• Lactate Dehydrogenase
• LDH Inhibitors
• NAD-mimetic Prodrugs

One of the most common phenotypes observed in tumors is the so-called Warburg effect: tumor cells generate energy via glycolysis even under normoxic conditions. This feature confers a selective advantage not only by promoting acidosis, uncontrolled proliferation, and metastasis, but also by enabling immune evasion. L-lactate is a key metabolite involved in this process. Once considered merely a waste product, it has recently gained significant attention due to its role as an important energy source for cancer cells and its involvement in chemoresistance. Elevated intracellular lactate levels have been associated with the phenomenon of 'lactylation' of both histone and non-histone proteins, suggesting a critical epigenetic role that includes p53 silencing and modulation of DNA repair mechanisms. The enzyme responsible for the production of this oncometabolite is lactate dehydrogenase (LDH). This NAD-dependant oxidoreductase is composed of four subunits arranged in various combinations of the H and M subunits. The main isoform involved in the conversion of pyruvate to lactate is the LDH5, a homotetrameric construct containing four subunits of LDHA. Given its crucial role in cancer cell metabolism, the identification of selective inhibitors of LDHA has become an urgent goal, with the aim of reducing tumor invasiveness, metastatic potential, and uncontrolled proliferation. However, this remains a significant challenge due to the ubiquitous expression of LDHA in human tissues, as well as the structural characteristics of the enzyme, including its deep and narrow active site and highly polar cofactor-binding pocket. Regarding the compounds synthesized so far, most were designed to compete with the cofactor NADH and/or pyruvate at their respective binding sites. However, major limitations have been reported, including poor cell permeability due to high polarity, and a lack of selectivity of the resulting inhibitors.
Our work focused on the synthesis of innovative molecules that act as cell-permeable bioprecursor of the NAD cofactor, exploiting enzymes of NAD salvage pathway for their metabolic activation. This strategy would lead to the intracellular generation of dinucleotide-based LDHA inhibitors, which, by competing with endogenous NADH at the cofactor-binding site, could reduce enzymatic activity and lactate production.
Our goal was to synthesize the adenine dinucleotides of gliocidin (compound 2.1a) which had previously shown to be a good substrate of the two enzymes that are responsible first for the
conversion of nicotinamide into nicotinamide-mononucleotide, nicotinamide
phosphoribosyltransferase (NAMPT), and then for the subsequent formation of NAD+, nicotinamide nucleotide adenylyltransferase 1 (NMNAT1). This way a “fake” NAD-cofactor is generated in cells. However, this compound was only tested as an inhibitor of inosine 5′-monophosphate dehydrogenase (IMPDH2), an enzyme involved in de novo guanine nucleotide biosynthesis. We instead hypothesized that this NAD-analogue could also inhibit LDHA.
In addition, our research also focused on the synthesis of reverse amide derivative of gliocidin (compound 2.3b), which had already been studied as an efficient inhibitor of IMPDH2 as well.
During our research it was found out how this compound possessed substantial differences in terms of reactivity and stability.
Following their synthesis, compounds 2.1a and 2.3b were evaluated for antiproliferative activity in MIA PaCa-2 human pancreatic cancer cells using the Alamar Blue (resazurin) assay and for reduction of lactate production in the same cancer cells.