Study of human lactate dehydrogenase-based microreactors for anticancer drugs screening

Cancer is among the leading causes of death worldwide. The International Agency for Research on Cancer estimates that in twenty years the rates of incidence and mortality of cancer will have an increase of 63% and 74%, respectively. Currently, the main types of cancer treatments are surgical intervention, chemotherapy, radiotherapy and immunotherapy. Each of these therapeutic approaches has advantages and disadvantages, dependent on the specific tumor type, its size, the presence of metastasis and the inter-patient variability. Beyond its genetic basis, it is crucial to recognize cancer also as a metabolic disease. Consequently, a promising antitumoral strategy could be to focus on the lactate dehydrogenase (LDH) enzyme, given its pivotal role in tumor metabolism. LDH has a tetrameric structure comprising LDH-A and LDH-B subunits. Notably, LDH-A is overexpressed in certain solid tumors. This condition drives a fundamental shift in the metabolic pathway, known as the Warburg effect, from oxidative phosphorylation towards aerobic glycolysis, promoting the proliferation of malignant cells. Given this, a valid antitumoral approach could be the inhibition of LDH-A. This necessitates the identification of compounds capable of selectively modulating LDH-A activity. However, the screening of novel compounds is typically a long and expensive process. Consequently, developing an LDH-based biosensor presents a feasible approach to reduce time and cost associated with the screening phase. Before developing the biosensor prototype, a detailed understanding of the enzyme's behaviour, alone and when interacting with known inhibitors, is fundamental. Therefore, the present work investigates LDH from a kinetic perspective. To further characterize the enzyme's properties, these studies are complemented by molecular docking simulations. The initial kinetic tests are performed on the sole LDH at a temperature of 37° and in a pH 7.4 solution buffer. Kinetic curves and their derived kinetic parameters are calculated by varying either the substrate concentration (at a fixed cofactor concentration of 0.23 mM) or the cofactor concentration (at a fixed substrate concentration of 1.63mM). The measurement starts when the enzyme is introduced in the solution. The variation in absorbance at 340 nm due to NADH oxidation is detected by UV-vis spectrophotometer and this measurement is then used to determine the reaction rate. In these conditions the resulting values of Vmax and Km are 861 µmol min-1 and 0.147 mM at a fixed cofactor concentration. While Vmax and Km are equal to 891 µmol min-1 and 0.034 mM at a fixed substrate concentration. Complementing this analysis, molecular docking simulations are performed to investigate the binding of LDH with pyruvate (substrate) and NADH (cofactor). These simulations reveal that pyruvate forms hydrogen bonds with Methionine and Glutamine residues of the enzyme, while NADH interacts via hydrogen bonds with Arginine, Serine, Threonine, Histidine, and Asparagine residues. The same kinetic analysis and simulations are then performed to characterize LDH’s behaviour with five known inhibitors: Galloflavin, Oxamate, FX-11, Gossypol and NHI-2. Since LDH will be immobilized within the biosensor for post-test retrieval, the aforementioned experiments on the free enzyme are crucial. They establish a benchmark to evaluate any changes in enzyme behaviour after encapsulation.