Integrative Structural Characterization of Candida glabrata Phosphoglycerate Kinase by Small-Angle X‑ray Scattering...

Candida glabrata is the second leading cause of mortality in immunocompromised patients hospitalized for invasive candidiasis (IC). Several drugs have been available to treat this disease for decades, such as polyenes, azoles, echinocandins, flucytosine, and, in critical cases, amphotericin B. However, these antifungals' constant and routine use have led to the development of resistance mechanisms, making the design and development of new drugs indispensable. The first step for the design and subsequent synthesis of a new chemical molecule as a potential antifungal is the identification of new therapeutic targets. In that pathway, our working group has identified moonlight-like cell wall proteins (CWPs) in different Candida species that can act as potential antifungal targets. One of these moonlight-like CWPs is phosphoglycerate kinase (Pgk) from C. glabrata. Once Pgk was identified as a potential therapeutic target in different human pathogens, the first step to perform drug design against this moonlight-like CWP was the elucidation of the three-dimensional (3D) structure since the 3D structure is key to understanding the interactions between a drug candidate and its target at the molecular level. In the present work, we aimed to elucidate the 3D structure of C. glabrata Pgk. To elucidate the 3D structure of this protein, the recombinant protein was expressed, purified, and structurally resolved by means of a structural analysis by small-angle X-ray scattering (SAXS). Additionally, in order to evaluate its potential as a therapeutic target, we have performed molecular docking studies and enzymatic activity assays with pure Pgk using known antifungals amphotericin B, nystatin, and fluconazole and with the new plausible drugs, such as nilotinib and netupitant. Our results showed some similarities and differences with orthologous Pgk proteins from other organisms, which was expected since Pgk has been observed to have evolved in the kingdoms of life. Molecular docking studies showed that Pgk interacts with all of the compounds tested. In enzyme activity assays, a change in the kinetic parameter Km on the enzyme Pgk was observed in response to its interaction with nilotinib, netupitant, and amphotericin B. Thus, our results allow us to propose Pgk from C. glabrata as a possible therapeutic target against candidiasis. We consider it essential to design and develop new molecules specifically targeting this enzyme, which will contribute to a decrease in mortality associated with IC and improve the patient's quality of life.