Triosephosphate isomerase as a therapeutic target against trichomoniasis

Highlights

• We screened a library of ∼500,000 compounds in silico, to identify selective compounds against TvTIM.

• We identified the compound: (3,3′-{[4-(4-morpholinyl)phenyl]methylene}bis(4-hydroxy-2H-chromen-2-one) (A4) as potential drug.

• We determined an IC₅₀ of 47 μM of the A4 compound, with favorable toxicity results (in theoretical and experimental assays).

• This finding represents a promising alternative to fight trichomoniasis.

Abstract

Trichomoniasis is the most common non-viral sexually transmitted infection, caused by the protozoan parasite Trichomonas vaginalis, affecting millions of people worldwide. The main treatment against trichomoniasis is metronidazole and other nitroimidazole derivatives, but up to twenty percent of clinical cases of trichomoniasis are resistant to these drugs. In this study, we used high-performance virtual screening to search for molecules that specifically bind to the protein, triosephosphate isomerase from T. vaginalis (TvTIM). By in silico molecular docking analysis, we selected six compounds from a chemical library of almost 500,000 compounds. While none of the six inhibited the enzymatic activity of recombinant triosephosphate isomerase isoforms, one compound (A4; 3,3′-{[4-(4-morpholinyl)phenyl]methylene}bis(4- hydroxy-2H-chromen-2-one) altered their fluorescence emission spectra, suggesting that this chemical might interfere in an important non-glycolytic function of TvTIM. In vitro assays demonstrate that A4 is not cytotoxic but does have trichomonacidal impact on T. vaginalis cultures.

With these results, we propose this compound as a potential drug with a new therapeutic target against Trichomonas vaginalis.

Introduction

The extracellular protozoan parasite Trichomonas vaginalis is the causative agent of trichomoniasis, the most common non-viral sexually transmitted disease (STD) [[1], [2], [3], [4], [5]]. There are approximately 270 million cases of trichomoniasis annually worldwide (WHO 2020). Imidazole derivatives, such as metronidazole and tinidazole, are the most common treatment, but this family of drugs was developed more than 60 years ago, and multiple resistance mechanisms have developed. Currently, up to 20 % of trichomoniasis cases are resistant to imidazole derivatives. Therefore, it is necessary to develop new drugs with different mechanisms of action [[4], [5], [6], [7], [8], [9]].

Trichomonas vaginalis has a highly repetitive genome, including many metabolic enzymes, such as the glycolytic enzyme triosephosphate isomerase (TvTIM), encoded by two functional genes; TvTIM1 and TvTIM2 [10]. The amino acid sequences of the two TvTIMs demonstrate a 98.4 % identity, and both isoforms share common structural and catalytic characteristics. Furthermore, their crystal structures are almost identical homodimers [11,12]. An additional function of TvTIM has been proposed due to gene duplication: the TvTIM protein on the surface of Trichomonas vaginalis interacts with extracellular matrix and basement membrane proteins such as laminin and fibronectin, which may have a role as virulence factors. TvTIM has also been identified in vesicles secreted by this parasite [13].

The dual location and function of TvTIM isoforms (in the glycolytic pathway and on the membrane surface) implies this multifunctional protein is an excellent new target for drug development against Trichomonas vaginalis [[13], [14], [15], [16]]. Previously, we identified a selective compound against TvTIM, which only inhibited the TvTIM2 recombinant protein without affecting in vitro T. vaginalis cultures [15]. In this study, we used a different high-throughput virtual screening methodology to select chemical compounds capable of binding to TvTIM. Our goal was to evaluate the effects of these selected compounds on the glycolytic activity in interactions that may alter the structure of TvTIM, thereby altering some functions of this protein in T. vaginalis cultures in vitro and demonstrating a probable trichomonacidal effect. These results will assist in the rational development of drugs against Trichomonas vaginalis that act via a different mechanism than the standard drugs.