Proteoglycan-degrading enzymes engineered for enhanced tumor microenvironment interaction in renal cell carcinoma

This work optimized proteoglycan-degrading enzymes through targeted mutagenesis to enhance their interaction with the tumor microenvironment in Renal Cell Carcinoma (RCC). A comprehensive mutagenesis approach identified 60 key mutations significantly improving enzymatic activity, stability, and structural integrity. When compared to Wild Type (WT) enzyme, a remarkable increase in specific activity by 35 % (p < 0.001) and a considerable decrease in the Km values for hyaluronidases from 2.5 mM to 1.5 mM (p < 0.05), as a result of these modifications. Computational methods are then employed to analyze the active site of the enzymes to detect potential residues that may alter. These computational techniques include molecular docking and protein structure prediction. The structural models of the enzymes are created by utilizing homology modeling and crystallography. These models demonstrate the spatial arrangement of the amino acid enzymes. It also illustrated the specific mutations to improve the potential of enzymes to relate to the Extracellular Matrix (ECM) of tumors. The computational screening methods effectively predicted how the modifications impact enzyme catalytic efficiency and stability. The modified enzymes retained 85 % of the enzyme activity, while the WT retained 60 %. Thus, the modified enzymes demonstrated better thermal stability than WT. Vitro test analyses show that the proteoglycan breakdown was significantly reduced by 70 % (p < 0.001), and for effective proteoglycan breakdown, hyaluronidase concentration is needed. This work proposed a novel therapeutic approach called proteoglycan-degrading enzymes for the treatment of RCC. These proteoglycan-degrading enzymes are more stable and effective for treating RCC, as demonstrated in the outcomes. Customized proteoglycan-degrading enzymes make the therapy more effective. The effective breakdown of the tumor's ECM in RCC models establishes this customized proteoglycan-degrading enzyme. These enzymes are effective for this customized cancer treatment as they improve stability, activity, and interaction with the TME.