Date of Award

Spring 5-18-2024

Degree Type

Thesis

Degree Name

MS Chemistry

Department

Chemistry and Biochemistry

Advisor

Gregory R Wiedman, PhD

Committee Member

Cosimo Antonacci, PhD

Committee Member

Wyatt R Murphy, PhD

Keywords

Flippase inhibitor, antimicrobial peptide, drug resistance, drug synergy, Cryptococcus neoformans, P4-ATPase flippase, Cdc50

Abstract

The synthesis, characterization, and evaluation of antifungal activity of peptides FQ15 and FG10 will be conducted to address the hypothesis. The hypothesis of this thesis is that a peptide with similar properties to AS15 would have similar activity against Cryptococcus neoformans. This study addresses the challenge of drug- resistant microbes by developing inhibitors targeting lipid flippase and exploring their potential as antimicrobial agents against Cryptococcus neoformans. Chapter 1 provides a historical context of the background on the development of antimicrobials and common antifungal treatments, as well as an introduction about C. neoformans and its resistance mechanism. Chapter 2 introduces the materials and the experimental methods used for the drug discovery of the antifungal peptide designed to target the fungus pathogen C. neoformans. Chapter 3 reviews the preliminary results and discussion. Lastly, Chapter 4 is the conclusion of this study.

FQ15 and FG10 peptides demonstrated efficacy against C. neoformans in vitro. C. neoformans is a challenging and threatening fungal pathogen that is highly resistant to caspofungin drug, which poses a significant potential threat to cause severe meningitis in immunocompromised individuals like those with HIV/AIDS. This ongoing study aimed to investigate antifungal properties, focusing on exploring the elements within CDC50 loop region of the fungal pathogen C. neoformans to develop potential flippase inhibitors to design peptides as antifungal agents. Previous research has highlighted the essential role of Cryptococcus lipid translocase (flippase) in caspofungin resistance mechanism. 3B lab members focused on identifying peptides resembling the original AS15 peptide with similar biophysical characteristics, including hydrophobicity, net charge, isoelectric point, and molecular weight within the CDC50 loop sequence to demonstrate antifungal efficacy. Flippase inhibitors show promise in combating drug-resistant microbes.

Solid phase peptide synthesis (SPPS) was utilized to synthesize 15 amino acid-long sequences, FQ15 and FQ15MA, and 10 amino- acid long sequences, FG10 and FG10MA. The utilization of Mass Spectroscopy (MS) and High-Performance Liquid Chromatography (HPLC) confirmed the detection and isolation of the synthesized peptides. HPLC played a role in analyzing peptide detection and fractions isolation. Adding myristic acid on the N- terminus of the same amino acid sequence was done to explore its potential as an antimicrobial agent, and it showed efficacy at higher concentrations. FQ15MA demonstrated an MIC of 16 μg/mL against H99 and FG10MA demonstrated an MIC of 8 μg/mL against H99 wild type and 64 μg/mL against ΔCDC50 mutant, and a fractional inhibitory concentration (FIC) index value of less than 0.5 when combined with caspofungin.

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