School of Chemistry - Research Publications
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ItemA Family of Dual-Activity Glycosyltransferase-Phosphorylases Mediates Mannogen Turnover and Virulence in Leishmania Parasites(CELL PRESS, 2019-09-11)Parasitic protists belonging to the genus Leishmania synthesize the non-canonical carbohydrate reserve, mannogen, which is composed of β-1,2-mannan oligosaccharides. Here, we identify a class of dual-activity mannosyltransferase/phosphorylases (MTPs) that catalyze both the sugar nucleotide-dependent biosynthesis and phosphorolytic turnover of mannogen. Structural and phylogenic analysis shows that while the MTPs are structurally related to bacterial mannan phosphorylases, they constitute a distinct family of glycosyltransferases (GT108) that have likely been acquired by horizontal gene transfer from gram-positive bacteria. The seven MTPs catalyze the constitutive synthesis and turnover of mannogen. This metabolic rheostat protects obligate intracellular parasite stages from nutrient excess, and is essential for thermotolerance and parasite infectivity in the mammalian host. Our results suggest that the acquisition and expansion of the MTP family in Leishmania increased the metabolic flexibility of these protists and contributed to their capacity to colonize new host niches.
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ItemDiscovery of N-Aryloxypropylbenzylamines as Voltage-Gated Sodium Channel NaV1.2-Subtype-Selective Inhibitors(WILEY-V C H VERLAG GMBH, 2019-03-05)We previously reported that a lipophilic N-(4'-hydroxy-3',5'-di-tert-butylbenzyl) derivative (1) of the voltage-gated sodium channel blocker mexiletine, was a more potent sodium channel blocker in vitro and in vivo. We demonstrate that replacing the chiral methylethylene linker between the amine and di-tert-butylphenol with an achiral 1,3-propylene linker (to give (2)) maintains potency in vitro. We synthesized 25 analogues bearing the 1,3-propylene linker and found that minor structural changes resulted in pronounced changes in state dependence of blocking human NaV 1.2 and 1.6 channels by high-throughput patch-clamp analysis. Compared to mexiletine, compounds 1 and 2 are highly selective NaV 1.2 inhibitors and >500 times less potent in inhibiting NaV 1.6 channels. On the other hand, a derivative (compound 4) bearing 2,6-dimethoxy groups in place of the 2,6-dimethyl groups found in mexiletine was found to be the most potent inhibitor, but is nonselective against both channels in the tonic, frequency-dependent and inactivated states. In a kindled mouse model of refractory epilepsy, compound 2 inhibited seizures induced by 6 Hz 44 mA electrical stimulation with an IC50 value of 49.9±1.6 mg kg-1 . As established sodium channel blockers do not suppress seizures in this mouse model, this indicates that 2 could be a promising candidate for treating pharmaco-resistant epilepsy.
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ItemThe Metabolite Repair Enzyme Phosphoglycolate Phosphatase Regulates Central Carbon Metabolism and Fosmidomycin Sensitivity in Plasmodium falciparum(AMER SOC MICROBIOLOGY, 2019-12-10)Members of the haloacid dehalogenase (HAD) family of metabolite phosphatases play an important role in regulating multiple pathways in Plasmodium falciparum central carbon metabolism. We show that the P. falciparum HAD protein, phosphoglycolate phosphatase (PGP), regulates glycolysis and pentose pathway flux in asexual blood stages via detoxifying the damaged metabolite 4-phosphoerythronate (4-PE). Disruption of the P. falciparumpgp gene caused accumulation of two previously uncharacterized metabolites, 2-phospholactate and 4-PE. 4-PE is a putative side product of the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase, and its accumulation inhibits the pentose phosphate pathway enzyme, 6-phosphogluconate dehydrogenase (6-PGD). Inhibition of 6-PGD by 4-PE leads to an unexpected feedback response that includes increased flux into the pentose phosphate pathway as a result of partial inhibition of upper glycolysis, with concomitant increased sensitivity to antimalarials that target pathways downstream of glycolysis. These results highlight the role of metabolite detoxification in regulating central carbon metabolism and drug sensitivity of the malaria parasite.IMPORTANCE The malaria parasite has a voracious appetite, requiring large amounts of glucose and nutrients for its rapid growth and proliferation inside human red blood cells. The host cell is resource rich, but this is a double-edged sword; nutrient excess can lead to undesirable metabolic reactions and harmful by-products. Here, we demonstrate that the parasite possesses a metabolite repair enzyme (PGP) that suppresses harmful metabolic by-products (via substrate dephosphorylation) and allows the parasite to maintain central carbon metabolism. Loss of PGP leads to the accumulation of two damaged metabolites and causes a domino effect of metabolic dysregulation. Accumulation of one damaged metabolite inhibits an essential enzyme in the pentose phosphate pathway, leading to substrate accumulation and secondary inhibition of glycolysis. This work highlights how the parasite coordinates metabolic flux by eliminating harmful metabolic by-products to ensure rapid proliferation in its resource-rich niche.
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ItemGram scale preparation of clozapine N-oxide (CNO), a synthetic small molecule actuator for muscarinic acetylcholine DREADDs(ELSEVIER SCIENCE BV, 2018)Chemogenetics uses engineered proteins that are controlled by small molecule actuators, allowing in vivo functional studies of proteins with temporal and dose control, and include Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). One major class of DREADDs are mutated muscarinic receptors that are unresponsive to acetylcholine, and are activated by administration of clozapine N-oxide (CNO). However, CNO is available in only small amounts and large scale studies involving animals and multiple cohorts are prohibitively expensive for many investigators. The precursor, clozapine, is also expensive when purchased from specialist suppliers. Here we report: •A simple extraction method of clozapine from commercial tablets;•A simple preparation of CNO from clozapine, and for the first time its single-crystal X-ray structure; and•That the CNO prepared by this method specifically activates the DREADD receptor hM3Dq in vivo. This method provides large quantities of CNO suitable for large-scale DREADD applications that is identical to commercial material.