Functional partnership between carbonic anhydrase and malic enzyme in promoting gluconeogenesis in Leishmania major

Abstract

Leishmania has a remarkable ability to proliferate under widely fluctuating levels of essential nutrients, such as glucose. For this, the parasite is heavily dependent on its gluconeogenic machinery. One perplexing aspect of gluconeogenesis in Leishmania is the lack of the crucial gene for pyruvate carboxylase (PC). PC-catalyzed conversion of pyruvate to oxaloacetate is a key entry point through which gluconeogenic amino acids are funneled into this pathway. The absence of PC in Leishmania thus raises question about the mechanism of pyruvate entry into the gluconeogenic route. In the present study, we report that this task is accomplished in Leishmania major through a novel functional partnership between its mitochondrial malic enzyme (LmME) and carbonic anhydrase 1 (LmCA1). Using a combination of pharmacological inhibition studies with genetic manipulation, we show that both of these enzymes are necessary for promoting gluconeogenesis and supporting parasite growth under glucose-limiting conditions. Functional cross-talk between LmME and LmCA1 was evident when it was observed that the growth retardation caused by inhibition of any one of these enzymes could be protected to a significant extent by overexpressing the other enzyme. We also found that, although LmCA1 exhibited constitutive expression, the LmME protein level was strongly upregulated under low glucose conditions. Notably, both LmME and LmCA1 were found to be important for survival of Leishmania amastigotes within host macrophages. Taken together, our results indicate that LmCA1 by virtue of its CO 2 concentrating ability stimulates LmME-catalyzed pyruvate carboxylation, thereby driving gluconeogenesis through the pyruvate-malate-oxaloacetate bypass pathway. Additionally, our study establishes LmCA1 and LmME as promising therapeutic targets.

Trypanosoma cruzi Malic Enzyme Is the Target for Sulfonamide Hits from the GSK Chagas Box

“…22 Aiming to help filling this gap, we decided to test Chagas Box compounds 18 against the T. cruzi malic enzyme, a key enzyme in energetic metabolism and a promising target for Chagas disease under investigation in our group. 17,24,25 T. cruzi Malic Enzymes are Inhibited by Small Molecules from the Chagas Box. Some of the previously reported ME inhibitors 17 share common structural features with the compounds present in the Chagas Box, 18 which prompted us to screen this collection for inhibitors of cytosolic and mitochondrial T. cruzi ME isoforms (Figure 1A and B).…”

“…In recent decades, there has been a growing trend for the use of phenotypic-based screening to identify bioactive molecules, particularly in drug discovery programs for infectious diseases. − As such, this strategy has been employed for the identification of compounds active against protozoan parasites. ,, Although successful for identifying novel hits with distinct chemical scaffolds, an intrinsic drawback of this approach is the lack of information about the primary mode of action of the selected molecules, creating the need for target deconvolution or identification . Aiming to help filling this gap, we decided to test Chagas Box compounds against the T. cruzi malic enzyme, a key enzyme in energetic metabolism and a promising target for Chagas disease under investigation in our group. ,, …”

Probing benzothiadiazine-1,1-dioxide ethylene glycol derivatives against Leishmania: synthesis and in vitro efficacy evaluation

“…The aromatic protons of the benzothiadiazine-1,1-dioxide synthon, were found in the sequence doublet (δ 7.81, J 7.8 Hz, H-8), triplet (δ 7.68, J 7.8 Hz, H-7), doublet (δ 7.33, J 7.6 Hz, H-5), and triplet (δ 7.47, J 7.6 Hz, H-6) in all the synthesised derivatives (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). The formation of the target compounds was also con rmed by all the derivatives having the characteristic singlet at δ 7.95 ppm which indicated the acidic proton on H-3.…”

“…In the third step, the N-2 atom of the benzothiadiazine-1,1-dioxide scaffold 1 was deprotonated using potassium carbonate (K 2 CO 3 ). This was followed in situ by nucleophilic substitution (SN) with various brominated ethylene glycol chain intermediates to afford the targeted benzothiadiazine-1,1-dioxide derivatives (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) in poor to excellent yields (17 to 81%) after ltration and puri cation by column chromatography (DCM:MeOH, 19:1) (Scheme 1; Step 3).…”

“…A promising drug target that is under investigation is the carbonic anhydrase (CA) enzyme [12,14]. To date, seven families of CA have been described in literature, namely α-, β-, γ-, δ-, ζ-, ηand θ-CA [12].…”