ABSTRACT 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 th...