Predicting of novel homoserine dehydrogenase inhibitors against <i>Paracoccidioides brasiliensis</i>: integrating <i>in silico</i> and <i>in vitro</i> approaches
Creators: name: Tartari, Jovana Chiapetti
name: Khan, Asif
name: da Silva Andrade, João Gabriel
name: Rodrigues, Francielli Abigail Vilugron
name: Bueno, Paulo Sérgio Alves
name: de Souza Lima, Diego
name: Canduri, Fernanda
name: de Freitas Gauze, Gisele
name: Kioshima, Érika Seki
name: Seixas, Flavio Augusto Vicente
Publisher: name: Taylor & Francis
Year: 2024
Subjects: subject: Biochemistry
subject: Pharmacology
subject: Biotechnology
subject: Chemical Sciences not elsewhere classified
subject: Biological Sciences not elsewhere classified
subject: Information Systems not elsewhere classified
subject: Cancer
subject: Infectious Diseases
subject: FOS: Health sciences

description: <b>Aim:</b> To search for potential inhibitors to homoserine dehydrogenase (HSD) in <i>Paracoccidioides brasiliensis</i> the causative agent of paracoccidioidomycosis, an infection with a high mortality rate in Brazil. <b>Materials &amp; methods:</b> The enzyme was modeled and used in the virtual screening of the compounds. The library was first screened by the Autodock, in which 66 molecules were better ranked than substrate, and then, also evaluated by the Molegro and Gold programs. <b>Results:</b> The HS23 and HS87 molecules were selected in common by the three programs, and ADME/Tox evaluation indicates they are not toxic. The molecular dynamics of <i>Pb</i>HSD bonded to ligands showed stable complexes until 50 ns. To validate the results, compounds were purchased for assays of minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC), synergic profile with Amphotericin B (AmB) and cytotoxicity. The two molecules presented MIC of 32 μg/ml and MFC of 64 μg/ml against the <i>P. brasiliensis</i> (strain Pb18). They also showed synergistic activity with AmB and a lack of toxicity against Hela and Vero cell lines. <b>Conclusion:</b> These results suggest that the HS23 and HS87 are promising candidates as <i>Pb</i>HSD inhibitors and may be used as hits for the development of new drugs against paracoccidioidomycosis. Paracoccidioidomycosis (PCM) is an infection that represents a significant public health concern in South America, with a high mortality rate. The enzyme Homoserine dehydrogenase (HSD) is essential for fungal survival so, targeting HSD with inhibitors could provide a novel therapeutic approach against PCM. The recommended therapy for PCM with azoles can last up to 24 months, which increases costs and is a cause of treatment abandonment, therefore, research for specific therapies against this disease is relevant. This study used computational simulations such as virtual screening and molecular dynamics to identify potential inhibitors for Homoserine Dehydrogenase from <i>Paracoccidioides brasiliensis</i> (<i>Pb</i>HSD). The compounds identified by different <i>in silico</i> approaches were further evaluated for their antifungal activity and cytotoxicity. Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC) assays were performed to evaluate the efficacy of the selected compounds. Cytotoxicity toward mammalian cells was also verified. The synergistic effects of both compounds when combined with Amphotericin B, the reference medicine to treat PCM, were also evaluated. The computational simulations selected two compounds, HS23 and HS87, that were used in bench assays, where both presented fungicidal activity (MIC of 32 μg/ml and MFC of 64 μg/ml) against <i>P. brasiliensis</i> (strain Pb18), the most virulent. Both compounds presented synergism with Amphotericin B, with the sum of the fractional inhibitory concentration of 0.312 for HS23 and 0.375 for HS87. Both compounds were non-toxic for Vero or HeLa cells, till the limit of the assay (512 μg/ml). The <i>in silico</i> results obtained by computational simulation were confirmed through <i>in vitro</i> assays, demonstrating that computational approaches are efficient tools for the discovery of new enzymatic inhibitors. Targeting enzymes of the amino acid biosynthesis pathway may be a strategy to overcome microbial resistance since there are no antifungals on the market that focus on this mechanism. Compounds with antifungal activity are rare compared with those with antimicrobial action. So, the results of this work could represent an important contribution to the improvement of the pharmacological arsenal against fungal infections.