Identification via a Parallel Hit Progression Strategy of Improved Small Molecule Inhibitors of the Malaria Purine Uptake Transporter that Inhibit Plasmodium falciparum Parasite Proliferation.

Emerging resistance to current antimalarial medicines underscores the importance of identifying new drug targets and novel compounds. Malaria parasites are purine auxotrophic and import purines via the Plasmodium falciparum equilibrative nucleoside transporter type 1 (PfENT1). We previously showed that PfENT1 inhibitors block parasite proliferation in culture. Our goal was to identify additional, possibly more optimal chemical starting points for a drug discovery campaign. We performed a high throughput screen (HTS) of GlaxoSmithKline's 1.8 million compound library with a yeast-based assay to identify PfENT1 inhibitors. We used a parallel progression strategy for hit validation and expansion, with an emphasis on chemical properties in addition to potency. In one arm, the most active hits were tested for human cell toxicity; 201 had minimal toxicity. The second arm, hit expansion, used a scaffold-based substructure search with the HTS hits as templates to identify over 2000 compounds; 123 compounds had activity. Of these 324 compounds, 175 compounds inhibited proliferation of P. falciparum parasite strain 3D7 with IC50 values between 0.8 and ∼180 µM. One hundred forty-two compounds inhibited PfENT1 knockout (pfent1Δ) parasite growth, indicating they also hit secondary targets. Thirty-two hits inhibited growth of 3D7 but not pfent1Δ parasites. Thus, PfENT1 inhibition was sufficient to block parasite proliferation. Therefore, PfENT1 may be a viable target for antimalarial drug development. Six compounds with novel chemical scaffolds were extensively characterized in yeast-, parasite-, and human-erythrocyte-based assays. The inhibitors showed similar potencies against drug sensitive and resistant P. falciparum strains. They represent attractive starting points for development of novel antimalarial drugs.

AMTB, a TRPM8 channel blocker: evidence in rats for activity in overactive bladder and painful bladder syndrome.

The activation of the TRPM8 channel, a member of the large class of TRP ion channels, has been reported to be involved in overactive bladder and painful bladder syndrome, although an endogenous activator has not been identified. In this study, N-(3-aminopropyl)-2-{[(3-methylphenyl) methyl]oxy}-N-(2-thienylmethyl)benzamide hydrochloride salt (AMTB) was evaluated as a TRPM8 channel blocker and used as a tool to evaluate the effects of this class of ion channel blocker on volume-induced bladder contraction and nociceptive reflex responses to noxious bladder distension in the rat. AMTB inhibits icilin-induced TRPM8 channel activation as measured in a Ca(2+) influx assay, with a pIC(50) of 6.23. In the anesthetized rat, intravenous administration of AMTB (3 mg/kg) decreased the frequency of volume-induced bladder contractions, without reducing the amplitude of contraction. The nociceptive response was measured by analyzing both visceromotor reflex (VMR) and cardiovascular (pressor) responses to urinary bladder distension (UBD) under 1% isoflurane. AMTB (10 mg/kg) significantly attenuated reflex responses to noxious UBD to 5.42 and 56.51% of the maximal VMR response and pressor response, respectively. The ID50 value on VMR response was 2.42 +/- 0.46 mg/kg. These results demonstrate that TRPM8 channel blocker can act on the bladder afferent pathway to attenuate the bladder micturition reflex and nociceptive reflex responses in the rat. Targeting TRPM8 channel may provide a new therapeutic opportunity for overactive bladder and painful bladder syndrome.