| Oxidative phosphorylation of mitochondria is the main source of energy for normal differentiated cells.However,tumor cells showed more dependency on glycolysis,which is known as the Warburg effect.Aerobic glycolysis provides a variety of biosynthetic intermediates to fuel the rapid proliferation of cancer cells.One of the intermediate of aerobic glycolysis,3-phosphoglycerate（3-PG）,is a substrate of the serine synthesis pathway.Serine is essential for the synthesis of nucleotides,lipids,and proteins,thereby promoting cell proliferation.The enzyme 3-Phosphoglycerate dehydrogenase（PHGDH）is the first and only rate-limiting enzyme in the serine synthesis pathway.Human PHGDH contains 533 amino acids and four domains: the substrate binding domain（SBD）,the nucleoside binding domain（NBD）,the allosteric substrate binding domain（ASB）,and the Aspartate kinase,Chorismate mutase and Tyr A regulatory domain（ACT）.PHGDH catalyzes the conversion of 3-PG to 3-phosphohydroxypyruvate（3-PHP）in the presence of oxidized coenzyme NAD+.Two downstream enzymes phosphoserine aminotransferase（PSAT1）and phosphoserine phosphatase（PSPH）catalyze the conversion of 3-PHP to L-serine.Plenty of studies have shown that PHGDH is highly expressed in liver cancer,breast cancer,melanoma and several other tumors.The overexpression is closely correlated with low survival rate and poor prognosis.Downregulation of PHGDH expression has been demonstrated to inhibit the proliferation of cancer cells,but exerts little effect on normal cells.In addition,PHGDH also catalyzes the formation of D-2-hydroxyglutarate（D-2HG）,an oncometabolite,thereby promoting the tumor progression.Thus,PHGDH has spurred a huge interest in designing anti-cancer drugs in recent years.Several small molecule inhibitors of PHGDH have been developed by different research groups.However,there are several general limitations of reported PHGDH inhibitors,e.g.,poor anti-cancer activity and unclear binding site（s）.Therefore,further development of these and new PHGDH inhibitors is necessary.To this regard,we firstly purified PHGDH protein,and established a screening system against the PHGDH enzyme activity.We then screened for potential PHGDH inhibitors based on a compound library consisting of 3,100 small molecules.Two small molecules oridonin and withaferin A were identified as PHGDH inhibitors through sequential screenings.The half-inhibition concentration(IC50)of oridonin was slightly stronger than that of withaferin A and three reported inhibitors.In cells expressing high level of PHGDH,oridonin is much more potent than the reported inhibitors.Isothermal calorimetric titration（ITC）analysis showed that oridonin directly bound to PHGDH.In addition,oridonin inhibited the PHGDH enzyme activity in a time-dependent manner: the longer the pre-incubation time,the stronger the inhibition effect.Owing to the presence of an α-β-unsaturated ketone group in oridonin,it was speculated that oridonin may covalently bind to PHGDH.To further study the interaction mechanism between PHGDH and oridonin,the structure of PHGDH-oridonin complex were analyzed via co-crystallization.The structure showed that the α-β-unsaturated ketone group of oridonin could covalently bind to the sulfydryl of cysteine 18（C18）.Besides,oridonin formed two hydrogen bonds with the side chain of E297,and several hydrophobic interactions with adjacent PHGDH residues.It was found that the binding of oridonin resulted in a larger and more exposed substrate binding pocket of PHGDH,compared with the structure that contained no inhibitor.Interestingly,the binding of oridonin also caused a shifting of arginine 54（R54）,which is involved in substrate binding.To verify whether R54 movement reduced substrate affinity,two mutants C18 W and R54 A were designed,mimicking the state of oridonin-bound PHGDH.The Lineweaver-Burk double reciprocal curves showed that the substrate binding affinity of C18 W and R54 A decreased by 3.9 and 8.1 folds respectively,compared with wild type（WT）.The above data supports an allosteric inhibition mechanism of oridonin,i.e.,the covalent C18 binding shifted R54,and decreased the substrate binding affinity of PHGDH.When C18 was mutated into serine（S）,its iso-steric amino acid,the enzyme activity was significantly reduced.However,it could still be inhibited by oridonin.This result suggested that oridonin possibly bound to multiple sites in PHGDH.To comprehensively study the binding sites,a qualitative mass spectrometry was performed.The result showed six modified cysteines: C18,C19,C48,C254,C281,and C295.The above sites were separately mutated into tryptophan（W）,the largest amino acid,to mimic the binding of oridonin.Except two insoluble mutants,the enzyme activities of other mutants were completely lost.This result suggests that the modification of these cysteines by oridonin could inhibit the enzyme activity of PHGDH.Furthermore,it was speculated that these cysteines were differentially modified by oridonin in terms of binding priority and occupancy in solution environment.A substrate competition assay showed that increasing the concentration of 3-PG by 100-folds resulted in about 7-folds elevation of the oridonin IC50,while increasing the concentration of NAD+ by 200-folds only slightly increased the IC50 of oridonin.These results suggested that at the domain level,modification of SBD was more important in oridonin-mediated inhibition on PHGDH,compared with the modification of NBD.To analyze the inhibition contribution by each cysteine,they were individually mutated into serine,and the sensitivity of these mutants against oridonin was measured.The results showed that the cysteine-to-serine mutations in SBD,including C18 S,C19S,C48 S,and C295 S,were barely active in enzyme activity.As a result,the sensitivity of the SBD mutants against oridonin could not be further analyzed.In contrast,the cysteine-to-serine mutations in NBD did not change the enzyme activity profoundly,and were similarly sensitive to oridonin as compared with WT PHGDH.Therefore,oridonin inhibited PHGDH activity mainly through binding to the cysteines located in SBD;although it could also bind to NBD cysteines at high concentration.In the above study,it was found that cysteine-to-tryptophan mutations significantly decreased the solubility of PHGDH.Several experiments were conducted to explore whether oridonin treatment reduced the stability of PHGDH.A differential scanning fluorescence（DSF）assay showed that oridonin decreased the thermal stability of PHGDH.Furthermore,a glutaraldehyde（GTA）cross-linking assay and a gel filtration chromatography assay showed that oridonin treatment induced the polymerization of PHGDH.Likewise,a precipitation assay showed that high concentration of oridonin induced the aggregation and precipitation of PHGDH.In conclusion,cysteine modification of PHGDH decreased the stability of PHGDH protein,promoted its aggregation and precipitation at high concentrations.To demonstrate the direct binding between oridonin and PHGDH in cells,a cellular thermal shift assay（CETSA）was performed in MDA-MB-468 cells.The result showed that oridonin treatment abruptly decreased the thermal stability of endogenous PHGDH,indicating that oridonin directly bound to PHGDH in cells.In addition,oridonin dose-dependently decreased the protein level of endogenous PHGDH in MDA-MB-468 cells.Furthermore,the proteasome inhibitor MG132 rescued the oridonin-induced reduction of PHGDH level,suggesting that oridonin promoted the degradation of PHGDH through the proteasome pathway.Moreover,it was found that oridonin inhibited the size and number of colony formation.In agreement,Ed U incorporation assay showed that oridonin inhibited DNA synthesis of MDA-MB-468 cells.Additionally,oridonin treatment induced the cleavage of poly ADP-ribose polymerase 1（PARP1）and the apoptosis of MDA-MB-468 cells.Gene mutation is one of the main causes of drug resistance in cancer cells.In the COSMIC database,the vast majority of PHGDH mutations were missense mutations.We selected eight missense mutants,including D62 H,A66T,R90 K,R230H,R236 H,R247W,P327 L,and A373 T to analyze their enzymatic activities as well as their sensitivity against oridonin.It was found that the enzyme activities of R90 K and R230 H mutants were obviously enhanced,while the enzyme activities of the other six mutants were significantly decreased.The enzyme activities of R236 H and A373 T mutants were completely lost.Furthermore,it was found that oridonin effectively suppressed the enzyme activity of all mutants.These results indicated that the above mutations did not change the inhibition potency of oridonin.In summary,oridonin was identified as a novel PHGDH inhibitor with submicromolar inhibitory activity through an in vitro screen.The crystal structure of PHGDH-oridonin complex revealed that oridonin formed a covalent bond with C18 of PHGDH,thereby allosterically inhibited the enzyme activity.Furthermore,mass spectrometry analysis showed more oridonin-bound cysteines in PHGDH.The cysteines located in SBD were preferentially modified by oridonin,compared with the NBD cysteines.Moreover,oridonin decreased the thermal stability of PHGDH and induced the aggregation and precipitation of PHGDH.In addition,oridonin not only directly bound to endogenous PHGDH,but also promoted its degradation through the proteasome.Moreover,oridonin efficiently inhibited the enzyme activities of PHGDH cancer mutants.The significance of this study is as following.Firstly,oridonin was identified as a novel potent inhibitor of PHGDH.Secondly,the crystal structure of PHGDHcovalent inhibitor complex was obtained for the first time,which further revealed a novel allosteric inhibition mechanism of oridonin on PHGDH.Thirdly,new characteristics of PHGDH protein was revealed by assessing the effects of cysteine modification on the enzyme activity,providing more information for development of covalent inhibitors.Fourthly,the molecular mechanism of oridonin-mediated degradation of PHGDH enlightens protein degradation as a new strategy for the development of PHGDH inhibitors. |
