Design of a Helical-Stabilized, Cyclic, and Nontoxic Analogue of the Peptide Cm-p5 with Improved Antifungal Activity

Fidel E Morales Vicente; Melaine González-Garcia; Erbio Diaz Pico; Elena Moreno-Castillo; Hilda E Garay; Pablo E Rosi; Asiel Mena Jimenez; Jose A Campos-Delgado; Daniel G Rivera; Glay Chinea; Rosemeire C L R Pietro; Steffen Stenger; Barbara Spellerberg; Dennis Kubiczek; Nicholas Bodenberger; Steffen Dietz; Frank Rosenau; Márcio Weber Paixão; Ludger Ständker; Anselmo J Otero-González

doi:10.1021/acsomega.9b02201

Design of a Helical-Stabilized, Cyclic, and Nontoxic Analogue of the Peptide Cm-p5 with Improved Antifungal Activity

ACS Omega. 2019 Nov 5;4(21):19081-19095. doi: 10.1021/acsomega.9b02201. eCollection 2019 Nov 19.

Authors

Fidel E Morales Vicente^{1

2

3}, Melaine González-Garcia⁴, Erbio Diaz Pico², Elena Moreno-Castillo¹, Hilda E Garay², Pablo E Rosi⁵, Asiel Mena Jimenez¹, Jose A Campos-Delgado³, Daniel G Rivera¹, Glay Chinea², Rosemeire C L R Pietro⁶, Steffen Stenger⁷, Barbara Spellerberg⁷, Dennis Kubiczek⁸, Nicholas Bodenberger⁸, Steffen Dietz⁸, Frank Rosenau⁸, Márcio Weber Paixão³, Ludger Ständker⁹, Anselmo J Otero-González⁴

Affiliations

¹ General Chemistry Department, Faculty of Chemistry and Center for Natural Products Research, Faculty of Chemistry, University of Havana, Zapata y G, 10400 La Habana, Cuba.
² Synthetic Peptides Group, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, 10600 La Habana, Cuba.
³ Center of Excellence for Research in Sustainable Chemistry (CERSusChem), Department of Chemistry, Federal University of São Carlos-UFSCar, São Paulo 13565-905, Brazil.
⁴ Center for Protein Studies, Faculty of Biology, University of Havana, 25 and I, 10400 La Habana, Cuba.
⁵ Department of Inorganic Chemistry, Analytical and Physical Chemistry, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires C1428EGA, Argentina.
⁶ Laboratory of Pharmaceutical Biotechnology, Department of Drugs and Medicines, School of Pharmaceutical Sciences, UNESP, Araraquara 14800-900, Brazil.
⁷ Institute of Medical Microbiology and Hygiene, University Clinic of Ulm, Robert Koch Str. 8, Ulm D-89081, Germany.
⁸ Institute of Pharmaceutical Biotechnology, Ulm University, James-Frank-Ring N27, 89081 Ulm, Germany.
⁹ Core Facility for Functional Peptidomics, Ulm Peptide Pharmaceuticals (U-PEP), University Ulm, Faculty of Medicine, Ulm University, 89081 Ulm, Germany.

Abstract

Following the information obtained by a rational design study, a cyclic and helical-stabilized analogue of the peptide Cm-p5 was synthetized. The cyclic monomer showed an increased activity in vitro against Candida albicans and Candida parapsilosis, compared to Cm-p5. Initially, 14 mutants of Cm-p5 were synthesized following a rational design to improve the antifungal activity and pharmacological properties. Antimicrobial testing showed that the activity was lost in each of these 14 analogues, suggesting, as a main conclusion, that a Glu-His salt bridge could stabilize Cm-p5 helical conformation during the interaction with the plasma membrane. A derivative, obtained by substitution of Glu and His for Cys, was synthesized and oxidized with the generation of a cyclic monomer with improved antifungal activity. In addition, two dimers were generated during the oxidation procedure, a parallel and antiparallel one. The dimers showed a helical secondary structure in water, whereas the cyclic monomer only showed this conformation in SDS. Molecular dynamic simulations confirmed the helical stabilizations for all of them, therefore indicating the possible essential role of the Glu-His salt bridge. In addition, the antiparallel dimer showed a moderate activity against Pseudomonas aeruginosa and a significant activity against Listeria monocytogenes. Neither the cyclic monomer nor the dimers were toxic against macrophages or THP-1 human cells. Due to its increased capacity for fungal control compared to fluconazole, its low cytotoxicity, together with a stabilized α-helix and disulfide bridges, that may advance its metabolic stability, and in vivo activity, the new cyclic Cm-p5 monomer represents a potential systemic antifungal therapeutic candidate.