Discovery Of NAD~+ Competitive Phosphoglycerate Dehydrogenase Inhibitors And Investigation Of Antitumor Activities

The programmed metabolism has also been described as one of the new hallmarks of cancer.It has been well recognized that genetic mutation or abnormal expression of metabolic enzymes could induce metabolic reprogramming of tumor cells,providing therapeutic vulnerabilities for cancer therapy.Phosphoglycerate dehydrogenase(PHGDH),a rate-limiting enzyme in the serine synthesis pathway(SSP),catalyzes the conversion of 3-phosphoglycerin to 3-phosphohydroxypyruvate to participate in the serine synthesis,thereby supporting the biosynthesis of macromolecules in cells.Recent studies have shown that PHGDH gene is amplified or overexpressed in breast cancer,lung cancer and other type of cancers and plays a key role in the occurrence and development of these cancers.As such,PHGDH has become one of the most investigated targets in cancer metabolism.The reported PHGDH inhibitors are mainly divided into two classes,orthosteric inhibitors and allosteric inhibitors,which all suffer from drawbacks:1)Orthosteric inhibitors,despite the substantial enzymatic activity,are mostly ineffective in cells;2)Allosteric inhibitors,though mostly less potent than orthosteric inhibitors at the enzymatic level,are more effective in cells yet their binding sites are mostly elusive.At present,none of PHGDH inhibitors has entered the stage of clinical research.Given the apparent limitation of current PHGDH inhibitors,this thesis aims to discover new PHGDH inhibitors with better activity,and overcome the bottleneck of poor cell activity of current inhibitors,provide new molecular skeleton for the research and development of PHGDH inhibitors.To this end,we adopted two strategies,in-house compound library screen and computer-aided drug design,to identify new PHGDH inhibitors.With these efforts,we obtained two lead compounds GDD-04-35and FKR-06-92 that showed the promise in the primary screen.We further evaluated the antitumor activities of these compounds and investigated the mechanism of action of the compounds.In the first part of this thesis,we evaluated the compound GDD-04-35 derived from the in-house compound library screen.At the molecular level,GDD-04-35 has an enzyme activity inhibitory effect on PHGDH equivalent to the positive drug CBR-5884.At the cellular level,the inhibitory effect of GDD-04-35 on the growth of tumor cells is significantly better than that of positive drugs.The detection of intracellular drug concentration suggests that this advantage may be related to the longer time of intracellular drugs.To understand how GDD-04-35 inhibited PHGDH,we carried out a competition assay of GDD-04-35 against the substrate 3-phosphoglycerate(3-PG)or the co-factor NAD~+.The result revealed a competitive mode of inhibition with NAD~+.To further clarify the binding site of GDD-04-35 on PHGDH,we did the co-crystallization of GDD-04-35 and PHGDH,which confirmed its interaction with asparagine(Asp)175 residue inside NAD~+binding pocket of PHGDH.Then,through the enzyme activity inhibition experiment of GDD-04-35 on PHGDH protein mutated at the key binding site,it was confirmed that GDD-04-35 exerted the enzyme activity inhibition effect by binding to site 175.Next,we preliminarily explored the mechanism of the compound at the cellular level and confirmed that gdd-04-35 reduced the de novo synthesis of glucose derived serine in tumor cells and caused G1 phase arrest of tumor cells.In addition,among a spectrum of tumor cell lines with different genetic background,GDD-04-35 showed the selective antitumor activity towards tumor cells with PHGDH amplification and PHGDH overexpression.Finally,we also showed that GDD-04-35 exhibited good antitumor activity in vivo.The second part of this thesis evaluated another compound FKR-06-92 that was discovered by computer-aided drug design with further structural modification.FKR-06-92 showed potent enzymatic inhibitory activity against PHGDH.Substrate competition assay and molecular docking revealed it as a NAD~+-competitive inhibitor of PHGDH.FKR-06-92 inhibited the de novo synthesis of serine,induced cell cycle arrest and apoptosis and selectively inhibited the growth of PHGDH-dependent cancer cells.Finally,we compared GDD-04-35 and FKR-06-92 to provide further guidance for the research and development of PHGDH inhibitors.GDD-04-35 and FKR-06-92 are NAD~+-competitive inhibitors in interaction mode,and both bind to D175 residue of PHGDH.In addition,both inhibit PHGDH enzyme activity and tumor cell growth,and both show selective inhibition on tumor cells with high PHGDH expression.However,in general,GDD-04-35 showed slightly stronger PHGDH inhibitory activity and antitumor effect than FKR-06-92.By comparing the structures of the two compounds,we found that although the sources of the compounds are different and the skeleton of the compounds is different,both of them form hydrogen bonds with PHGDH through a sulfonamide group and an amide group.At the same time,the spatial distance between the sulfonamide group and the amide group is also similar in the two compounds,which may indicate the great potential of developing PHGDH inhibitors with these functional groups as the core.As for the difference of their activity,it may be related to the difference of branched chain,suggesting that the structural modification of branched chain can be an effective strategy to further improve the activity of these compoundsIn conclusion,this thesis took two independent approaches to discover two new PHGDH inhibitors that showed promise in anticancer activity and overcome the bottleneck of poor cell activity of current PHGDH competitive inhibitors.We discovered two lead compoiunds that show promise in inhibiting PHGDH activity and selectively inhibit the growth of cancer cells with high PHGHD expression.Our findings provide basis for the development PHGHD inhibitors.