An Antiparasitic Compound from the Medicines for Malaria Venture Pathogen Box Promotes Leishmania Tubulin Polymerization

Abstract

The few frontline antileishmanial drugs are poorly effective and toxic. To search for new drugs for this neglected tropical disease, we tested the activity of compounds in the Medicines for Malaria Venture (MMV) “Pathogen Box” against Leishmania amazonensis axenic amastigotes. Screening yielded six discovery antileishmanial compounds with EC50 values from 50 to 480 nM. Concentration–response assays demonstrated that the best hit, MMV676477, had mid-nanomolar cytocidal potency against intracellular Leishmania amastigotes, Trypanosoma brucei, and Plasmodium falciparum, suggesting broad antiparasitic activity. We explored structure–activity relationships (SAR) within a small group of MMV676477 analogs and observed a wide potency range (20–5000 nM) against axenic Leishmania amastigotes. Compared to MMV676477, our most potent analog, SW41, had ∼5-fold improved antileishmanial potency. Multiple lines of evidence suggest that MMV676477 selectively disrupts Leishmania tubulin dynamics. Morphological studies indicated that MMV676477 and analogs affected L. amazonensis during cell division. Differential centrifugation showed that MMV676477 promoted partitioning of cellular tubulin toward the polymeric form in parasites. Turbidity assays with purified Leishmania and porcine tubulin demonstrated that MMV676477 promoted leishmanial tubulin polymerization in a concentration-dependent manner. Analogs’ antiparasitic activity correlated with their ability to facilitate purified Leishmania tubulin polymerization. Chemical cross-linking demonstrated binding of the MMV676477 scaffold to purified Leishmania tubulin, and competition studies established a correlation between binding and antileishmanial activity. Our studies demonstrate that MMV676477 is a potent antiparasitic compound that preferentially promotes Leishmania microtubule polymerization. Due to its selectivity for and broad-spectrum activity against multiple parasites, this scaffold shows promise for antiparasitic drug development.

Inhibiting parasite proliferation using a rationally designed anti‐tubulin agent

“…We identified a compound that specifically alters parasite MTs and inhibits parasite replication and host cell invasion. Parabulin, which inhibits growth of protozoan MTs, complements the recent identification of a tubulin‐binding compound that selectively promotes growth of Leishmania and Trypanosoma (kinetoplastid) MTs to specifically inhibit parasite replication (Ullah et al , 2020). By pairing structural and biochemical studies with drug design and Toxoplasma assays, we confirm the potential of rationally developed MT‐targeting agents as anti‐parasitic treatments.…”

Optimization of a Pyrimidinone Series for Selective Inhibition of Ca2+/Calmodulin-Stimulated Adenylyl Cyclase 1 Activity for the Treatment of Chronic Pain

“…The pyrazole-pyrimidine substructure has been recently reported in only one other manuscript to possess antileishmania properties via promotion of microtubule polymerization. 75 Moreover, three analogues tested for activity against four human kinases relevant to offtarget toxicity displayed moderate-to-no inhibition at a single dose of 10 μM. This would suggest that kinase inhibition may not preclude this scaffold from advancement; however, the kinases tested are not exhaustive and additional experiments are necessary to understand the potential impact the molecules may have on the human kinome.…”

Multistage and transmission-blocking tubulin targeting potent antimalarial discovered from the open access MMV Pathogen Box

“…Recently, MMV676477 has been shown to inhibit Leishmania replication by stabilizing microtubule assembly [24]. To examine whether the same phenomenon occurs in Plasmodium , MMV676477 and its analogues were first screened virtually against the Pf αI/ Pf β-tubulin heterodimer.…”