Steven G. Van Lanen, Ph.D. - US grants

DESCRIPTION (provided by applicant): The goal of the proposed research is to study the functionality of the domains of the iterative type I enediyne C-1027 polyketide synthase (PKS). Conserved domains will be analysized by site-directed mutagenesis of the proposed active sites, and the mutants characterized by in vivo analysis. It is hypothesized the PKS contains an unusual acyl carrier protein (ACP) domain that requires a unique phosphopantetheinyl transferase (PPTase), which is proposed to also be a domain found within the PKS. The function of the putative ACP and PPTase domain will be analyzed by a thorough in vivo and in vitro analysis, which includes mutant generation of the proposed active sites. Finally, it is proposed that the regiochemistry of enediyne formation is controlled by the PKS. This will be tested by the creation of hybrid PKSs from C- 1027, which contains a 9-carbon enediyne core, and calicheamicin, which contains a 10-carbon enediyne core. The results will provide insights into the programming rules of enediyne biosynthesisand will allow for the generation of "unnatural" natural enediynes with predicted chemistry.

DESCRIPTION (provided by applicant): The emergence of multiple drug resistant pathogens is becoming problematic worldwide, and the discovery of new antibiotics continues to decline. The broad, long-term objective of this proposal is to discover, characterize, and develop a new structural class of antibiotics-nucleoside antibiotics-that target bacterial translocase I involved in peptidoglycan cell wall biosynthesis. The specific aims of this proposal are (I) to characterize the assembly and incorporation of the aminoribosyl moiety found within the family of lipopeptidyl-nucleoside antibiotics and (II) to functionally and mechanistically characterize enzymes catalyzing novel or unusual biochemical reactions represented herein by a new family of serine hydroxymethyltransferase-like enzymes that are hypothesized to catalyze an aldol-type condensation to form unusual nonproteinogenic amino acids. Specific Aim I and II will be achieved by using the robust genetic system developed for the lipopeptidyl nucleoside A-90289-producing strain for in vivo studies employing gene inactivation and cross-complementation, and recombinant proteins will be exploited for functional and mechanistic studies in vitro. The results will establish a new mechanism for incorporating ribosyl units into natural product scaffolds and will establish a paradigm for the entry into high-carbon nucleoside antibiotics. The results will be essential for our long-term goals of searching for new nucleoside antibiotics using genetic information (a genotype-to-chemotype approach), will allow for the structural diversification of the parent scaffolds using combinatorial biosynthesis and total synthesis, and will provide the basis for the design of second generation antibiotics with improved biocompatibility and pharmacological properties. PUBLIC HEALTH RELEVANCE: Diseases caused by multidrug resistant bacteria are becoming a significant threat to human health worldwide. The goal of this proposal is to study nucleoside antibiotics that represent a new structural class of antibiotics, have a different mode of action than clinically used antibacterial drugs, and are in general not toxic to animals.

DESCRIPTION (provided by applicant): The emergence of multiple drug resistant pathogens is becoming problematic worldwide, and the discovery of new antibiotics continues to decline. The broad, long-term objective of this proposal is to discover, characterize, and develop a new structural class of antibiotics-nucleoside antibiotics-that target bacterial translocase I involved in peptidoglycan cell wall biosynthesis. The specific aims of this proposal are (I) to characterize the assembly and incorporation of the aminoribosyl moiety found within the family of lipopeptidyl-nucleoside antibiotics and (II) to functionally and mechanistically characterize enzymes catalyzing novel or unusual biochemical reactions represented herein by a new family of serine hydroxymethyltransferase-like enzymes that are hypothesized to catalyze an aldol-type condensation to form unusual nonproteinogenic amino acids. Specific Aim I and II will be achieved by using the robust genetic system developed for the lipopeptidyl nucleoside A-90289-producing strain for in vivo studies employing gene inactivation and cross-complementation, and recombinant proteins will be exploited for functional and mechanistic studies in vitro. The results will establish a new mechanism for incorporating ribosyl units into natural product scaffolds and will establish a paradigm for the entry into high-carbon nucleoside antibiotics. The results will be essential for our long-term goals of searching for new nucleoside antibiotics using genetic information (a genotype-to-chemotype approach), will allow for the structural diversification of the parent scaffolds using combinatorial biosynthesis and total synthesis, and will provide the basis for the design of second generation antibiotics with improved biocompatibility and pharmacological properties.

New antibiotics are needed, particularly those that can be considered as new chemical entities and have novel targets relative to the current, clinical armament of antibiotics. Highly modified nucleoside antibiotics that inhibit bacterial translocase I (TL1) involved in cell wall biosynthesis fit both these descriptions, and have excellent potential in part because they are (i) nanomolar inhibitors of TL1, (ii) inhibit a target that has been proven to be essential for the survival of most, if not all, bacteria, (iii) are effective antibiotics in both in vitro and in vivo models, and (iv) have no apparent toxicity in mice. We have defined the biosynthetic mechanism leading to the core disaccharyl-nucleoside structure of several promising nucleoside antibiotics including A-90289 from Streptomyces sp. SANK 60405, muraminomicin from Streptosporangium sp, and muraymycin from Streptomyces sp. LL-AA896 using a combined in vivo and in vitro approach. The results have defined a multi-enzyme pathway highlighted by divergence from the primary building block UMP and reconvergence to form the core nucleoside. This data was utilized to scan the wealth of genomic information to identify a new lead antibiotic, sphaerimicin, which was isolated and revealed to share the nucleoside core structure but have several unique features including a dihydroxylated piperidine ring of unknown origin. We will now accomplish the following specific aims: (i) to define the mechanism for the attachment of the 3-amino-3-carboxypropyl (3A3CP) moiety that generates the last, shared intermediate in the biosynthesis of A-90289, muraminomicin, muraymycin, and sphaerimicin, which is hypothesized to occur via a new enzyme strategy catalyzed by a pyridoxal-5?-phosphate-dependent protein and (ii) to delineate the biosynthetic mechanism for divergence from the last, shared intermediate to generate unique, nucleoside core scaffolds that are further decorated by fatty acids, polyketides, nonribosomal peptides, and/or saccharides. A biosynthetic mechanism for the diazepanone ring for A-90289 and the highly unusual fused piperidine ring system in sphaerimicin will be defined.

Tuberculosis (TB)?which is primarily caused by the bacterial pathogen Mycobacterium tuberculosis (Mtb)?is an ancient disease that remains one of the deadliest communicable diseases worldwide. A paramount concern heading into the future is the rapid rise in drug-resistant TB. The World Health Organization estimated 480,000 cases of TB with 190,000 deaths in 2014 with resistance to the first-line anti-TB drugs isoniazid and rifampicin. Furthermore, totally drug-resistant Mtb has now been documented in multiple countries including the United States. The capuramycin family of glycosylated nucleoside antibiotics are excellent candidates for anti-TB drug discovery and development because they (i) are considered new chemical entities with several unusual structural features compared to all antibiotics including clinical anti-TB drugs, (ii) target a novel and essential enzyme (translocase 1; TL1) in cell wall biosynthesis, (ii) have exceptional anti-Mtb activity in vitro and in vivo, (iv) are bactericidal and kill Mtb faster than any first-line anti-TB drug in vitro, and (v) have no toxicity. Our primary objectives in this proposal are to define a biosynthetic mechanism for the assembly of the unusual unsaturated hexuronic acid component in capuramycins (Aim 1) and establish complementary chemical (via neoglycorandomization; Aim 2) and biosynthetic (via native and nonnative glycosyltransferases; Aim 3) platforms for rapidly generating novel hexuronic acid-substituted capuramycins that can be screened for TL1 inhibition, anti-Mtb activity, and improved pharmacological properties. Additionally, these novel capuramycin analogues will be screened as potential substrates or inhibitors of the phosphotransferase CapP, which covalently modifies capuramycin as a strategy of self-resistance within the producing strain and is potentially a widespread resistance mechanism. It is expected that, upon completion of the aims, a new biosynthetic mechanism for sugar incorporation and modification will be defined. Furthermore, the completion of the aims will provide the first practical, comprehensive strategy to rapidly interrogate/modulate the fundamental features of capuramycin core pharmacophore, which will not only be important for the clinical development of capuramycin but can be applied to other glycosylated nucleoside antibiotics, of which dozens are now known with diverse biological activities.

ABSTRACT The 10-membered enediynes [exemplified by calicheamicin (CLM), esperamicin (ESP) and dynemicins (DYN)] are arguably among the most renowned natural products (NPs) discovered to date by virtue of their unprecedented complex molecular architectures, notable anticancer and anti-infective potencies and, in the case of CLM, demonstrated clinical utility. The current study builds on a longstanding collaborative effort of achievement and discovery relating to key aspects of 10-membered enediyne biosynthesis as well as parallel innovative efforts to co-opt key biosynthetic catalysts for synthetic applications. The studies put forth will take advantage of this strong foundation and a powerful combination of genetic, biochemical, chemical and protein structural tools to elucidate the remaining unusual biosynthetic transformations and to exploit select catalysts for enediyne non-native modification. Specifically, aims 1 and 2 will focus on extending our understanding of the fundamental steps of enediyne core biosynthesis common to CLM/DYN/ESP, DYN anthraquinone biosynthesis and a selected set of unique tailoring reactions (CLM/ESP thiosugar sulfur installation and aminopentose N- alkylation, ESP C6-hydroxylation and O-glycosylation). In parallel, aim 3 will focus on tactical structural studies to augment both aims 1 and 2 and the structural study of ?unknowns? to facilitate functional annotation. Additional studies in aim 2 with key catalysts and corresponding non-native substrates are designed to assess the potential for strategic installation of chemoselective handles to enable novel approaches for facile, mild bioconjugation of CLM to tumor-targeting mAbs (in collaboration with Pfizer).

SARS-CoV-2 is a positive-sense, single-stranded RNA [(+)ssRNA] virus that relies on its RNA-dependent RNA polymerase (RdRp) for survival. Within the months of April and May, five groups have independently reported the production of recombinant SAR-CoV-2 RdRp and a preliminary activity assessment of this essential enzyme. Included in these studies was direct evidence that the triphosphate version of the nucleoside analogue remdesivir, which has received Emergency Use Authorization from the Food and Drug Administration to treat SARS-CoV-2, is incorporated in place of ATP into the growing RNA oligonucleotide, ultimately leading to chain termination. Other nucleobase and nucleoside analogues including EIDD-1931 and favipiravir, the latter of which has been approved in Japan to treat the (-)ssRNA influenza virus, have demonstrated promise as therapeutic agents against SARS-CoV-2 by likely interfering with RNA metabolism via inhibition or processing as alternative substrates for RdRp. This and other data suggest that RdRp is an excellent target for the discovery and development of novel SARS-CoV-2 therapeutics. However, one of the major bottlenecks in exploiting RdRp as a drug target is the relatively low throughput activity-based assays that are costly, prone to interference, and/or lack flexibility in the experimental design. The primary objective of this proposal is to develop a broadly applicable RdRp activity-based assay that will be used for antiviral drug discovery efforts. Our specific aim is to establish a novel, real-time assay using a five-enzyme coupled system with a colorimetric readout. The new assay will be directly compared to the traditional polyacrylamide gel electrophoresis and a liquid scintillation proximity end- point assay, and further validated with high resolution mass spectrometry. It is expected that, by accomplishing this aim, the assay will enable a thorough biochemical characterization of SARS-CoV-2 RdRp and, for the first time, enable the testing of synthetic compound and natural product libraries to identify inhibitors, alternative substrates, modulators, or effectors of SARS-CoV-2 RdRp activity in a high throughput screening format. Notably, the strategy implemented herein is expected to complement on-going structural-based anti-SARS-CoV-2 discovery efforts. Finally, the activity-based assay can be readily adapted for RdRp from other (+)ssRNA and (- )ssRNA viruses in an effort to identify therapeutics against a broad spectrum of pandemic-causing viruses.