# Integrative Structural Characterization of Candida glabrata Phosphoglycerate Kinase by Small-Angle X‑ray Scattering and AlphaFold: Implications for Therapeutic Targeting in Candidiasis

## TL;DR

This study explores the 3D structure of a potential antifungal target in Candida glabrata, using X-ray and computational methods to support drug development for candidiasis.

## Contribution

The study provides the first structural characterization of C. glabrata phosphoglycerate kinase (Pgk) using SAXS and AlphaFold, supporting its role as a novel antifungal target.

## Key findings

- C. glabrata Pgk shows structural similarities and differences compared to orthologs from other organisms.
- Molecular docking revealed interactions between Pgk and tested antifungals and potential new drugs.
- Enzymatic assays showed changes in kinetic parameters when Pgk interacted with nilotinib, netupitant, and amphotericin B.

## Abstract

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.