Identifying purine nucleoside phosphorylase as the target of quinine using cellular thermal shift assay

Abstract

Mechanisms of action (MoAs) have been elusive for most antimalarial drugs in clinical use. Decreasing responsiveness to antimalarial treatments stresses the need for a better resolved understanding of their MoAs and associated resistance mechanisms. In the present work, we implemented the cellular thermal shift assay coupled with mass spectrometry (MS-CETSA) for drug target identification inPlasmodium falciparum, the main causative agent of human malaria. We validated the efficacy of this approach for pyrimethamine, a folic acid antagonist, and E64d, a broad-spectrum cysteine proteinase inhibitor. Subsequently, we applied MS-CETSA to quinine and mefloquine, two important antimalarial drugs with poorly characterized MoAs. Combining studies in theP. falciparumparasite lysate and intact infected red blood cells, we foundP. falciparumpurine nucleoside phosphorylase (PfPNP) as a common binding target for these two quinoline drugs. Biophysical and structural studies with a recombinant protein further established that both compounds bind within the enzyme’s active site. Quinine binds to PfPNP at low nanomolar affinity, suggesting a substantial contribution to its therapeutic effect. Overall, we demonstrated that implementation of MS-CETSA forP. falciparumconstitutes a promising strategy to elucidate the MoAs of existing and candidate antimalarial drugs.

Thermal proteome profiling for interrogating protein interactions

“…; or metabolite (preprint:Saei et al, 2018;Dziekan et al, 2019;Sridharan et al, 2019b)], genetic [e.g., gene knock-outBanzhaf et al, 2020)], or enzymatic (preprint:Saei et al, 2018) perturbations; or different cell states [different phase of the cell cycleDai et al, 2018), or growth phase;Fig 2]…”

Derivatives of the antimalarial drug mefloquine are broad spectrum antifungal molecules with activity against drug-resistant clinical isolates

“…As alluded to in the introduction, the mechanism of action for MEF as an anti-malarial drug has remained enigmatic despite its many years of use in the clinical setting. Recent proposals for its mechanism of action either as an antimalarial drug or as part of repurposing to other indications include: lysosomal/vacuole inhibitor (39), disruptor of mitochondrial proton motive force (40), protein translation inhibitor (41), and purine nucleoside phosphorylase (PNP) inhibitor (42). The latter two proposals involve specific target proteins, a subunit of 80S ribosome and the PNP enzyme.…”

Drug screens of NGLY1 Deficiency worm and fly models reveal catecholamine, NRF2 and anti-inflammatory pathway activation as clinical approaches

“…The expression of interferon genes is constitutively up-regulated in Ngly1 -/murine embryonic fibroblasts (Yang et al 2018). It's tempting to speculate further that hyperactive innate immunity responses contribute to larval growth arrest and developmental delay in NGLY1-deficient animals and that these antiinflammatory responses are dampened by inhibitors of DNA synthesis, specifically purine biosynthetic enzymes targeted by the fly repurposing hits thioguanosine and the dihydrofolate reductase-like inhibitor pyrimethamine (Dziekan et al, 2019), and possibly the diaminopteridinecontaining novel lead compounds that resemble pyrimethamine. Even more so because the same mechanism of action appears to have been revealed by parallel repurposing and discovery screens.…”