for subsequent S. coelicolor genome microarray analysis. Phenotypic profiling (e.g. growth rate, antibiotic biosynthesis, morphological characteristics) will be performed for each isogenic strain. Ill. Construction of recombinant S. coelicolor strains with engineered regulatory gene::gfp fusions to study at the proteomic level temporal and spatial expression patterns for secondary metabolism. From Specific Aims I - Ill, combine Boolean modeling with data from genomic microarray, mutant phenotype profiling and GFP expression analysis to decode the primary network of regulatory circuits in S. coelicolor secondary metabolism. With these methods established, apply high throughput approaches to additional regulatory systems identified using the methods of Aims I -Ill to establish the detailed layered regulatory network involved in control of antibiotic metabolic pathway gene expression in the S. coelicolor genome.

Although a lung is a portal of entry for this M. tuberculosis and chronic pneumonia is the most common disease caused by M. tuberculosis infection, the specific nature of the pulmonary host response to this pathogen is relatively unknown. The central goal of the studies outlined in this proposal is to determine whether components of the early pulmonary immune response are critical determinants for resistance or susceptibility to disease caused by M. tuberculosis. The specific aims will address the role of TNF-alpha, beta chemokines and the B7/CD28/CTLA4 costimulation pathway in the initiation and amplification of the pulmonary host response to aerosol infection with M. tuberculosis. Preliminary studies using transgenic mice with a local inhibition of TNF-alpha in the lung (SPCTNFRIIFc mice) indicate that blockade of TNF-alpha selectively in the lung results in early deaths, severe lung pathology and an alteration of antigen specific immunity. These studies also indicate that transgenic mice with local inhibition of the immune response in the lung are a useful model to study pulmonary host defense. Further studies are proposed to compare the response that develops in the lung vs. regional lymph nodes in wildtype and SPCTNFRIIFc mice. To investigate the role of beta chemokines in the pulmonary host defense, transgenic mice that secrete a virally encoded inhibitor of all chemokines has been generated. The phenotype of these mice will be determined then used to investigate the role of beta chemokines in the initiation of the pulmonary host response to M. tuberculosis. To investigate the role of the B7/CD28/CTLA4 costimulation pathway in the initiation and amplification of the pulmonary host response to M. tuberculosis, transgenic mice that secrete an inhibitor of the B7/CD28/CTLA4 co stimulation pathway will be generated, characterized then used in experiments examining the immune response to M. tuberculosis. To determine the relative importance of the intrapulmonary component of the immune response vs the systemic immune response, in each of the project specific aims, mice will be included with both local inhibition of the immune response and systemic inhibition. In the studies examining the pulmonary host response to M. tuberculosis, infections will occur via the aerosol route because inhalation is the usual route of infection in humans. The primary endpoints will include bacterial burden, lung histology, survival and the phenotypic and functional characteristics of the immune response. The proposed studies should advance the existing knowledge of the specific nature of the pulmonary immune response to M. tuberculosis, will directly test the role of these mediators in host defense against M. tuberculosis, suggest important targets for novel therapies and elucidate role of specific mediators of the host immune response that are critical for the induction of an antigen specific immune response by candidate vaccines.

Social support is a resource that allows people to live healthier and more productive lives; it becomes especially important in times of rapid change. Change creates stress with which people need to cope, and they often turn to others for advice, information, and understanding. Social support has long been known to promote psychological health and to protect against the adverse health effects of stress. Yet, in conceptualizing social support, researchers have inadvertently adopted a Western definition that emphasizes explicit efforts to extract or provide help or comfort. The proposed research builds on several preliminary studies showing that Asians and Asian-Americans are significantly less likely than European-Americans to seek such explicit social support for coping with stress, because their social relations may be disrupted by so doing. Using multiple methodologies, such as survey and physiological measures, the proposed studies will examine the use of explicit versus implicit social support (which we define as drawing on the awareness and/or company of supportive others without explicitly requesting or receiving support vis-a-vis a specific stressful event) and explore cultural differences in their use and physiological/psychological impact on managing stress. The intellectual merit of the proposed research stems from its ability to 1) broaden our conceptual understanding of social support by exploring the stress-reducing benefits of implicit as well as explicit social support and 2) broaden our understanding of cultural differences in how social support is extracted, experienced, and utilized to reduce adverse physiological and emotional responses to stress. The broader impact of the work stems from its challenge to existing Western conceptualizations of social support and its potential to enlighten the social support experiences of currently under-represented populations. As such, it has the ability to inform social support interventions with multicultural populations.

DESCRIPTION (provided by applicant): Despite remarkable progress, an understanding of the molecular mechanisms, catalytic activities, kinetic properties, substrate specificity and protein-protein recognition in both natural and hybrid PKSs remains limited. This renewal application of a highly productive collaborative program proposes to employ the versatile and well-characterized Streptomyces Venezuela pikromycin PKS, as well as the erythromycin, tylosin, curacin and bryostatin pathways which were the subjects of expanded detailed analysis during the previous cycle of support and are now poised for major new progress. These systems each bear fascinating biochemical features that will expand our understanding of the specificity and structural characteristics that lead to biological activity within and between natie and hybrid PKS modules. Our objectives and approach will focus on assessing the molecular details of polyketide chain initiation, elongation, keto group processing, and termination that lea to the remarkable chemical diversity of polyketide natural products. Detailed biochemical analysis, along with X-ray and cryoEM structural biology, and molecular dynamics approaches will be applied to probe substrate specificity. Moreover, synthetic chemistry of natural and near-natural substrates will be employed to develop chemoenzymatic approaches to enable pursuit of our long term objective of engineering PKS systems that efficiently generate novel structures with significant potential as therapeutic agents. Specific aims include: I. Molecular analysis of bacterial modular polyketide synthases. We will design and employ natural and unnatural synthetic substrates and extender units to explore selectivity and tolerance in chain loading, elongation and processing in the terminal modules of Pik (modules 5 and 6), DEBS (modules 5 and 6), Tyl (modules 6 and 7), and select Cur PKS modules. II. Develop mutational strategies to engineer modular PKSs with greater catalytic efficiency toward unnatural substrates. A high-throughput bioactivity-based screen will be developed to assess the efficiency of mutant PKS modules for improved activity toward target unnatural substrates. III. Molecular analysis of bacterial symbiont trans-AT modular PKSs and ?ranching. We will explore the protein recognition determinants for trans-AT interactions, substrate selectivity, and structure and function using synthetic substrates, biochemical analysis, x-ray crystallography, cryoEM, and FT-ICR MS. In addition, a proof-of-concept method will be developed to interrogate biochemical function using bryostatin (Bry) PKS modules 3 and 4 and BryP/surrogate trans-ATs and ?ranching enzymes.

[unreadable] DESCRIPTION (provided by applicant): Marine cyanobacteria are extraordinarily rich in their production of biologically-active and structurally- unique natural products. A number of these secondary metabolites or their derivatives are lead compounds n drug development programs aimed at providing new therapies to treat cancer, bacterial infections, inflammatory responses, and in crop protection to kill harmful microbial pathogens and insects. Isolation and structural analysis of marine and terrestrial cyanobacterial natural products has provided access to an unusually large number of mixed non-ribosomal peptide synthetase/polyketide synthase (NRPS/PKS) systems. The corresponding metabolic systems are comprised of an intriguing set of complex multifunctional proteins that along with allied enzymes generate structurally complex molecules via a modular multi-step process. Over the past several years the Sherman and Gerwick laboratories have developed a complementary program to clone and characterize the biosynthetic pathways of novel cyanobacterial secondary metabolites that possess significant potential for biotechnological applications. A full understanding of the molecular mechanisms, catalytic activities, kinetic properties, and substrate specificities within cyanobacterial biosynthetic pathways is just beginning to unfold. The proposed research will build upon our studies of the curacin and jamaicamide metabolic systems, two distinct yet related pathways that are genetically characterized and poised for detailed biochemical studies. This detailed genetic and biochemical understanding will facilitate the design of new biosynthetic systems that harness the growing potential of cyanobacterial secondary metabolism. Despite considerable gains over the past few years, the full promise of cyanobacterial natural products to yield new lead compounds for development as useful Pharmaceuticals, will only be realized by closing a series of key gaps in knowledge and technology. Solving these challenges will require development and optimization of genetic and biochemical methods that allow us to 1) utilize unique secondary metabolite enzymes for creation of novel small molecules, 2) manipulate cyanobacterial natural product gene clusters to produce analog structures. The specific aims are: 1. To investigate biochemically unique aspects of the curacin (Cur), and jamaicamide (Jam) biosynthetic pathways including formation of the cyclopropane ring, cis-alkene formation, and termination in Cur, and chain initiation, vinyl chloride formation and termination in Jam. 2. Perform bioassays on new compounds resulting from Specific Aim 1 including evaluation for inhibition of tubulin polymerization and binding site specificity, biochemical assays relevant to cancer, and in house screens at U-M and SIO relevant to anti-microbial activity and neurotoxicity, respectively. [unreadable] [unreadable] [unreadable]

The under-representation of minorities in academic institutions is a persistent social problem. One social psychological explanation for this chronic situation is that individuals in minority groups face the additional psychological burden of stereotype threat; that is, they must contend with negative stereotypes about their group that can hamper their performance and lead to disidentification from academic domains. In past social psychological research, self-affirmation has been shown to attenuate this stereotype threat. The present research seeks to explore the underlying mechanisms of how and why self-affirmation attenuates stereotype threat and improves academic performance. The basic premise of this proposal is that when dealing with a stressor, people must not only contend with solving the problem at hand, but additionally, they must also deal with the broader psychological implications of success and failure for their self-evaluation. These additional psychological concerns, exacerbated when an individual is a member of a negatively stereotyped group, can be attenuated when alternative self-resources are affirmed through the process of self-affirmation. To examine the dynamic nature of how individuals sustain motivation under threat, a field study is proposed with children in a middle school composed of students of varying races and ethnicities. The field study will feature: 1) a self-affirmation intervention that has been demonstrated to reduce stereotype threat and improve performance in similar settings; 2) the repeated measurement of potential mediators including daily perceptions of stereotype threat, collective threat, self-esteem, collective self-esteem, academic identification and self-efficacy; 3) the observation of academic performance (i.e., classroom quizzes and examinations). It is proposed that self-affirmation can lead to improved academic performance among stereotype threatened individuals by buffering self-worth against daily stressors and stereotype threat. By utilizing repeated measurements of potential mediators, this research will examine on a within-person level whether days in which students perceive stereotype threat are associated with reductions in self-esteem and academic motivation, and whether self-affirmation can attenuate this link between perceptions of stereotype threat and self-evaluation. The goal of this research is to explore and better understand how drawing on alternative self-resources can enable individuals to maintain motivation and improve performance in the face of negative group stereotypes and psychological threat more generally. The present research has the potential to benefit educational practices by elucidating the mechanisms by which stereotype threat can be reduced and academic motivation sustained.

[unreadable] DESCRIPTION (provided by applicant): Cytochrome P450s are one of the most widely distributed classes of enzymes in nature, catalyzing the oxidation of a broad range of natural product and xenobiotic small molecules. Although hundreds of P450 hydroxylases have been examined in the oxidative metabolism of xenobiotics and steroids, only a small number have been studied in bacterial secondary metabolism, especially in macrolide antibiotic biosynthetic pathways. In most of these pathways, hydroxylation(s) occurs in the late stages of biosynthesis after formation of the macrolide by the polyketide synthase (PKS). In addition to significant increases in biological potency, hydroxylation provides potential sites for chemical modification and further enhancement of anti-infective activity. Thus, the creation of novel macrolide analogs through combinatorial biosynthesis and chemoenzymatic synthesis warrants a concomitant effort towards the development of macrolide monooxygenases with broad substrate specificity. The aim of the proposed work is to develop a thorough understanding of the substrate flexibility and functionality of the cytochrome P450-PikC macrolide monooxygenase. Our recent structure determination of the enzyme has provided fascinating new insights into its catalytic mechanism and ability to generate several products by hydroxylation of the 12-membered ring macrolide YC-17 and the 14-membered ring macrolide narbomycin. This information will direct protein engineering efforts to better understand the function and positional specificity of the enzyme, as well as its ability to catalyze hydroxylation or epoxidation reactions. Moreover, we plan to investigate the unprecedented desosamine sugar-mediated anchoring of macrolides within the PikC binding domain to develop engineered monooxygenases with versatile substrate selectivity. Specific Aim 1. Determination of the PikC structure and the mechanism(s) of hydroxylation of narbomycin and YC-17. Specific Aim 2. Explore the role of macrolide sugar-mediated anchoring on substrate binding and catalytic activity of PikC. Specific Aim 3. Engineering of novel macrolide hydroxylases. [unreadable] [unreadable] [unreadable]

Although public interest in genes and genotyping is increasing, much of the public discourse on genetics centers on the simplistic notion that there is a clear gene that can be directly linked to specific psychological or behavioral tendencies (e.g. ?The Shyness Gene?). This simplistic understanding of the role of genes can be particularly problematic when it is associated with group differences, such as cultural and racial differences, as such a view can lead to thinking that many observed psychological and behavioral differences are innate and fixed. The influence of genes on the shaping of everyday behaviors is far from simple and how social and cultural factors impact the behavioral expression of genes is still largely unknown. The present research examines how culture might influence the way in which particular genes lead to specific patterns of behaviors. Previous research has found that there are large differences in how people rely on social support to cope with their stress. That is, European Americans tend to seek social support more explicitly and directly than Asians/Asian Americans, who prefer more indirect and implicit social support. Previous research has also found that particular genes (e.g., serotonin transporter promoter polymorphism) can influence psychological predispositions (e.g., how strongly a person reacts to stress). Building on these findings, this research examines whether people with the same genetic predisposition actually behave in largely different manners, if they are exposed to different cultural norms and expectations. The research will thus investigate whether and how culture might diversify the psychological and behavioral expression of genes. More specifically, the present research will examine: 1) how specific genes are linked to psychological proneness to stress reactivity and social affiliation; 2) how culture interacts with these specific genes to produce the culturally divergent ways in which people use social support; and 3) how the culturally specific patterns of social support behavior of Asians change as they acculturate to the U.S. The studies will combine genetic analysis with multiple psychological methods, such as large survey design, lab experiment, and daily diary.

Bridging the fields of psychology, biology, and anthropology, this program of research aims to create a new interdisciplinary theoretical framework for understanding cross-cultural and cross-ethnic behavioral variation. This program of research, an international collaboration between researchers in the United States and the Republic of Korea, will also foster opportunities for researchers and students to be exposed to and trained in theoretical and technical approaches in the different disciplines. Finally, this program of research will contribute to a scientific pool of knowledge that can be utilized in educating the public regarding the role of genes in the determination of human behaviors and promoting a more sophisticated understanding of group differences.

Acid-Amino-Acid Ligases; Acids; Anabolism; Anti-Infective Agents; Anti-Infective Drugs; Anti-Infectives; Anti-infective Preparation; AntiInfective Drugs; AntiInfectives; Antiinfective Agents; Assay; B. anthracis; Bacillus anthracis; Bioassay; Biochemical; Biologic Assays; Biological Assay; Biological Factors; Chemicals; Class; Collaborations; Dehydratases; Development; Enzymes; Factor, Biologic; Family; Future; Generalized Growth; Genetic; Genomics; Growth; High Throughput Assay; Hydrases; Hydro-Lyases; In Vitro; Infection; Iron Chelates; Iron Chelating Agents; Kinetic; Kinetics; Laboratories; Mammals, Mice; Mice; Michigan; Murine; Mus; Natural Products; Pathogenesis; Pathway interactions; Peptide Synthetases; Proteins; Research Design; Role; Siderochromes; Siderophores; Study Type; Tissue Growth; Universities; Virulence; Work; acid aminoacid ligase; anthracis; base; biosynthesis; communicable disease control agent; gene product; high throughput screening; in vivo; inhibitor; inhibitor/antagonist; macrophage; member; new therapeutics; next generation therapeutics; novel therapeutics; ontogeny; pathway; peptide synthase; petrobactin; social role; study design

DESCRIPTION (provided by applicant): The International Biodiversity Conservation Group (ICBG) program links three key issues, including human health, biodiversity conservation, and economic development by encouraging programs in the U.S. and programs in countries with high biodiversity to form integrated research teams. The ICBG described in this application involves programs located in the U.S. and Costa Rica that will cooperate to: Improve human health through the discovery of bioactive natural products from Costa Rica's rich biodiversity using ecologically-driven approaches. Contribute to the development of a bioenergy program toward discovery of cellulases and other enzymes for applications in biofuel production. Focus natural product and biosynthetic enzyme-related research on unexplored and under-explored microorganisms such as marine bacteria and insect microbial endosymbionts. Improve the research capacity and economic opportunities for Costa Rica and contribute to its National Biodiversity Strategy through gathering data for its biodiversity inventory, intensive screening of its natural products in medically relevant assays, high throughput testing of its hydrolytic enzymes, sharing of resources, clear benefit-sharing, and training of students and visiting scientists. These broad aims will be pursued through three Associate Programs located both in Costa Rica at the National Institute of Biodiversity (INBio), and the U.S. at Harvard Medical School (HMS) and the University of Michigan (U-M). The Associate Programs will conduct the pre-clinical research to discover, isolate, evaluate and develop therapeutic agents from natural products. Their collection programs, which will be coupled with genetic and phenotypic analyses, will expand Costa Rica's biodiversity inventory for microorganisms. Workshops and scientific exchanges will provide training opportunities. A bioenergy research program on sugar hydrolase discovery and commercial development is proposed as well as a program to harness enzymes from natural product biosynthesis (e.g., thioester hydrolases and decarboxylases) with application in liquid fuel production (biodiesel).

DESCRIPTION (provided by applicant): The International Biodiversity Conservation Group (ICBG) program links three key issues, including human health, biodiversity conservation, and economic development by encouraging programs in the U.S. and programs in countries with high biodiversity to form integrated research teams. The ICBG described in this application involves programs located in the U.S. and Costa Rica that will cooperate to: Improve human health through the discovery of bioactive natural products from Costa Rica's rich biodiversity using ecologically-driven approaches. Contribute to the development of a bioenergy program toward discovery of cellulases and other enzymes for applications in biofuel production. Focus natural product and biosynthetic enzyme-related research on unexplored and under-explored microorganisms such as marine bacteria and insect microbial endosymbionts. Improve the research capacity and economic opportunities for Costa Rica and contribute to its National Biodiversity Strategy through gathering data for its biodiversity inventory, intensive screening of its natural products in medically relevant assays, high throughput testing of its hydrolytic enzymes, sharing of resources, clear benefit-sharing, and training of students and visiting scientists. These broad aims will be pursued through three Associate Programs located both in Costa Rica at the National Institute of Biodiversity (INBio), and the U.S. at Harvard Medical School (HMS) and the University of Michigan (U-M). The Associate Programs will conduct the pre-clinical research to discover, isolate, evaluate and develop therapeutic agents from natural products. Their collection programs, which will be coupled with genetic and phenotypic analyses, will expand Costa Rica's biodiversity inventory for microorganisms. Workshops and scientific exchanges will provide training opportunities. A bioenergy research program on sugar hydrolase discovery and commercial development is proposed as well as a program to harness enzymes from natural product biosynthesis (e.g., thioester hydrolases and decarboxylases) with application in liquid fuel production (biodiesel).

Dr. Heejung Kim and colleagues (University of California, Santa Barbara) investigate the role of culture, as a form of social environment, in the behavioral expression of genes. Specifically, in this research, they examines the dynamic interplay of socio-cultural and genetic factors, and their effects on socio-emotional processes such as emotional support seeking, emotion regulation, and emotional attention, in order to understand the psychological and biological mechanisms underlying emotional responses. The proposal will focus on the oxytocin receptor polymorphism (OXTR), a genetic locus thought to be associated with socio-emotional sensitivity, and its role in social behavior in East Asian and U.S. cultural contexts. The researchers examine whether OXTR variants, as well as experimentally manipulated oxytocin levels, are related to the ability to accurately detect others' mental and emotional states, and whether this ability is related to the tendency of individuals to engage in culturally appropriate social behaviors.

Most conversations about the role of genes typically center on the idea that a gene is linked to a particular behavior. In the context of cultural, racial, gender, or other group differences, this simplistic understanding about the role of genes can lead to thinking that group differences are fixed or immutable. The model of gene-culture interaction advanced in this research provides a more sophisticated perspective by specifying the pathway by which socio-cultural factors can shape the behavioral outcomes of genetic predispositions. Identifying the psychological and biological mechanisms of gene-culture interaction will advance the public understanding of the complex but fascinating interaction between "nature" and "nurture" that produces diversity in human behavior. The work will also support the training and education of students.

The purpose of this research, largely undertaken in collaboration with logicians, is to study operator algebras using a recent variant of classical model theory that interacts well with the structures of functional analysis. A basic goal is to determine how well this "continuous" model theory distinguishes analytic structures, and which properties it captures. Among the applications is new information about isomorphisms and embeddings between structures and their ultrapowers, as these can be readily reinterpreted using continuous model theory. The project also develops the continuous version of many model theoretic concepts, such as stability, primeness, quantifier elimination, model companions, and existential closedness, both for general logical theorems and new paradigms in functional analysis. Up to this point the emphasis has been on tracial von Neumann algebras, but the approach is expected to be fruitful for C*-algebras, subfactors, algebras equipped with an automorphism, etc.

The past few years have seen several surprising applications of logical techniques in functional analysis, mostly coming from combinatorial and descriptive set theory. This project aims to import concepts from model theory, the study of the relation between mathematical objects and their logical properties. Given an object, suppose one knows the answer to all the questions that can be formulated in a specific way (first-order syntax) -- how well does one understand the object? In general one cannot recover the object itself, but this "logical information" is relevant to many lines of research, and only recently has an appropriate version of model theory been applied to operator algebras. This project will continue to develop the ties between logic and functional analysis, in particular supporting travel for researchers and training for graduate students. The results should offer insights into active topics with applications to other branches of mathematics, for instance the Connes Embedding Problem and logical stability.

DESCRIPTION (provided by applicant): We seek renewal of a highly productive multiple-PI research program involving the analysis and engineering of a broad class of monooxygenases from diverse natural product pathways. Cytochrome P450s are one of the most widely distributed groups of enzymes in nature, catalyzing the oxidation of natural product and xenobiotic small molecules. Although hundreds of P450s have been examined in the oxidative metabolism of xenobiotics and steroids, only a small number have been studied in bacterial secondary metabolism, especially in macrolide antibiotic biosynthetic pathways. In most of these systems, hydroxylation and/or epoxidation reactions occur in the late stages of biosynthesis after macrolide formation by the polyketide synthase (PKS). In addition to significant increases in biological potency, hydroxylation provides potential sites for chemical modification and further enhancement of bioactivities. Thus, the creation of novel macrolide analogs through in vivo metabolic engineering and in vitro chemoenzymatic synthesis warrants a concomitant effort towards the development of monooxygenases with defined substrate specificities. The aim of the proposed work is to expand our understanding of the substrate flexibility and functionality of a range of P450 monooxygenases from macrolide and select other natural product systems. Our progress over the first period of support has provided fascinating new insights into the molecular mechanisms of these biocatalysts, and their ability to generate novel products by hydroxylation, and epoxidation of natural and unnatural substrates. This information will direct protein engineering/substrate engineering efforts to better understand the function and positional specificity of the enzyme, as well as its ability to catalyze a range of oxidative reactions. Our program brings complementary approaches of synthetic chemistry to create diverse substrates, biochemistry to investigate and develop engineered monoxygenases with versatile substrate selectivity, and X-ray and NMR- based methods to obtain high resolution structural information for mechanistic understanding of these remarkable proteins. Specific Aim 1. Assess the impact of steric, electronic and directing group factors on catalytic promiscuity in the P450 PikC using a series of synthetic analogs of the natural macrolide substrate YC-17. Employ X-ray and solution NMR based structural biology approaches to gain detailed insights into binding parameters, protein-substrate dynamics, and the mechanistic basis for regio- and stereochemical specificity of natural and unnatural substrates. Specific Aim 2. Expand access to diverse synthetic substrates for a range of new P450 enzymes to investigate regio- and stereochemical details of monooxygenase-catalyzed hydroxylation and epoxidation reactions. Specific Aim 3. Pursue biochemical and structural studies of mixed-function iterative P450 enzymes to analyze substrate specificity and kinetics, as well as to investigate binding and catalytic mechanisms. PUBLIC HEALTH RELEVANCE: The studies proposed will broaden our knowledge of an important class of enzymes whose catalytic capabilities lead to important new medicinal agents in the form of natural product antibiotics and anticancer drugs. This new information will be used to generate novel biologically active compounds for the discovery and development of new pharmaceutical agents to fight human diseases.

With this award, the Chemistry of Life Processes Program is supporting an International Collaboration in Chemistry (ICC) collaborative between Professors David H. Sherman of the University of Michigan and Roberto G. S. Berlinck of the Universidade de Sao Paulo, Brazil (supported by the Sao Paulo Research Foundation (FAPESP)). The objective of this research effort is to investigate fundamental aspects of alkaloid natural product assembly and tailoring by marine fungi derived from Brazilian biodiversity resources. The fungal alkaloid natural products are among the most under-explored from a genetic and biochemical point of view. The University of Michigan will conduct total genome sequencing, bioinformatic mining, and annotation to identify target natural product biosynthetic systems from selected marine fungi. Molecular genetic and biochemical approaches will be employed collaboratively to understand the basis for construction of the core polycyclic ring systems, as well as oxidative modifications and structural rearrangements to elaborate the complex suíte of molecules within this fascinating class of metabolites. The group in Brazil will work on manipulating growth condition to maximize production of key biosynthetic intermediates and final products to facilitate an understanding of fundamental genetic and physiological factors involved in augmenting secondary metabolism and output of natural products.

The mechanisms for creating chemical diversity in these pathways are often unique compared to bacterial and plant systems. Thus, there are great opportunities to gain new knowledge that can lead to pathway engineering and advance the fields of chemical biology and medicinal chemistry. The Broader Impacts of the proposal are multi-factorial and include expanding the genomic and biochemical basis for fungal alkaloid natural product diversification and training of students and postdoctoral fellows in cross-disciplinary aspects of natural product sciences. The PI at the University of Michigan plans to conduct workshops in Brazil to promote training in natural product pathway engineering and synthetic biology applications.

DESCRIPTION (provided by applicant): This revised grant application is focused on experimental studies to elucidate the biosynthesis of three biogenetically related families of natural products: (1) the Paraherquamides, Asperparalines, Malbranchemaides, Marcfortines and Chrysogenamide (the monooxopiperazines); (2) the Stephacidins, Notoamides, Waikialoids and Brevianamides (the dioxopiperazines); and (3) the Citrinadins, Citrinalins, Cyclopiamines and PF1270 alkaloids (the decarbonylated alkaloids). The goals are to exploit the powerful synergies of total synthesis, whole genome sequencing, bioinformatics analysis, genome mining, functional expression and X-ray crystallography of biosynthetic enzymes to fully elucidate the corresponding biosynthetic pathways to all three families of alkaloids. I. Paraherquamides, Asperparalines, Malbranchemaides, Marcfortines and Chrysogenamide: the Monooxopiperazines. Provocative evidence has been elucidated in our laboratory indicating that this class of alkaloids, are constructed by a rare biosynthetic intramolecular [4+2] cycloaddition reaction resulting from the reductive release of a Trp-Pro (or Trp-Pip) dipeptide amino-aldehyde from a NRPS module that upon reverse prenylation, suffers a cascade of cyclization, dehydration, tautomerization and intramolecular Diels-Alder cycloaddition to construct the monooxopiperazine bicyclo[2.2.2]diazaoctane ring system common to this family. Through the use of total chemical synthesis of isotopically labeled intermediate metabolites, genome mining, biosynthetic gene cluster identification and functional expression of biosynthetic enzymes, key features of the biosynthetic pathways to these complex secondary metabolites will be experimentally elucidated. In a multi-PI relationship and sub-award with Prof. David Sherman's laboratory (University of Michigan), we are actively engaged in the high-resolution elucidation of the entire biosynthetic pathway to these biomedically significant alkaloids. II. Stephacidins, Notoamides, Waikialoids and Brevianamides: The Dioxopiperazines. The dioxopiperazine family of bicyclo[2.2.2]diazaoctane alkaloids are constructed by a net oxidative transformation of a fully prenylated dioxopiperazine substrate. We have discovered that two orthologous species of Aspergillus produce the opposite enantiomers of Stephacidn A and Notoamide B. This fascinating enantiodivergent biogenesis will be further evaluated using bioinformatics analysis by a new collaborator, Prof. Martin Kreitman, a renowned evolutionary geneticist, to determine the evolutionary mechanisms that resulted in this rare production of opposite enantiomers of these complex alkaloids. III. Citrinadins, Citrinalins, Cyclopiamines and PF1270 Alkaloids: The Decarbonylated Alakloids. As a natural out-growth of our work on the Paraherquamide family of prenylated indole alkaloids, we propose to initiate a new project to study the total synthesis and biosynthesis of these structurally related alkaloids that appear to have arisen biogenetically from the reductive decarbonylation of bicyclo[2.2.2]diazaoctane progenitors. Here also, we shall deploy the powerful synergies of total synthesis, whole genome sequencing, bioinformatics analysis, genome mining and functional expression of biosynthetic enzymes to fully elucidate the corresponding biosynthetic pathways to all three families of alkaloids. In all three sub-projects, total synthesis of the natural products and isotopically-labeled biosynthetic intermediates and probe molecules will be utilized to confirm pathway transformations. New chemical entities generated in this program, either from the biological sources or through chemical synthesis, will be extensively screened and evaluated for biological activities at the Univ. of Michigan Center for Chemical Genomics, the National Human Genome Research Institute, and to Prof. Sachiko Tsukamoto (Japan) for analysis of biological activity using a series of biochemical and cell-based assays relevant to cancer and parasitic disease targets. Additional collaborators include: Prof. Sachiko Tsukamoto, Kumamoto University, Japan; Prof. Jens Frisvad, Technical University, Denmark; Prof. Martin Kreitman, University of Chicago; and Prof. Janet Smith, University of Michigan.

Ebola is one of the deadliest contagious diseases to emerge into the public consciousness in recent years. Accompanied by truly gruesome and deadly images, it has been a source of much fear, xenophobia, and social division across the world. While it makes obvious sense to increase some vigilance and engage in self-protective health behaviors, most health experts maintain that the actual risk of contracting Ebola is extremely low, at least outside of the directly affected regions in West Africa. Yet, the degree to which people respond to the potential risk is vastly disproportionate to the actual risk. Thus, in many parts of the world, the actual costs of Ebola are not the disease itself, but the negative psychological and behavioral effects - both individual and social - that are motivated by fear. Fear and social division are not only consequences of Ebola, but they have the potential to transform and perpetuate negative social consequences. Although the US is currently at low risk for Ebola contagion, the potential costs of individual distress as well as restrictive group protection behaviors are real. The present research proposes a theoretically-based intervention to attenuate these negative effects. Moreover, beyond the current Ebola concern, the knowledge gained from this research will be relevant to other, continually occurring, international threats of contagious diseases by potentially mitigating their psychological and social costs. This project was submitted in response to NSF 15-006 Dear Colleague Letter on the Ebola Virus.

This research aims to understand the psychological mechanism of fear-driven individual and social consequences. Drawing from the knowledge-base of social, cultural, and health psychology, two studies using large nationally representative samples will focus on two distinct responses to disease-related fear: 1) anxiety and stress that are detrimental to individual well-being; and 2) manifestations of group-protection to minimize risk. The project will investigate whether cultural orientation (individualism vs. collectivism within the US) will influence which psychological response set is most likely. The prediction is that individualists will be more concerned with self-protection, and thus, are more likely to experience disproportionate anxiety and stress, whereas collectivists will be more concerned with group protection. Based on this model, the proposed research will also test the effectiveness of psychological interventions (self- and group-affirmation) to address the threat and reduce the psychological and social costs of the specter of Ebola.

? DESCRIPTION (provided by applicant): This proposed MIRA project employs a range of multi-disciplinary approaches toward the discovery and analysis of natural products and the biosynthetic pathways that assemble and modify complex metabolites. The proposal covers three areas that have been supported by NIGMS during the past 20 years. Each has been articulated as a Grand Challenge designed to complement our accomplishments and continue to push forward vigorously to discover new knowledge and offer solutions with high potential for improving human health. Grand Challenge I of this MIRA application is based on the exciting momentum of a highly productive and collaborative program lead by my group that focuses on the pikromycin (Pik), erythromycin (DEBS), tylosin (Tyl), curacin (Cur) and bryostatin (Bry) pathways whose detailed analysis has been further developed during the previous cycle of support. These systems each bear fascinating biochemical features that will expand our understanding of substrate selectivity, and structural characteristics that enable functional activity within and between native and engineered polyketide synthase/non-ribosomal peptide synthetase modules. Grand Challenge II of this proposal focuses on studies relating to natural product pathway tailoring enzymes. A fundamental aspect of structural diversification in secondary metabolism involves oxidative processes that contribute significantly to biological activity. This can be readily appreciated in a number of important molecules that are clinical therapeutic agents, or show significant potential as drug leads. Based on the important successes in our research relating to P450 substrate and enzyme engineering over the past four years, we have been emboldened to expand our work in exciting new directions. This includes plans to investigate a range of P450 monoxygenases that catalyze iterative oxidative processes. We will also investigate monooxygenases that catalyze C-C coupling involving substrates in both inter- and intramolecular oxidation reactions, including aromatic, alkyl and alkenyl functional groups. One of the most underexplored, yet very important classes of tailoring enzyme includes the acyl/peptidyl carrier protein dependent monooxygenases, and we propose to explore mechanisms of selectivity and proceed with efforts to expand their substrate recognition and biocatalytic properties. Grand Challenge III focuses on natural product discovery and pathway engineering. We have established the technologies and bioinformatics capabilities to readily assemble and mine genomic, and metagenomic datasets from diverse microbiome populations toward natural product gene cluster discovery, which is now poised for heterologous expression in amenable microbial hosts. The next wave of progress will rely on ready identification of the most novel pathways, and our ability to express them using facile synthetic biology methods. We plan to attack these problems with utmost energy and determination to gain access to important compounds with valuable medicinal properties.

PROJECT SUMMARY A significant limitation in natural product drug discovery is the inability to effectively convert sequence information to new compounds in a rapid and high throughput fashion. The long-term objective of this project is to systematically address this issue with the goal of re-establishing the once vibrant pipeline of environmentally-derived natural compounds, with a special emphasis on discovering new antibiotics. The final vision is the development of a microfluidics biosynthetic platform capable of generality, speed, throughput, and economy in natural product pathway reconstitution and compound discovery. The goal of the current R21 application is captured in the following specific aim: To generate the complex natural product antibiotic erythromycin A using cell-free biosynthesis. By doing so, precedent will be set for the ability to produce such compounds using a purely in vitro approach. This and additional preliminary data collected through the proposed work will then be the basis for continual research towards a cell-free biosynthetic platform capable of precise engineering and ultra-high throughput. As a result, the platform will allow unprecedented access to the broadest range of new chemical entities, including novel antibiotic compounds, while being unencumbered by the constraints of a cellular host.

Understanding the basic human motives that drive behavior has been a question of great historical significance in psychology. Yet, an integrative theory, allowing for different sociocultural and individual factors that might prioritize one kind of motive over another, is still missing. This project aims to develop a theoretical model of the sociocultural determinants of human motives. To develop a powerful testbed, with strong broader impacts, the research examines why and how individual actions are motivated in response to environmental challenges. Environmental crises such as pollution or the depletion of natural resources pose some of the greatest collective challenges faced by society today. Responses of individuals to these challenges have critical consequences. By understanding what drives people's actions in this context, progress will be made in addressing a fundamental question about human behavior. The research is expected to have wide impact across a number of scholarly disciplines, with important implications for public policy, education and philanthropy.

The theory proposes that different sociocultural and individual factors orient people towards more internal and personal reasons or toward more external and social reasons for their actions. The factors that orient people toward more internal and personal reasons for their actions include individualistic values, high socioeconomic status, and social belonging. The factors that orient people toward more external and social reasons for their actions include collectivistic values, low socioeconomic status, and social isolation. The research examines the extent to which environmental beliefs versus perceived environmental norms drive pro-environmental behaviors, and how this differs for people from a wide range of personal experiences and backgrounds. The explanatory role of sense of control and attention allocation is also investigated. The studies use diverse methodologies including a nationally representative study of Americans, a laboratory behavioral study, a longitudinal study of daily environmental behaviors, and a community field experiment at local grocery stores assessing shopping behavior. The new theoretical model can also be used to understand why and how individual actions are motivated in response to a broad range of collective challenges, including widespread infectious disease and the threat of terrorism.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

Abstract We are requesting a Research Supplement to Promote Diversity in Health-Related Research to complement the parent grant R35 GM118101 entitled, Discovery and Characterization of Natural Product Systems. The supplement funds are proposed to support Maria Luisa Adrover- Castellano, who is a first year Ph.D. student in the Program of Chemical Biology at the University of Michigan. Maria has the potential and ability to contribute significantly to the overarching goals in the parent grant aimed at understanding the selectivity and specificity employed by modular polyketide synthases (PKSs). As a Latina, Maria is enthusiastic about contributing to the diversity of the university while serving as a role model for future generations of young scientists. Maria's Puerto Rican upbringing exposed her to unique life and cultural experiences, allowing her to offer different points of view. Maria is committed to bringing this diverse perspective to enrich the overall community using her scientific research as an inspiration and powerful tool. We have together developed a plan for her research and growth as a scientist during the Ph.D. tenure. The goals are directed towards the synthesis of unnatural substrates for polyketide synthase (PKS) modules found in the pikromycin biosynthetic pathway. These can be utilized to interrogate different modules, particularly the terminal two PKS monomodules, PikAIII (module 5) and PikAIV (module 6) to produce unnatural macrolactones via biocatalytic transformations. The macrolactones produced through this work can be further converted to their active, antimicrobial counterparts through biotransformations that append a glycosyl group and catalyze regio- and stereospecific oxidations. As a final step, Maria plans to investigate the antibiotic activity of these new macrolides using both an in vitro ribosome inhibition assay, and to determine their relative minimum inhibitory concentrations (MICs) using whole cell bioassays against human pathogenic bacteria. Ultimately, this project relates to the design and development of new macrolide antibiotics, a key objective of the R35 GM118101 grant.

Current combined antiretroviral therapies (cART) suppress viral levels in the blood but do not eradicate reservoirs of cells harboring integrated copies of HIV proviral genomes. These cells persist in part because the provirus maintains a latent state that evades the immune response and viral cytopathic effect. Approaches to clear reservoirs by reactivating latent cells have provided evidence that latency can be reversed in vivo, however reversal of latency alone has not been sufficient to reduce latent reservoirs. Efforts are now in place to couple latency reactivation with strategies to eradicate the infected cells ? such as by design and activation of more efficacious anti-HIV cytotoxic T lymphocytes (CTLs). Another key player is Nef, an accessory protein encoded by HIV, which is a primary focus of our proposed research. Because Nef inhibits the activity of anti- HIV CTLs, a potent inhibitor of this protein would help achieve HIV eradication. One of the main functions of Nef is the down-modulation of major histocompatibility complex class I encoded proteins (MHC-I), masking infection from the host immune system and allowing HIV infected cells to persist. Combination therapy with latency antagonists plus Nef inhibitors could act synergistically to clear HIV reservoirs. To date, no Nef inhibitor has achieved potent restoration of MHC-I in the presence of Nef. We developed a high-throughput assay to identify inhibitors of Nef-mediated MHC-I downregulation, and a screen of natural product extracts (NPEs) yielded 10 hits with Nef inhibitory activity. We identified a number of related compounds, as the active component in several of these extracts. The pure natural products potently restore surface expression of MHC- I in the presence of Nef without inhibiting its other activities. We tested a number of structurally related compounds within this natural product family and identified two that possess pM to nM potencies in human primary cells. Based on this strong preliminary data, we believe that further enhancing the Nef inhibitory activity of these molecules through analog development will yield a safe anti-Nef drug. Therefore, we plan to (A) optimize these inhibitors by further separating and characterizing the anti-Nef effect from off-target activities to identify a lead drug candidate for development and (B) determine the mechanism by which the inhibitor disrupts Nef-mediated MHC-I downmodulation so that optimization can be conducted more intelligently. These goals will be achieved through the following specific aims: (1) Conduct lead compound structural optimization to improve pharmaceutical properties. (2) Perform a detailed functional analysis of all promising analogs to identify ideal lead compounds and (3) Determine the mechanism by which the natural product-derived inhibitor disrupts Nef-mediated MHC-I downmodulation including target identification and biochemical studies. From this work, we expect to generate a new class of compounds that are potent Nef inhibitors with high pharmaceutical potential. The addition of Nef inhibitory compounds to current cART cocktails is expected to enhance immune clearance of viral reservoirs, leading to the long-elusive HIV cure.

ABSTRACT TB treatment is an enigma of ineffectiveness. Current TB chemotherapy rapidly kills nearly all bacteria within two weeks, yet tolerable treatment failure rates are only achieved after 6 months, and even then, ~5% of cases relapse. Our recent work shows that bacterial factors associated with small MIC shifts are important predictors of treatment outcome, but the driving forces behind those shifts are unknown. This project unites three labs, with highly complementary expertise, around interrogating carefully curated M. tuberculosis clinical isolates with leading edge approaches in genetics, metabolism, gene regulation and network-based modeling to reveal fundamental new knowledge about how TB responds to front-line drugs. The direct result of this effort will be a suite of candidate biomarkers with great potential to personalize treatment duration by predicting treatment outcome and greatly simplify TB drug trials, as well as novel drug targets to improve outcomes and shorten therapy. These translational aims will be pursued in future studies, using the insights, strains and tools developed in the program.

HTS Targeting HIV-1 Protease Autoprocessing for First in Class Drug Discovery Project Summary This proposal is in response to PAR-17-438: Assay development and screening for discovery of chemical probes or therapeutic agents (R01). The goal of this project is to carry out high-throughput screens and follow-up characterizations to identify molecules inhibiting HIV-1 protease (PR) autoprocessing with modes of action (MOAs) different from the currently available protease inhibitors (PIs). In the infected cell, HIV-1 PR is initially synthesized as part of the Gag-Pol polyprotein precursor with its proteolysis activity tightly suppressed. During late stage of virion production, the precursor self-catalyzes the cleavage reactions that lead to liberation of the free mature PR in a temporospatially regulated fashion. The FDA-approved HIV-1 PIs primarily target the catalytic site of the mature PR and they are significantly less effective at suppressing precursor-mediated autoprocessing, suggesting that these two forms of HIV-1 PR are enzymatically different. Also, the emergence of PI resistant strain in patients treated with PI-containing combination antiretroviral therapy (cART) is an ongoing problem that diminishes treatment efficacy, which warrants the need for new therapeutics. This project seeks to find novel autoprocessing inhibitors targeting the precursor at regions not recognized by the currently available PIs. Towards this goal, we have established a cell-based functional assay that has faithfully recapitulated the autoprocessing phenotypes observed with proviral constructs. This assay has also, for the first time, made it possible to screen for autoprocessing inhibitors by HTS using AlphaLISA (amplified luminescent proximity homogeneous assay ELISA) technology. Our pilot screens of ~26K small molecule compounds displayed satisfactory performance with Z? factors >0.45, S/N ratios >10, and hit rates <0.1% although no confirmed hit was identified. Therefore, we plan to screen a collection of natural product extracts (40K extracts, 5-25 compounds per extract, totaling ~0.6 million chemicals) with a wild type and two PI resistant precursors in collaboration with Dr. David Sherman at University of Michigan Life Sciences Institute (Aim 1). In parallel, we will team up with Drs. Thomas Chung and Ian Pass at Sanford Burnham Prebys Medical Discovery Institute to screen their ~350K small molecule library (Aim 2). These HTS campaigns will hopefully identify a handful confirmed compounds that will be subjected to a battery of established secondary and tertiary assays (Aim 3) in order to find novel autoprocessing inhibitors that are different from the current HIV-1 PIs in their MOA. This next generation of therapeutic probes, when used in combination with the current PIs, will implement a new therapeutic approach: targeting a vital enzyme (HIV-1 protease) at two distinct functional states (precursor and mature PR) and at different regions (non-catalytic and catalytic sites) at the same time. Such a strategy is expected to drastically increase difficulty (genetic barrier) for HIV-1 to evolve viable strains simultaneously resistant to inhibitors from both classes to resist the resistance.

ABSTRACT The rapid spread of multi-drug resistance has created a great need for new combination therapies to treat a variety of conditions, including infectious diseases and cancer. In one pressing example, multidrug resistant tuberculosis (TB) affects about 500,000 people each year and novel drug regimens are sorely needed. However, identifying new regimens has been daunting in part due to the inability to prioritize among a very large number of possible drug combinations. To address this need, we have generated an experimentally grounded, machine learning algorithm, INDIGO-MTB, which predicts the synergy or antagonism of TB drug combinations with high accuracy. Here we propose to adapt INDIGO-MTB into a multifactorial pipeline to dissect combinatorial drug efficacy and drive preclinical regimen development for TB. We will build in and validate the ability to predict drug interactions under stressful environmental conditions that mimic TB infection, and extract molecular mechanisms of drug interactions. We will then combine synergy and efficacy measurements to create new regimen rankings, which we will validate both in vitro and in a mouse model of TB infection. Altogether, our work will establish a tool for rapid assessment of TB drug combinations and a framework for applying this approach to other conditions where new multidrug therapies are needed.