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Antifungal, Antimycobacterial, Protease and α‒Amylase Inhibitory Activities of a Novel Serine Bifunctional Protease Inhibitor from Adenanthera pavonina L. Seeds

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Abstract

Antifungal resistance poses a significant challenge to disease management, necessitating the development of novel drugs. Antimicrobial peptides offer potential solutions. This study focused on extraction and characterization of peptides from Adenanthera pavonina seeds with activity against Candida species, Mycobacterium tuberculosis, proteases, and α-amylases. Peptides were extracted in phosphate buffer and heated at 90°C for 10 min to create a peptide rich heated fraction (PRHF). After confirming antimicrobial activity and the presence of peptides, the PRHF underwent ion exchange chromatography, yielding retained and non-retained fractions. These fractions were evaluated for antimicrobial activity and cytotoxicity against murine macrophages. The least toxic and most active fraction underwent reversed-phase chromatography, resulting in ten fractions. These fractions were tested for peptides and antimicrobial activity. The most active fraction was rechromatographed on a reversed-phase column, resulting in two fractions that were assessed for antimicrobial activity. The most active fraction revealed a single band of approximately 6 kDa and was tested for inhibitory effects on proteases and α-amylases. Thermal stability experiments were conducted on the 6 kDa peptide at different temperatures followed by reassessment of antifungal activity and circular dichroism. The 6 kDa peptide inhibited yeasts, M. tuberculosis, human salivary and Tenebrio molitor larvae intestine α-amylases, and proteolytic activity from fungal extracts, and thus named ApPI. Remarkably, ApPI retained antifungal activity and conformation after heating and is primarily composed of α-helices. ApPI is a thermally stable serine protease/α-amylase inhibitor from A. pavonina seeds, offering promise as a foundational molecule for innovative therapeutic agents against fungal infections and tuberculosis.

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Data Availability

Data will be made available on request.

Abbreviations

PRHF:

Peptide‒rich heated fraction

IC50 :

Half maximal inhibitory concentration

MTT:

3‒(4,5‒Dimethylthiazol‒2‒yl)‒2,5‒diphenyl‒tetrazolium bromide

CFU:

Colony forming unit

OD:

Optical density

U:

Unit activity

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Acknowledgements

This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Brasil—(CAPES)—Finance Code 001. We acknowledge the financial support of the Brazilian agencies CNPq and FAPERJ (E-26/202.760/2018-Bolsa). We acknowledge Luiz Carlos Dutra de Souza and Valeria Miguelote Kokis for their technical support.

Funding

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Finance Code 001, Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro, E-26/202.760/2018-Bolsa.

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Authors and Affiliations

  1. Laboratório de Fisiologia e Bioquímica de Microrganismos, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos dos Goytacazes, 28013-602, RJ, Brazil

    Rodrigo da Silva Gebara, Marciele Souza da Silva, Valdirene Moreira Gomes & André de Oliveira Carvalho

  2. Laboratório de Biologia do Reconhecer, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos dos Goytacazes, 28013-602, RJ, Brazil

    Sanderson Dias Calixto, Thatiana Lopes Biá Ventura Simão, Elena Lassounskaia & Jorge Hudson Petretski

  3. Laboratório de Química e Função de Proteinas e Peptídeos, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos dos Goytacazes, 28013-602, RJ, Brazil

    Ana Eliza Zeraik

  4. Laboratório de Produtos Bioativos, Universidade Federal do Rio de Janeiro, Macaé, 27933-378, RJ, Brazil

    Michelle Frazão Muzitano

Authors
  1. Rodrigo da Silva Gebara
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  2. Marciele Souza da Silva
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  6. Elena Lassounskaia
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  7. Michelle Frazão Muzitano
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  8. Jorge Hudson Petretski
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  9. Valdirene Moreira Gomes
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  10. André de Oliveira Carvalho
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Contributions

Gebara RS conceived, performed and wrote the manuscript. Silva MS performed the enzyme inhibition assays. Calixto SD performed the cytotoxicity assay. Simão TLBV performed the antimycobacterial assay. Zeraik AE performed the circular dichroism assay, Elena Lassounskaia conceived the antimycobacterial assay. Muzitano MF conceived the cytotoxicity assay. Petretski JH conceived the zymography analysis. Gomes VM revised the manuscript. Carvalho AO conceived and designed the experiments, acquired the fundings, and wrote and revised the manuscript.

Corresponding author

Correspondence to André de Oliveira Carvalho.

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Highlights

• ApPI, a new thermally stable serine protease/α‒amylase inhibitor from A. pavonina.

• ApPI inhibits Candida albicans and Mycobacterium tuberculosis.

• ApPI inhibits serine proteases extracted from Saccharomyces cerevisiae and C. albicans.

• ApPI retains antimicrobial activity after heating at 130 °C for 30 min.

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da Silva Gebara, R., da Silva, M.S., Calixto, S.D. et al. Antifungal, Antimycobacterial, Protease and α‒Amylase Inhibitory Activities of a Novel Serine Bifunctional Protease Inhibitor from Adenanthera pavonina L. Seeds. Probiotics & Antimicro. Prot. (2023). https://doi.org/10.1007/s12602-023-10194-z

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