LAUSR

Significance There is an urgent need for alternative antimalarials with the emergence of artemisinin-resistant malaria parasites. Blocking sugar uptake in Plasmodium falciparum by selectively inhibiting the hexose transporter P. falciparum hexose transporter 1 (PfHT1) kills the blood-stage parasites without affecting the host cells, making PfHT1 a promising therapeutic target. Here, we report the development of a series of small-molecule inhibitors that simultaneously target the orthosteric and the allosteric binding sites of PfHT1. These inhibitors all exhibit selective potency on the P. falciparum strains over human cell lines. Our findings establish the basis for the rational design of next-generation antimalarial drugs. Artemisinin-resistant malaria parasites have emerged and have been spreading, posing a significant public health challenge. Antimalarial drugs with novel mechanisms of action are therefore urgently needed. In this report, we exploit a “selective starvation” strategy by inhibiting Plasmodium falciparum hexose transporter 1 (PfHT1), the sole hexose transporter in P. falciparum, over human glucose transporter 1 (hGLUT1), providing an alternative approach to fight against multidrug-resistant malaria parasites. The crystal structure of hGLUT3, which shares 80% sequence similarity with hGLUT1, was resolved in complex with C3361, a moderate PfHT1-specific inhibitor, at 2.3-Å resolution. Structural comparison between the present hGLUT3-C3361 and our previously reported PfHT1-C3361 confirmed the unique inhibitor binding-induced pocket in PfHT1. We then designed small molecules to simultaneously block the orthosteric and allosteric pockets of PfHT1. Through extensive structure–activity relationship studies, the TH-PF series was identified to selectively inhibit PfHT1 over hGLUT1 and potent against multiple strains of the blood-stage P. falciparum. Our findings shed light on the next-generation chemotherapeutics with a paradigm-shifting structure-based design strategy to simultaneously target the orthosteric and allosteric sites of a transporter.