Click chemistry-facilitated comprehensive identification of proteins adducted by antimicrobial 5-nitroimidazoles for discovery of alternative drug targets against giardiasis

Abstract

Giardiasis and other protozoan infections are major worldwide causes of morbidity and mortality, yet development of new antimicrobial agents with improved efficacy and ability to override increasingly common drug resistance remains a major challenge. Antimicrobial drug development typically proceeds by broad functional screens of large chemical libraries or hypothesis-driven exploration of single microbial targets, but both strategies have challenges that have limited the introduction of new antimicrobials. Here, we describe an alternative drug development strategy that identifies a sufficient but manageable number of promising targets, while reducing the risk of pursuing targets of unproven value. The strategy is based on defining and exploiting the incompletely understood adduction targets of 5-nitroimidazoles, which are proven antimicrobials against a wide range of anaerobic protozoan and bacterial pathogens. Comprehensive adductome analysis by modified click chemistry and multidimensional proteomics were applied to the model pathogen Giardia lamblia to identify dozens of adducted protein targets common to both 5'-nitroimidazole-sensitive and-resistant cells. The list was highly enriched for known targets in G. lamblia, including arginine deiminase, α-tubulin, carbamate kinase, and heat shock protein 90, demonstrating the utility of the approach. Importantly, over twenty potential novel drug targets were identified. Inhibitors of two representative new targets, NADP-specific glutamate dehydrogenase and peroxiredoxin, were found to have significant antigiardial activity. Furthermore, all the identified targets remained available in resistant cells, since giardicidal activity of the respective inhibitors was not impacted by resistance to 5'-nitroimidazoles. These results demonstrate that the combined use of click chemistry and proteomics has the potential to reveal alternative drug targets for overcoming antimicrobial drug resistance in protozoan parasites.

Characterization of Metronidazole-Resistant Giardia intestinalis Lines by Comparative Transcriptomics and Proteomics

“…The created radical is highly reactive, in contrast to semi-quinones and other natural substrates of the oxidoreductases ( Supplementary Figure 7 ). MTZ radicals cause damage in a non-targeted manner ( Uzlikova and Nohynkova, 2014 ; Dingsdag and Hunter, 2018 ; Lauwaet et al, 2020 ), especially to the DNA ( Figure 8 ). In the presence of oxygen, radical formation is reduced by futile cycling ( Figure 8 ).…”

“…Despite its long and widespread use, it is still not fully understood how MTZ exerts cytotoxicity on bacteria and protozoa, since numerous target molecules have been identified but their relative importance for cell killing remains unclear ( Lauwaet et al, 2020 ). The drug is assumed to enter cells via passive diffusion, although recent studies suggest involvement of active mechanisms ( Dingsdag and Hunter, 2018 ).…”

Role of Click Chemistry in Organic Synthesis

“…Agrihari et al introduced CuAAC catalyzed synthesis of p-tert-butylcalix [4] arene linked benzotriazolyl dendrimers using CuSO 4 .5H 2 O and NaN 3 to prepare N-1, N-2 type 6 fold compounds in good yields [89]. The synthesized compounds were evaluated for in vitro and in vivo anti-bacterial studies against a range of microbes and demonstrated good biological potential.…”

“…These vast characteristics make click chemistry a powerful tool that paves a path in several fields of research viz. designing of drugs and lead structures [1][2][3][4]. Therefore the synergy between these disciplines has given rise to an area of intense research activity.…”

Gold(I) Phosphine Derivatives with Improved Selectivity as Topically Active Drug Leads to Overcome 5-Nitroheterocyclic Drug Resistance in Trichomonas vaginalis

“…Oral drug administration leads to clinical and microbiological cure in the majority of cases, but treatment failures occur in a significant fraction of patients. Surveillance studies have found Mz resistance in 2.4–9.6% of isolates in the United States, 2.2% in Spain, and 17.4% in Papua New Guinea. , Mz and other nitroheterocyclic drugs are prodrugs that must be reduced to reactive intermediates by microbial reductases before they form adducts that inactivate different cellular targets . Consequently, Mz resistance is mediated by diminished expression or function of major drug-reducing pathways, including pyruvate/ferredoxin oxidoreductase and ferredoxin, − thioredoxin reductase (TrxR), flavin reductase, and alcohol dehydrogenase. − These redox pathways normally allow the parasite to cope with oxidative stresses encountered under microaerophilic growth conditions in the host.…”