David H. Sherman - US grants

Polyketide-derived metabolites are found in both procaryotes (mainly actinomycetes) and eucaryotes where they play an amazing variety of functional roles. Many of these compounds have important biological activities and have been developed as antibiotics and chemotherapeutic agents in both human and veterinary medicine. The importance of these compounds is increasing as the need arises to develop agents which effectively combat recalcitrant infectious diseases, and in the development of small molecules which mimic or inhibit the activity of natural hormones and immunomodulators. The primary aim of the proposed research is to understand the molecular mechanisms controlling carbon- chain construction in the biosynthesis of polyketide antibiotics. Molecular genetic, biochemical and chemical approaches will be used to obtain information on the functional role of each enzyme comprising the polyketide synthase (PKS) including; condensing enzymes, acyl carrier proteins, acyl- and terminal transferases. Molecular genetic studies have revealed a close relationship between fatty acid and polyketide biosynthesis, and a model will be developed to investigate the mechanistic similarities and differences of these important biosynthetic systems. In order to establish an effective experimental model, we will clone and sequence the fatty acid synthase (FAS) genes of Streptomyces coelicolor. Although we expect the molecular genetic details will have important comparative value, this work will be pursued with the goal of establishing the S. coelicolor FAS as a universal indicator system to study individual components of PKS and FAS gene clusters. The versatility of the approach is provided by the ease of structural determination of fatty acids compared to complex secondary metabolites. In this respect, the invariant reductive cycle that occurs in fatty acids compared to complex secondary metabolites. In this respect, the invariant reductive cycle that occurs in fatty acid biosynthesis provides an enormous strategic advantage. Of primary importance is the question of carbon chain length determination, and the effect of altering activity of PKS condensing enzyme on chain length control in polyketide antibiotic biosynthesis. Hybrid FAS/PKS gene clusters will be constructed using trans-complementation and gene replacement technology. Structural analysis of the fatty acids produced will allow us to analyze the precise functional contributions of individual PKS genes in the hybrid system. Overall, this work should provide important insight into the molecular genetic and biochemical relationships within the FAS and PKS systems in Streptomyces. Moreover, it is hoped that this work will provide an important theoretical and experimental base for the rational production of novel polyketide-derived metabolites using molecular genetic technology.

Polyketide-derived metabolites are found in both procaryote's (mainly actinomycetes) and eucaryotes where they display an amazing diversity of functional roles. Many of these compounds have important biological activities and have been developed as antibiotics and chemotherapeutic agents in both human and veterinary medicine. The importance of these compounds is increasing as the need arises to develop agents which effectively combat recalcitrant infectious diseases, and in the discovery of small molecules which mimic or inhibit the activity of natural hormones and immunomodulators. The primary aim of the proposed research is to understand the molecular mechanisms controlling carbon-chain construction in the biosynthesis of polyketide-derived macrolide antibiotics. Molecular genetic, biochemical and bioorganic chemical approaches will be used to obtain information on the functional role of each catalytic domain comprising the multifunctional (type I) polyketide synthase (PKS) including; condensing enzymes, acyl and terminal transferases, dehydrases, keto and enoylreductases, and acyl carrier proteins. Molecular genetic studies have revealed a close relationship between multifunctional fatty acid and polyketide synthases, and a model will be developed to dissect the mechanistic similarities and differences of these important biosynthetic systems. In order to establish an effective experimental model, we will investigate the methymycin (met) type I PKS of Streptomyces venezuelae. Although we expect the molecular genetic details will have important comparative value, this work will be pursued with the goal of establishing the S. venezuelae PKS as a novel system to study individual functional domains of macrolide-producing multifunctional PKSs. The versatility of the overall investigation is provided by the cross- disciplinary nature of the approach. Our initial efforts will include the overexpression and purification of the met multifunctional protein that mediates early biosynthetic steps including: carbon chain elongation, ketoreduction and dehydration. The ability to study these processes has only recently become possible due to technological advances that allow the overexpression of Streptomyces PKS proteins, and intact incorporation of chain elongation intermediates into macrolide antibiotics. Of primary importance is the mechanism of chain construction and functionalization of the nascent unsaturated fatty acid that is subsequently lactonized to form the macrolide molecule. Studies are designed to determine directly whether elaboration of the unsaturated fatty acid intermediate occurs linearly, as suggested by the deduced sequences of macrolide type I proteins. Structural analysis of the unsaturated fatty acids produced by in vitro conversion studies will allow us to analyze the precise functional contributions of individual PKS domains in the purified system. Overall, this work should provide important insight into the molecular genetic and biochemical relationships within type I PKS systems in Streptomyces and other actinomycetes. Moreover, it is hoped that this work will provide an important theoretical and experimental base for the rational production of novel macrolide metabolites using molecular genetic technology.

Metabolic engineering 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 amplification of rate-limiting steps to enhance production of valuable compounds. Since microbial metabolism is dynamically controlled by cellular events relating to growth and differentiation, rational pathway manipulation must consider a complex set of variables. Thus, the current challenge is metabolic engineering is to develop a comprehensive strategy that examines the temporal and spatial profiles that affect microbial biosynthetic potential. The primary aim of the proposed research is to develop a rational approach for metabolic engineering of secondary metabolite production using the cephamycin C biosynthetic pathway in Streptomyces clavuligerus as the model system. In this work molecular genetic, physiological and mathematical modeling approaches will be combined to obtain information on the temporal and spatial expression of rate- limiting enzymes and dclX, a recently discovered positive regulatory gene for this important class of natural products. A new in-vivo reporter will be employed by coupling promoter sequences and structural genes to the gene encoding green fluorescent protein. Temporal and spatial gene expression patterns in S. clavuligerus will be assessed and quantified using confocal microscopy. In addition, a regulable promoter system will be used to control expression of dclX and structural genes encoding rate-limiting enzymes. This unique tool will provide a strategy to investigate the effect of perturbing temporal expression patterns of key enzymes in this important metabolic system. Furthermore, a mathematical model will be developed to predict the effect of temporal perturbation of the biosynthetic machinery on cephamycin C biosynthesis. Overall, this work should provide important insight in to the temporal and spatial relationships of gene expression and protein localization within the beta-lactam pathways in Streptomyces and other microorganisms. Moreover, it is hoped that this work will provide an important theoretical and experimental base for the rational manipulation of complex metabolic pathways for novel metabolite production and improved efficiency. Please visit: http://www.cems.umn.edu/~hu-grp/gfp/nih.html)

The overall goal of this research program is to discovery new natural products that will lead to novel anti-cancer drugs. This will be accomplished by using two powerful new molecular genetic approaches for creation of complex natural products. First, combinatorial biology libraries will be made by harnessing extensive metabolic potential directly from culturable marine microorganisms. A sophisticated gene cloning and pathway manipulation system using cosmid and bacterial artificial chromosome-based vector systems will be used to generate natural products through heterologous expression in Streptomyces venzuelae. Microbial genomes from marine actinomycetes (provided by Wright and Crews), cyanobacteria (provided by Gerwick), myxobacteria and fungi (provided by Crews) will be accessed and libraries enriched for secondary metabolite biosynthetic gene clusters will be generated to express diverse natural product biosynthetic pathways for subsequent analysis in a broad array of "smart assays" developed at Novartis Pharmaceuticals (Bair). Second, combinatorial biosynthesis approaches will be used to engineer specific changes to biosynthetic pathways from the curacin A (Gerwick) and latrunculin (Crews) polyketide systems. In this work, polyketide synthase gene probes based on highly conserved sequence motifs will be used to isolate individual sets of overlapping cosmid clones that comprise the complete curacin A and latrunculin biosynthetic gene clusters for subsequent characterization at the molecular genetic level. This information is required to generate a wide range of molecular modifications for these important structures that have already been identified as promising anti-cancer drug leads. Generation of novel structures based on the curacin A and latrunculin core molecules will be accomplished through deliberate modification of genes and enzymes that specify choice of loading domain, extender units, keto group processing and termination of the curacin A and latrunculin chain elongation intermediates. A parallel effort will be pursued to discover, evaluate and engineer novel forms of natural product tailoring enzymes with flexible substrate specificity. Overall, we expect to generate a large number of exciting new natural products for development as novel anti-cancer agents using the powerful tools of microbial genomics, combinatorial biology, combinatorial biosynthesis, natural products chemistry and innovative anti-cancer drug screening assays systems.

The primary goal of this research program is to discover new natural products that will lead to novel anti-cancer drugs. Overall, we expect to generate a large number of exciting new natural products for development as novel anti-cancer agents using the powerful tools of microbial genomics, combinatorial biology, combinatorial biosynthesis, natural products chemistry and innovative anti-cancer drug screening assay systems. This will be accomplished by using two powerful molecular genetic approaches for creation of complex natural products along with sophisticated "smart assays" for molecular-based screening at Novartis Pharmaceuticals. Five research groups will work together to access unique marine microbial genomes, generate combinatorial biology and combinatorial biosynthesis libraries, culture and extract novel metabolites, screen extracts for anti-cancer biological activity and characterize new chemical entities. Project #1 is headed by Dr. Amy Wright at Harbor Branch Oceanographic Institution (HBOI) who will collect and characterize novel marine actinomycetes. Dr. Peter McCarthy at HBOI will direct the core facility and manage microbial genomic DNA preparation, fermentation and extraction for the combinatorial biology clones. Project #2 is headed by Dr. David H. Sherman at the University of Minnesota, the overall P.I., who will direct the combinatorial biology as well as combinatorial biosynthesis efforts for the entire program. The administrative core will also be managed by Dr. Sherman. Team #3 is headed by Dr. Kenneth Blair of the Oncology Department at Novartis Pharmaceuticals. His group will be responsible for high throughput screening using "smart assays" developed for anti-cancer drug discovery. Project #4, headed by Dr. William Gerwick at Oregon State University, will focus on isolation and characterization of novel marine cyanobacteria for generation of combinatorial biology libraries. His group will also participate in combinatorial biosynthesis work on the curacin A polyketide synthase. Project #5 of this highly collaborative and interactive effort, headed by Dr. Phillip Crews of U.C. Santa Cruz, will focus on isolation and identification of novel marine microbes, including sponge- derived actinomycetes, myxobacteria and fungi. His laboratory will provide genomic source material for combinatorial biology as well as for combinatorial biosynthesis on the latrunculin A polyketide synthase. Both the Wright, Gerwick and Crews laboratories will each provide natural products purification and structure elucidation support. In summary, unique compound structural diversity will be generated by this multi- disciplinary collaboration.

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 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.

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]

[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]

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 cryptophycins are a structurally diverse class of polyketide/non-ribosomal peptide natural products that possess potent anticancer activity. However, despite this impressive activity, the development of the cryptophycins into a beneficial cancer chemotherapeutic agent has suffered from clinically significant neurotoxicity that correlates with treatment. Nonetheless, the promising therapeutic spectrum of this natural product has motivated Acera Biosciences Inc. to pursue the discovery and development of a cryptophycin compound that that can re-enter clinical evaluation. Within this discovery effort, the company seeks to generate large, structurally diverse libraries of cryptophycin compounds for subsequent biological activity screening. Due to the complex chemical structure of the cryptophycin natural products, which features multiple stereocenters, a macrolactone ring, and a reactive 2-epoxide moiety, the generation of these compounds by traditional synthetic methods requires multiple steps, resulting in low overall yields and a high cost per compound. As such, these methods are not amenable to the rapid production of structurally diverse compounds. Accordingly, in this Phase I STTR proposal, Acera will develop a novel high throughput synthetic technology that will enable access to a diversity of cryptophycin analogues that can be rapidly screened for desirable pharmacological properties. This innovative synthetic platform seeks to utilize routine solid-phase synthesis for the construction of compound libraries containing linear cryptophycin intermediates. The hallmark of the proposed strategy involves chemoenzymatic transformation of the linear cryptophycin intermediates into mature, macrocyclic cryptophycin analogues. In particular, Acera Biosciences Inc. seeks to leverage the catalytic power of the cryptophycin thioesterase (Crp TE) and the cryptophycin epoxidase (CrpE) to specifically and efficiently transform the resin-bound intermediates to macrocyclic compounds bearing the key epoxide functional group. To establish the feasibility of this approach, this proposed research aims to incorporate structural diversity into the 3-chloro-O-methyl-tyrosyl cryptophycin synthon via substitution with 18 commercially available phenylalanine analogues. The resulting 18 novel cryptophycin compounds will be screened for anti-tumor activity and neurotoxicity using a multi-faceted preclinical drug development paradigm developed at Henry Ford Health System. A key feature of this strategy is that small quantities of compounds are required to identify an initial lead compound, making it an ideal complement to the high throughput chemoenzymatic production of cryptophycin analogues. Once proof-of-concept is established, research efforts in Phase II will apply this technology toward the generation of thousands of chemically diverse cryptophycin compounds that will be screened for desirable pharmacological activity. Identified lead compounds can then be licensed to pharmaceutical or biotechnology companies interested in expanding their anti-cancer development programs. PUBLIC HEALTH RELEVANCE: Cancer represents a significant global human health concern that justifies substantial research investments for the discovery and development of novel treatments. Cryptophycin is a known, potent anti-cancer compound that has been dropped from clinical testing due to intolerable side-effects. This proposed research seeks to develop a novel technology for the rapid generation of cryptophycin analogues that may display fewer side effects, thereby enabling cryptophycin to be utilized by physicians in the battle against this often deadly disease.

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).

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The outer membrane of Gram-Negative Bacteria is an important permeability barrier that consists of an asymmetric bilayer in which the inner leaflet is primarily composed of phospholipids and the outer leaflet is primarily composed of lipopolysaccharides (LPS). Outer membrane proteins (OMPs) of the ?-barrel family span the bilayer and serve as channels and transporters. Using a chemical genetic approach, we recently identified a multi-protein complex that spans the periplasm that is responsible for the assembly of LPS. We are currently pursuing biochemical and structural studies of these proteins in order to understand how the hydrophobic LPS molecule is transported to and assembled in the outer leaflet of the OM. We hope to identify whether there are general principles that guide the assembly of this membrane and to characterize these new protein targets for antibiotic development.

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.

? 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.

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.

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.