R01AI170906

Project Summary/Abstract

The available antifungal drugs against invasive fungal infections are limited due to the challenge in selectively killing eukaryotic pathogens without harming humans. Chitin synthases (CHSs) represent one of the few proven targets whose inhibition provides highly selective antifungal effects without any detectable toxicity to humans. CHSs are transmembrane processive glycosyltransferases (GTs) responsible for the biosynthesis of chitin, an essential polysaccharide component of the fungal cell wall.

Due to their essential function in fungal physiology, CHSs are targeted by naturally occurring peptidyl nucleoside (PN) antifungal agents. PNs exhibit in vivo activities against multiple endemic fungal pathogens without adverse effects on humans and exhibit a strong synergy with current FDA-approved antifungal drugs. However, their development has been slow because of only moderate antifungal activities against more clinically prevalent fungal pathogens such as Candida albicans and the absence of atomic-level understanding of CHS.

Our long-term goal is to provide a comprehensive understanding of CHS catalysis, regulation, and inhibition at the atomic level. The current application focuses on structural and mechanistic studies of the catalysis and inhibition of C. albicans CHS. C. albicans has four CHSs, of which CACHS1 and CACHS2 require simultaneous inhibition for fungicidal effects. While existing PNs potently inhibit CACHS2, they are much weaker against CACHS1 and thus exhibit only moderate antifungal activity against C. albicans. The molecular mechanism behind the difference in PN potency between CACHS1 and CACHS2 is currently unknown. For the future development of CHS-targeting anti-Candida agents, it is essential to understand their structural and mechanistic differences for both catalysis and inhibition.

As a preliminary study, we heterologously expressed and purified catalytically active CACHS1 and CACHS2 and solved the cryo-EM structures of CACHS2 in the apo-, substrate-bound, and PN (nikkomycin Z and polyoxin D)-bound forms. We also developed novel activity assays for the determination of chitin chain length and quantitation of both long insoluble chitin and short soluble chito-oligosaccharides and established a method for chemo-enzymatic synthesis of nikkomycin analogs.

Based on these developments, we propose to study the mechanism of chitin formation and extrusion by CACHS2 (Aim 1), the functional and structural basis of the lower susceptibility of CACHS1 to PNs as well as the synergy of using both CACHS1 and CACHS2 inhibitors (Aim 2), and the detailed and systematic structure-activity relationships of PNs (Aim 3). For Aims 2 and 3, clinical isolates of C. albicans and non-albicans Candida strains will also be included for inhibitor testing, increasing the translational potential of our research program.

The proposed research is significant because it will provide the molecular basis for future development of novel antifungals against a clinically unexploited target.

Duke University

TO ASSIST PUBLIC AND PRIVATE NONPROFIT INSTITUTIONS AND INDIVIDUALS TO ESTABLISH, EXPAND AND IMPROVE BIOMEDICAL RESEARCH AND RESEARCH TRAINING IN INFECTIOUS DISEASES AND RELATED AREAS, TO CONDUCT DEVELOPMENTAL RESEARCH, TO PRODUCE AND TEST RESEARCH MATERIALS. TO ASSIST PUBLIC, PRIVATE AND COMMERCIAL INSTITUTIONS TO CONDUCT DEVELOPMENTAL RESEARCH, TO PRODUCE AND TEST RESEARCH MATERIALS, TO PROVIDE RESEARCH SERVICES AS REQUIRED BY THE AGENCY FOR PROGRAMS IN INFECTIOUS DISEASES, AND CONTROLLING DISEASE CAUSED BY INFECTIOUS OR PARASITIC AGENTS, ALLERGIC AND IMMUNOLOGIC DISEASES AND RELATED AREAS. PROJECTS RANGE FROM STUDIES OF MICROBIAL PHYSIOLOGY AND ANTIGENIC STRUCTURE TO COLLABORATIVE TRIALS OF EXPERIMENTAL DRUGS AND VACCINES, MECHANISMS OF RESISTANCE TO ANTIBIOTICS AS WELL AS RESEARCH DEALING WITH EPIDEMIOLOGICAL OBSERVATIONS IN HOSPITALIZED PATIENTS OR COMMUNITY POPULATIONS AND PROGRESS IN ALLERGIC AND IMMUNOLOGIC DISEASES. BECAUSE OF THIS DUAL FOCUS, THE PROGRAM ENCOMPASSES BOTH BASIC RESEARCH AND CLINICAL RESEARCH. SMALL BUSINESS INNOVATION RESEARCH (SBIR) PROGRAM EXPANDS AND IMPROVES PRIVATE SECTOR PARTICIPATION IN BIOMEDICAL RESEARCH. THE SBIR PROGRAM INTENDS TO INCREASE AND FACILITATE PRIVATE SECTOR COMMERCIALIZATION OF INNOVATIONS DERIVED FROM FEDERAL RESEARCH AND DEVELOPMENT, TO INCREASE SMALL BUSINESS PARTICIPATION IN FEDERAL RESEARCH AND DEVELOPMENT, AND TO FOSTER AND ENCOURAGE PARTICIPATION OF SOCIALLY AND ECONOMICALLY DISADVANTAGED SMALL BUSINESS CONCERNS AND WOMEN-OWNED SMALL BUSINESS CONCERNS IN TECHNOLOGICAL INNOVATION. THE SMALL BUSINESS TECHNOLOGY TRANSFER (STTR) PROGRAM STIMULATES AND FOSTERS SCIENTIFIC AND TECHNOLOGICAL INNOVATION THROUGH COOPERATIVE RESEARCH AND DEVELOPMENT CARRIED OUT BETWEEN SMALL BUSINESS CONCERNS AND RESEARCH INSTITUTIONS, TO FOSTER TECHNOLOGY TRANSFER BETWEEN SMALL BUSINESS CONCERNS AND RESEARCH INSTITUTIONS, TO INCREASE PRIVATE SECTOR COMMERCIALIZATION OF INNOVATIONS DERIVED FROM FEDERAL RESEARCH AND DEVELOPMENT, AND TO FOSTER AND ENCOURAGE PARTICIPATION OF SOCIALLY AND ECONOMICALLY DISADVANTAGED SMALL BUSINESS CONCERNS AND WOMEN-OWNED SMALL BUSINESS CONCERNS IN TECHNOLOGICAL INNOVATION. RESEARCH CAREER DEVELOPMENT AWARDS SUPPORT THE DEVELOPMENT OF SCIENTISTS DURING THE FORMATIVE STAGES OF THEIR CAREERS. INDIVIDUAL NATIONAL RESEARCH SERVICE AWARDS (NRSAS) ARE MADE DIRECTLY TO APPROVE APPLICANTS FOR RESEARCH TRAINING IN SPECIFIED BIOMEDICAL SHORTAGE AREAS. IN ADDITION, INSTITUTIONAL NATIONAL RESEARCH SERVICE AWARDS ARE MADE TO ENABLE INSTITUTIONS TO SELECT AND MAKE AWARDS TO INDIVIDUALS TO RECEIVE TRAINING UNDER THE AEGIS OF THEIR INSTITUTIONAL PROGRAM.

93.855 - Allergy and Infectious Diseases Research

National Institute of Allergy and Infectious Diseases (NIAID) [HHS - NIH]

Durham, North Carolina 277103023 United States

Single Zip Code

NIH Research Project Grant (Parent R01 Clinical Trial Not Allowed) (PA-20-185)

Amendment Since initial award the total obligations have increased 382% from $639,109 to $3,081,067.