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 make 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 nonactive 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. 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 make 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 nonactive 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. The rate of glucose metabolism is elevated in different types of cancer that primarily utilize aerobic glycolysis, a phenomenon known as the Warburg effect (1Vander Heiden M.G. Cantley L.C. Thompson C.B. Understanding the Warburg effect: the metabolic requirements of cell proliferation.Science. 2009; 324: 1029-1033Crossref PubMed Scopus (8407) Google Scholar, 2Warburg O. On the origin of cancer cells.Science. 1956; 123: 309-314Crossref PubMed Scopus (8363) Google Scholar). An enhanced glucose metabolic rate is required to meet the increased energy needs and metabolite demands required to support rapid tumor progression (3Pedersen P.L. Warburg, me and Hexokinase 2: multiple discoveries of key molecular events underlying one of cancers' most common phenotypes, the "Warburg Effect", i.e., elevated glycolysis in the presence of oxygen.J. Bioenerg. Biomembr. 2007; 39: 211-222Crossref PubMed Scopus (345) Google Scholar). HK, the first enzyme in glucose metabolism, catalyzes the irreversible rate-limiting phosphorylation of glucose to glucose-6-phosphate (G6P). In addition to glycolysis, the HK reaction contributes to different pathways, including the tricarboxylic acid cycle and pentose phosphate pathway, for the synthesis of nucleotides, lipids, and amino acids required for rapid tumor growth (3Pedersen P.L. Warburg, me and Hexokinase 2: multiple discoveries of key molecular events underlying one of cancers' most common phenotypes, the "Warburg Effect", i.e., elevated glycolysis in the presence of oxygen.J. Bioenerg. Biomembr. 2007; 39: 211-222Crossref PubMed Scopus (345) Google Scholar, 4Patra The pentose phosphate and 39: PubMed Scopus Google Scholar, glycolysis and phosphorylation in and to PubMed Scopus Google Scholar). As an effective of glucose metabolism, HK for the inhibition of cancer growth and the development of anticancer HK with NTD and have been including the new domain known as is the size and a domain of of and regulatory in the and PubMed Scopus Google Scholar, the N- and C-terminal of PubMed Scopus Google Scholar, of and metabolic PubMed Scopus Google Scholar, M.G. of and as through PubMed Scopus Google Scholar). The of the NTD and is by and and of of and regulatory in the and PubMed Scopus Google Scholar, the N- and C-terminal of PubMed Scopus Google Scholar, of of with glucose and PubMed Scopus Google Scholar, The of PubMed Scopus Google Scholar, The of the of and and of its PubMed Scopus Google Scholar). a the active site is in a the of the and and are the residues at the beginning of the and of the NTD and The linker helix-α13 is the of the NTD and its active site to it to the The residues and are required to the on of glucose in for its on the of the N- and C-terminal of PubMed Scopus Google Scholar, The of the of and and of its PubMed Scopus Google Scholar, C.B. The of of new the of with glucose and PubMed Scopus Google Scholar, of the N- and C-terminal of 2009; PubMed Scopus Google Scholar). HK1 is in tissues and is the of and metabolic PubMed Scopus Google Scholar). On the other HK2 is and in types of it is required to glycolysis for tumor growth and glycolysis and phosphorylation in and to PubMed Scopus Google Scholar, cancers have aerobic PubMed Scopus Google Scholar, P.L. to to the "Warburg and a target for effective 2009; PubMed Scopus Google Scholar, Hexokinase is required for tumor initiation and and its is in of PubMed Scopus Google Scholar, Hexokinase tumor growth and by in PubMed Scopus Google Scholar). HK2 in and increased glycolysis and phosphorylation in and to PubMed Scopus Google Scholar). HK2 found to required for tumor initiation and in of and cancer Hexokinase is required for tumor initiation and and its is in of PubMed Scopus Google Scholar). In HK2 to the and its with the and tumor cell Hexokinase the of energy metabolism and of PubMed Scopus Google Scholar, of and PubMed Scopus Google Scholar, Hexokinase to is for cell and is by PubMed Scopus Google Scholar). in normal HK2 is found in tissues and at the in and tissues glycolysis and phosphorylation in and to PubMed Scopus Google Scholar, cancers have aerobic PubMed Scopus Google Scholar). The of HK2 for the and of a of tumor types and its in normal tissues make it an attractive target for the development of anticancer therapeutics Hexokinase is required for tumor initiation and and its is in of PubMed Scopus Google Scholar, Hexokinase tumor growth and by in PubMed Scopus Google Scholar, P.L. a key in the growth and of many cancers and an for PubMed Scopus Google Scholar, Thompson C.B. in cancer cell metabolism and PubMed Scopus Google Scholar, cell for PubMed Scopus Google Scholar, of of with in PubMed Scopus Google Scholar, of aerobic glycolysis and its key new for cancer PubMed Scopus Google Scholar, the of HK2 in cancer metabolism and PubMed Scopus Google Scholar). the most cancer is a with side due to its for cancer the of HK2 in cancer metabolism and PubMed Scopus Google Scholar). inhibitors of glucose metabolism have been as an effective for cancer glycolysis and phosphorylation in and to PubMed Scopus Google Scholar, the of HK2 in cancer metabolism and PubMed Scopus Google Scholar, of enzyme (HK2) tumor PubMed Scopus Google Scholar). to the of the active site residues of one of the in the design and development of anticancer inhibitors on the HK reaction is the of HK2 the other The of the of and and of its PubMed Scopus Google Scholar, Hexokinase is required for tumor initiation and and its is in of PubMed Scopus Google Scholar). therapeutics that target cancer growth will have to HK2 and with HK in normal this to the HK active site to the inhibition of isozymes. HK1 and HK2 that for the development of HK2-specific The is catalytically active in its to the the NTD is in HK2. We previously found that the size of the linker helix-α13 the activity of the NTD of HK2 The of the of and and of its PubMed Scopus Google Scholar). the enzyme with of the of the linker the NTD the of of the linker helix-α13 the activity of the NTD The of the linker helix-α13 to regulate the NTD activity of HK2 it an target for the development of specific inhibitors of active NTD with to the HK active In this we the of the linker helix-α13 residues on the activity of the NTD of HK2 to a regulatory to the of residues in the linker helix-α13 and its of on the NTD activity of HK2. We residues (D447, S449, and K451) at the beginning of the linker helix-α13 that regulate the activity of the NTD of HK2. residues have been found to the the NTD active the of the linker helix-α13 and its with the of the of HK1 and that of HK2 residues to in the NTD regulatory but these residues to the activity of the The NTD regulatory site of HK2 is a target for the design of anticancer therapeutics that reduce the rate of glycolysis in cancer through specific inhibition of the HK2. to and is a in is the NTD and of of with glucose and PubMed Scopus Google Scholar, The of PubMed Scopus Google Scholar, The of the of and and of its PubMed Scopus Google Scholar). The of the NTD and is among the HK of of with glucose and PubMed Scopus Google Scholar, for of the enzyme PubMed Scopus Google Scholar, and of glucose in the PubMed Scopus Google Scholar, of of a molecular for the of via and PubMed Scopus Google Scholar, The of with the new its of PubMed Scopus Google Scholar, of a of and selective PubMed Scopus Google and the active site is in a the and In HK, the glucose site is and the residues and in the NTD and The of the of and and of its PubMed Scopus Google Scholar). We previously that the linker helix-α13 is for the activity of the NTD and is of a The of the of and and of its PubMed Scopus Google Scholar). The linker helix-α13 the active site at the of the NTD, it to the is to the that the In this we the of residues of the linker helix-α13 on the activity of the NTD of HK2 to a regulatory in the design of anticancer therapeutics to reduce the rate of glycolysis in cancer through the inhibition of HK2. the of residues of the linker helix-α13 in the activity of the NTD of to the linker helix-α13 and its of the NTD and of HK2 are catalytically the rate of the NTD in the in the presence of to catalytically the HK2 and by as The of the of and and of its PubMed Scopus Google Scholar). The with to of the and The rate of HK2 at of glucose and the the of the linker helix-α13 with the and of the in the NTD The of the as to to the linker of a with of the linker helix-α13 is in is and by in HK1 and The enzyme that D447A and catalytically the of in the presence of in the and at nonactive site residues to the activity of NTD of HK2. the activity of D447A and amino acid The NTD activity in the presence of and and The amino acid and the but increased the size of the side for these the and The of the NTD activity of and that not the but the and is in the activity of the and and a specific the linker helix-α13 and for activity of the NTD of HK2. 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The of the of hydrogen of the and the of and of the linker helix-α13 increased the activity of the NTD in the and of the a with of the linker helix-α13 of been shown to for the of the side of amino acids in in PubMed Scopus Google Scholar). The increased the NTD activity by and the with and by and increased the NTD activity. the at and increased the NTD and are on the of the and As the linker helix-α13 with the first of the the are in an active with the of the and the NTD activity. 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The deuterium rate in the presence of glucose to the of the and in the and the of our is of the of as are in with glucose and of of with glucose and PubMed Scopus Google Scholar, The of PubMed Scopus Google Scholar, The of the of and and of its PubMed Scopus Google Scholar, C.B. The of of new the of with glucose and PubMed Scopus Google Scholar, for of the enzyme PubMed Scopus Google Scholar, of of a molecular for the of via and PubMed Scopus Google Scholar, The of with the new its of PubMed Scopus Google Scholar). this analysis the first of dynamics of the of The of the NTD of HK2 in the and NTD that is as a The NTD in a at of and in the presence of The deuterium by with a The with the to the deuterium for the in dynamics the and and to the the deuterium exchange increased to The enhanced the deuterium for the and with The of and for the NTD and with The of the and in the presence of glucose with at the linker helix-α13 the and NTD of HK2. to the and of the NTD and the of domains amino acid by residues and amino acid of the to NTD in the not the of as the for the in the to amino acid of the to the NTD of HK2. The of the NTD with dynamics, by glucose addition In the D447A and increased the deuterium for with the reduced with the for residues The increased dynamics of D447A and on residues the first of the The deuterium at residues in the at increased by and for D447A and In addition to the analysis on the NTD the the enzyme as a In the NTD D447A increased the of the by with of On the other deuterium of residues at the addition of glucose for and in the NTD and The in dynamics in the the addition of glucose is to the enzyme for as of active site residues the of for The of the NTD by D447A may increased dynamics of the of with the of glucose its and on the of the of deuterium at the for and D447A in the we the that exchange the of the NTD of HK2 and The of the increased on the of D447A with the the effect of on the dynamics of the in the of deuterium exchange by the of the In the the D447A increased the dynamics of residues and of the by and in the and and in the NTD increased the dynamics by but it by In the and are of the of the that is the on one side and and on the other D447A increased the of residues and by and in the and by and in the NTD increased the of these by and it by and the the enhanced dynamics in to the in the presence of D447A and In the the dynamics of that connects the of the with the of the increased by for D447A in the NTD and and by for in the In the dynamics of by in the in the D447A increased the deuterium for and by and in the NTD and to the first of the in the that is to linker helix-α13 on one side and and on the other the deuterium for these by in are multiple including the of the in the and the first of the linker The deuterium of is one of the among the NTD with for the of of the NTD and in the presence of The of increased by for D447A in the NTD and and by for and in the the dynamics of NTD The side of a hydrogen with the of the The enhanced dynamics of reduce the of the and the of with glucose for the of the nonactive site residues and to the activity of the NTD of HK2 due to increased dynamics the active the of the linker helix-α13 different in the NTD and the In the and at the D447A the that increased the of the linker helix-α13 with the by and in the NTD and and and not its in the and at the D447A not the of the linker helix-α13 with the but and reduced its by and the deuterium rate of the linker helix-α13 in the is with that of other with deuterium and and for the and in the and In the NTD the linker helix-α13 in the and for the and The dynamics of the linker helix-α13 may to its in the NTD and of the NTD active site are by in the linker helix-α13 The of the of and and of its PubMed Scopus Google Scholar). In D447A enhanced the dynamics of the linker reduce the of the NTD active to the of the NTD to a HK2 is in types of cancers but limited in normal tissues Hexokinase is required for tumor initiation and and its is in of PubMed Scopus Google as HK1 is the in normal of and metabolic PubMed Scopus Google Scholar). In addition to its in glycolysis, HK2 is for and for tumor initiation and in many types of cancers Hexokinase is required for tumor initiation and and its is in of PubMed Scopus Google Scholar, of of with in PubMed Scopus Google Scholar, Hexokinase the and of cell PubMed Scopus Google Scholar). HK2 is an attractive for the development of anticancer the is to the specific inhibition of glycolysis in cancer normal HK2 other The HK a and active site residues making the specific inhibition of HK2 a the NTD regulatory site of HK2 is a target for the specific inhibition of glycolysis in the NTD is in HKs the design of anticancer that the NTD of HKs activity the development of specific anticancer to the of the NTD and of the and residues of the are to the and of the NTD regulatory site and their in the activity of the analysis on and in the HK1 and HK2 isozymes. the activity in the with HK2 to its NTD but not for the HK1 as HK1 a The enzyme analysis of the activity to the for HK1 and HK2 The and of the activity for HK1 and the NTD regulatory site is a target for the specific inhibition of HK2 with limited on the of other HKs to the specific inhibition of glycolysis in different cancer types with HK2 In the and are in the of HK in the NTD, a with of HK2 and and of but hydrogen with of HK1 and In the and for the NTD but and not reduced the activity. of the the HK domains in the linker helix-α13 and at the of the NTD and The linker helix-α13 connects the NTD to the but is and not with the NTD by is due to of the linker helix-α13 in the the activity of the D447A and in the NTD The D447A that the with of the activity in the NTD and in the it the a hydrogen with of the activity in the NTD with of the activity in the the linker helix-α13 in the not have to NTD but the with are in the NTD activity of HK2. at to the dynamics and of the HK2 the of this analysis for the of and D447A The first are for and of the In the the increased dynamics of the D447A with the at the linker helix-α13 in addition to of the and of the NTD, the in the The enhanced dynamics for the D447A are to by of the and been shown to for NTD activity. In with the the at residues and of the of the NTD enhanced exchange at residues and of the Similar to the linker helix-α13 by residues on one of the in the of the NTD at residues and but not in the The enhanced dynamics in the of the D447A with is due to of the of with as at been shown to the NTD activity of HK2. the D447A of the and and the linker helix-α13 their in the The been on the that increased the NTD activity by The dynamics of NTD increased in the presence of with the due to the of its with of the linker helix-α13 dynamic D447A at the and linker helix-α13 and the active site and linker helix-α13 in a the and the enhanced dynamics by the D447A of the NTD active site The of the active site by D447A is by the the of and of the and of the NTD, The and increased a of for the to for In the of the with the but its In the of the linker helix-α13 the of the NTD and in the D447A as by the the domains The at the of and on the NTD, linker and The for with of for the with of a effect on the linker helix-α13 in the presence of with the enzyme The enhanced and increased of the active site the of D447A to the NTD the of and its to the activity of the NTD of HK2. The enhanced of the NTD in the presence of the D447A reduce its the of to the activity of the NTD of HK2. On the other the effect of on the linker helix-α13 its to the NTD the of the linker helix-α13 in the activity of the NTD of HK2. is an for many as cancer their metabolic to the rate of aerobic glycolysis in to the energy and metabolite demands of cancer (1Vander Heiden M.G. Cantley L.C. Thompson C.B. Understanding the Warburg effect: the metabolic requirements of cell proliferation.Science. 2009; 324: 1029-1033Crossref PubMed Scopus (8407) Google Scholar, P.L. Warburg, me and Hexokinase 2: multiple discoveries of key molecular events underlying one of cancers' most common phenotypes, the "Warburg Effect", i.e., elevated glycolysis in the presence of oxygen.J. Bioenerg. Biomembr. 2007; 39: 211-222Crossref PubMed Scopus (345) Google Scholar, 4Patra The pentose phosphate and 39: PubMed Scopus Google Scholar, glycolysis and phosphorylation in and to PubMed Scopus Google Scholar). The HK reaction is an step for the of glucose metabolism. The upregulation of HK2 in cancer is a to the elevated aerobic glycolysis and in and the expression of HK2 in specific normal tissues it an attractive target for the selective inhibition of glucose metabolism in cancer glycolysis and phosphorylation in and to PubMed Scopus Google Scholar, of and metabolic PubMed Scopus Google Scholar, cancers have aerobic PubMed Scopus Google Scholar, Hexokinase is required for tumor initiation and and its is in of PubMed Scopus Google Scholar, Hexokinase tumor growth and by in PubMed Scopus Google Scholar). The in the development of HK2 inhibitors that target cancer but limited with the HK1 of the and active site of HK the design of inhibitors that target HK2 with limited with the other is

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