Modulation of Antibacterial Activity and Cytotoxicity in Amphipathic Cyclic Peptide [R4W4] Using Histidine Substitution

Author

Ryan Stueber, Chapman UniversityFollow

Date of Award

Summer 8-2021

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Pharmaceutical Sciences

First Advisor

Dr. Rakesh Kumar Tiwari

Second Advisor

Dr. Keykavous Parang

Third Advisor

Dr. Aftab Ahmed

Abstract

Antibiotics have been the gold standard frontline defense against bacterial infections for decades. At the same time, these infecting bacteria have continued to evolve to resist developed antibiotics in an almost endless cycle. As such, we aim to take an alternative approach utilizing antimicrobial peptides (AMPs) as a means to end this cycle. [R4W4] is among known AMPs that demonstrated antimicrobial activity against methicillin-resistant staphylococcus aureus (MRSA) with a minimum inhibitory concentration (MIC) of 2.67 μg/mL. This peptide had notably effective antimicrobial activity, especially relative to linear (R4W4). However, it displayed a concerning level of cytotoxicity; eliciting a human embryonic kidney (HEK-293T) cell viability ≥ 80% as the concentration reached 15μM, beyond which cell viability sharply declines as low as 20 %. In previous studies, it was observed that substitution with histidine (H) into a tryptophan (W) and arginine (R) rich AMP resulted in a strong modulation of cell cytotoxicity.

In this study, we designed and synthesized four cyclic peptides ([W3HR4], [W4H3R], [W4H2R2], and [WH3R4]), in addition to the linear and cyclic variant of potent template AMP ((R4W4), and [R4W4]), to observe how these sequence modulations affected antibacterial activity and cytotoxicity. It was observed that [W4H2R2] had a lower MIC against both Gram-positive bacteria (e.g., MRSA, Staphylococcus aureus (SA)) at 3.1 μg/mL and for Gram-negative (E. coli) at 6.2 μg/mL, compared to peptide [R4W4], which had a MIC value of 6.2 μg/mL for SA and 25 μg/mL for E. coli. The hemolytic activity of [W4H2R2] against human red blood cells (hRBCs) ABSTRACT VII elicited 3.9% hemolysis at 62.5 μg/mL treatment when compared to that of [R4W4], which had 6.2% hemolysis. Lastly, [W4H2R2] retained a higher level of cell viability (99% when treating HEK-293 cells with 40 μg/mL) while at the same concertation [R4W4] retained 79.96% cell viability. These results strongly support the hypothesis that a H sequence substitution into peptide [R4W4] will modulate antibacterial activity and cytotoxicity. In particular, [W4H2R2] elicited higher potency antibacterial activity, sustained lower levels of hemolytic activity, and retained higher cell viability at higher concentrations when compared to the template peptide [R4W4].

Creative Commons License

This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

Recommended Citation

Stueber, RL. Modulation of Antibacterial Activity and Cytotoxicity in Amphipathic Cyclic Peptide [R4W4] Using Histidine Substitution. [master’s thesis]. Irvine, CA: Chapman University; 2021. https://doi.org/10.36837/chapman.000308