OF THE RESEARCH PLAN The studies to be pursued under the proposed extension fall within three general areas: i) enzyme catalyst engineering and modulation (theme 1); ii) selected platform applications (theme 2); and iii) a systematic study of the impact of glycosylation on fundamental drug properties (theme 3). The key tenets of each are briefly described below and parallel the consistently high level of significance and innovation reflected in past periods of support. The overarching significance stems from the anticipated advancements of universal principles for enzyme catalyst engineering/modulation, novel enabling strategies for synthesis and biosynthetic study, unique probes or leads of significance to diverse therapeutic areas, and a potential new conceptual framework for the general use of glycosylation in drug development. Anticipated drivers of innovation are both by design (particularly novel synthetic and assay strategies) and directed discovery (unprecedented glycosyltransferase modulators and probes/leads with high potential to lead to new understanding in the fundamentals of immunomodulation and/or inflammation). Studies directed toward enzyme catalyst engineering and modulation (theme 1) will focus on comparing the directed evolutionary paths among the structurally similar glycosyltransferases (GTs) OleD and OleI, assessing the transferability of beneficial mutations across OleD and OleI and identifying mutational hot spots within the atypical GTB-fold GT AmphD1. The comparative OleD/OleI studies will consist of: i) the evaluation of an ?OleI Loki,? which contains the 5 key mutations of structurally conserved residues that led to OleD Loki; and ii) a parallel epPCR-directed evolution approach for increasing OleI proficiency/permissivity using the same screens and mutational strategy that led to the discovery of enhanced OleD progeny. Key outcomes of this comparative study will be a potential universal blueprint for improving the proficiency/promiscuity of most GTB-fold GTs and improved GTs for select sugar-conjugation (D-Gal analogs) to support theme 3. The directed evolution of AmphD1 (and parallel proposed structure elucidation and structure-based engineering) will also follow our OleD precedent and offers both the potential to expand the universal GT engineering blueprint toward atypical GTs and to identify GT variants to dramatically simplify the targeted synthesis of the unique preclinical antifungal lead 2?-epi-amphotericin (theme 2). In addition, we will pursue the mechanistic study of small molecule inhibitors and activators identified via the ClNP-Glc-based HT screen - studies that hold high potential in the discovery of new paradigms for GT catalytic modulation that may extend to other enzyme families and/or have pharmacologic applications. Finally, while we remain enthusiastic about the uncharted frontier of enzyme modulation via controlled electrical fields, such technically challenging experiments require the physical presence of both the materials scientists and enzymologists and, unfortunately, our local collaborator (Prof. Hinds) recently relocated to the University of Washington. Thus, this has become a lower priority in the near term. Among the various selected platform applications (theme 2), we will direct the real-time NMR-based assay toward key and novel late-stage biosynthetic transformations - specifically, to understand the stepwise aminopentose N-alkylation reactions en route to enediynes and indolocarbazoles and the key steps of enediyne- associated thiosugar biosynthesis. The main outcome of these studies will be the elucidation of what is anticipated to be (based on bioinformatics) novel enzyme-catalyzed chemistries. In addition, we will continue the ongoing studies discussed within the progress report (immunomodulatory studies with macrolide neoglycosides, anti-Mtb studies with spectinomycin neoglycosides, and further advancing protein glycosylation studies) and initiate new combined (neo)glycorandomization diversification efforts toward a prioritized list of additional targets. Specifically, we seek to generate and evaluate (neo)glycorandomized libraries of a third or fourth generation ?- lactam antibiotic (primary objective to broaden antibacterial scope), rapamycin (primary objectives to assess mechanistic modulation and potency), and daptomycin (primary objectives to improve formulation, potency and/or antibacterial scope). For all targets selected, we have access to the necessary fundamental evaluative activity assays within my lab or via the Center of Pharmaceutical Research and Innovation (under my directorship) and these new initiatives hold promise for the discovery of new potential anti-infective, anticancer and/or immunomodulatory probes/leads and for advancing new tools for biopharmaceutical development. As highlighted in the progress report, we have also initiated arguably one of the first broad systematic studies of the impact of glycosylation on fundamental drug properties (theme 3), the strategic design of which will probe both the impact of the sugar and corresponding drug leaving group on key drug property principles. The corresponding in vitro and in vivo drug properties of the ?-glucosides will be evaluated by Vertex. In parallel, Prof. Leggas (co-I) will evaluate the in vitro and in vivo drug properties of a SN-38 10-O-?-glycoside sugar variant series from which the top 2-3 analogs will be selected for subsequent in vivo anticancer efficacy studies. These studies are anticipated to reveal fundamental insights regarding the impact of the sugar, drug and corresponding glycosidic bond as the basis of a potentially new conceptual framework for the general use of glycosylation as a transformative drug development tool. Program Director/Principal Investigator (Last, First, Middle): Thorson, Jon DETAILED BUDGET FOR INITIAL BUDGET PERIOD DIRECT COSTS ONLY FROM 07/01/2016 THROUGH 06/30/2017 List PERSONNEL (Applicant organization only) Use Cal, Acad, or Summer to Enter Months Devoted to Project Enter Dollar Amounts Requested (omit cents) for Salary Requested and Fringe Benefits NAME Thorson, Jon Leggas, Markos Zhang, Jianjun Hughes, Ryan Eckenrode, Joseph ROLE ON PROJECT Cal. Mnths Acad. Mnths Summer Mnths INST.BASE SALARY SALARY REQUESTED FRINGE BENEFITS PD/PI 2.40 183,300 36,660 9,929 Co-I 0.36 121,097 3,633 1,093 Postdoc 12 44,803 44,803 14,656 Grad Student 12 24,000 24,000 4,524 Grad Student 12 24,000 24,000 4,524 133,096 34,726 167,822 TOTAL 46,589 4,726 59,459 28,524 28,524 SUBTOTALS CONSULTANT COSTS EQUIPMENT (Itemize) SUPPLIES (Itemize by category) Tissue culture, cell biology, molecular biology, biochemical, chemical reagents, waste disposal Bioanalytical reagents/supplies LC/MS/MS maintenance Animal costs107,775 TRAVEL travel to scientific meetings3,000 INPATIENT CARE COSTS OUTPATIENT CARE COSTS ALTERATIONS AND RENOVATIONS (Itemize by category) OTHER EXPENSES (Itemize by category) graduate student tuition, publication costs. 16,666 CONSORTIUM/CONTRACTUAL COSTS DIRECT COSTS SUBTOTAL DIRECT COSTS FOR INITIAL BUDGET PERIOD (Item 7a, Face Page) $ 295,263 CONSORTIUM/CONTRACTUAL COSTS FACILITIES AND ADMINISTRATIVE COSTS TOTAL DIRECT COSTS FOR INITIAL BUDGET PERIOD $ 295,263 PHS 398 (Rev. 08/12 Approved Through 8/31/2015) OMB No. 0925-0001 Page Form Page 4
| Elshahawi, Sherif I; Cao, Hongnan; Shaaban, Khaled A et al. (2017) Structure and specificity of a permissive bacterial C-prenyltransferase. Nat Chem Biol 13:366-368 |
| Hughes, Ryan R; Shaaban, Khaled A; Zhang, Jianjun et al. (2017) OleD Loki as a Catalyst for Tertiary Amine and Hydroxamate Glycosylation. Chembiochem 18:363-367 |
| Zhang, Jianjun; Hughes, Ryan R; Saunders, Meredith A et al. (2017) Identification of Neuroprotective Spoxazomicin and Oxachelin Glycosides via Chemoenzymatic Glycosyl-Scanning. J Nat Prod 80:12-18 |
| Han, Lu; Singh, Shanteri; Thorson, Jon S et al. (2016) Loop dynamics of thymidine diphosphate-rhamnose 3'-O-methyltransferase (CalS11), an enzyme in calicheamicin biosynthesis. Struct Dyn 3:012004 |
| Cai, Wenlong; Wang, Xiachang; Elshahawi, Sherif I et al. (2016) Antibacterial and Cytotoxic Actinomycins Y6-Y9 and Zp from Streptomyces sp. Strain Gö-GS12. J Nat Prod 79:2731-2739 |
| Huber, Tyler D; Wang, Fengbin; Singh, Shanteri et al. (2016) Functional AdoMet Isosteres Resistant to Classical AdoMet Degradation Pathways. ACS Chem Biol 11:2484-91 |
| Huber, Tyler D; Johnson, Brooke R; Zhang, Jianjun et al. (2016) AdoMet analog synthesis and utilization: current state of the art. Curr Opin Biotechnol 42:189-197 |
| Elshahawi, Sherif I; Shaaban, Khaled A; Kharel, Madan K et al. (2015) A comprehensive review of glycosylated bacterial natural products. Chem Soc Rev 44:7591-697 |
| Singh, Shanteri; Michalska, Karolina; Bigelow, Lance et al. (2015) Structural Characterization of CalS8, a TDP-?-D-Glucose Dehydrogenase Involved in Calicheamicin Aminodideoxypentose Biosynthesis. J Biol Chem 290:26249-58 |
| Zhang, Jianjun; Ponomareva, Larissa V; Nandurkar, Nitin S et al. (2015) Influence of Sugar Amine Regiochemistry on Digitoxigenin Neoglycoside Anticancer Activity. ACS Med Chem Lett 6:1053-8 |
Showing the most recent 10 out of 31 publications
