Linker residues regulate the activity and stability of hexokinase 2, a promising anticancer target

Abstract

Hexokinase (HK) catalyzes the first step in glucose metabolism, making it an exciting target for the inhibition of tumor initiation and progression due to their elevated glucose metabolism. The upregulation of hexokinase-2 (HK2) in many cancers and its limited expression in normal tissues makes it a particularly attractive target for the selective inhibition of cancer growth and the eradication of tumors with limited side effects. The design of such safe and effective anticancer therapeutics requires the development of HK2-specific inhibitors that will not interfere with other HK isozymes. As HK2 is unique among HKs in having a catalytically active N-terminal domain (NTD), we have focused our attention on this region. We previously found that NTD activity is affected by the size of the linker helix-α13 that connects the N- and C-terminal domains of HK2. Three non-active site residues (D447, S449, and K451) at the beginning of the linker helix-α13 have been found to regulate the NTD activity of HK2. Mutation of these residues led to increased dynamics, as shown via hydrogen-deuterium exchange analysis and molecular dynamic simulations. D447A contributed the most to the enhanced dynamics of the NTD, with reduced calorimetric enthalpy of HK2. Similar residues exist in the C-terminal domain (CTD) but are unnecessary for HK1 and HK2 activity. Thus, we postulate these residues serve as a regulatory site for HK2, and may provide new directions for the design of anticancer therapeutics that reduce the rate of glycolysis in cancer through specific inhibition of HK2.

METTL3-mediated chromatin contacts promote stress granule phase separation through metabolic reprogramming during senescence

“…The two HK2 catalyzing domains convert glucose to Glucose-6-phosphate (G-6-P) that is a rate-limiting manner in glycolysis 29 , 30 . Point mutations at both N-terminal and C-terminal catalytic domains (D209A and D657A) of HK2 effectively diminish the enzymatic activity of HK2 29 , 31 .…”

“…The two HK2 catalyzing domains convert glucose to Glucose-6-phosphate (G-6-P) that is a rate-limiting manner in glycolysis 29 , 30 . Point mutations at both N-terminal and C-terminal catalytic domains (D209A and D657A) of HK2 effectively diminish the enzymatic activity of HK2 29 , 31 . To explore the metabolic consequences of HK2 upregulation in senescence, we conducted 13 C glucose isotope tracing in control and senescent cells with or without HK2 knockdown, and then rescued with ectopic expression of either wildtype or enzymatic activity inactive mutant HK2 (Fig.…”

The Promoting Role of HK II in Tumor Development and the Research Progress of Its Inhibitors

“…High-throughput screening expedites the identification of potential structures, resulting in significant time and cost savings. Precise drug development is enhanced by targeting specific residues [ 183 ], while drug repositioning and formulation modification are effective strategies to discover new drugs and minimize expenses and time consumption.…”

“…Moreover, they proposed that these residues serve as a regulatory site for HK II, which could be targeted to specifically inhibit the enzyme. This inhibition, in turn, could effectively reduce the rate of cancer glycolysis, offering a promising avenue for the development of novel anticancer drugs [ 183 ]. Additionally, in a study conducted by Ruiqi Li et al, mutations such as V431Cfs*26, L795Rfs*10, and A901Rfs*74 were found in cases of gastric adenocarcinoma (STAD), lung squamous carcinoma (LUSC), and endometrial carcinoma (UCEC).…”

Dimethyl sulfoxide reduces the stability but enhances catalytic activity of the main SARS‐CoV‐2 protease 3CLpro

“…The qPCR instrument was equipped with a Peltier‐based thermal system for uniform temperature ramping and thermal accuracy to ensure reproducibility of the data. The data were fitted to Boltzmann sigmoidal function using the Excel add‐on package XLfit (IDBS limited, Bridgewater, NJ), and the T m was calculated at the middle of the denaturation transition as described previously 42 . The T m value of 3CLpro was measured at different DMSO concentrations to determine its effect on the stability of the protease in a buffer containing 20 mM Hepes, pH 7.0, 150 mM NaCl, 1 mM EDTA, and 1 mM TCEP.…”