Desenvolvimento de um protocolo de simulação por dinâmica molecular para avaliação estrutural e energética de variantes da l-asparaginase humana

Abstract

L-asparaginase is an enzyme widely employed in the treatment of acute lymphoblastic leukemia (ALL) due to its ability to degrade L-asparagine (Asn), which is essential for the survival of leukemic cells. However, currently available formulations are derived from bacterial sources and are often associated with severe immune reactions. Although the human body possesses a homologous enzyme, hASNase1, its low catalytic activity precludes its direct clinical application. To overcome this limitation, mutations have been proposed to enhance its catalytic efficiency. This study aimed to develop a computational protocol, based on molecular dynamics (MD) simulations and structural analyses, to evaluate the structural and energetic effects of hASNase1 variants and to identify molecular features associated with increased catalytic activity. The systems were

built based on the native structure of the enzyme and simulated under physiological conditions. Structural stability was assessed through root mean square deviation (RMSD), while interatomic interaction potentials (PII) and binding free energy calculations were used to analyze the interactions between hASNase1 variants and Asn. MM/PBSA calculations revealed increasing binding affinities in the order D87S < D84N < A186V < D87N, consistent with substrate residence times in the catalytic site. Experimentally, catalytic activity increases of 17× (D87S), 19× (D84N), 50× (A186V), and 52× (D87N) were observed compared to the wild-type enzyme, confirming the effectiveness of the DM + MM/PBSA protocol in identifying high-performance variants. In conclusion, the DM and MM/PBSA protocol elucidated the structural determinants underlying the experimentally observed activity gains in hASNase1 variants, identifying D87N and A186V as promising candidates for an optimized human L-asparaginase. Understanding the relationship between structure and catalytic activity in hASNase1 variants reinforces the use of in silico approaches in the rational development of safer and more effective biopharmaceuticals for the treatment of ALL.