Background: Malaria is a devastating infectious disease that disproportionally threatens hundreds of millions of people in developing countries. In the history of anti-malaria campaign, chloroquine(CQ) has played an i...Background: Malaria is a devastating infectious disease that disproportionally threatens hundreds of millions of people in developing countries. In the history of anti-malaria campaign, chloroquine(CQ) has played an indispensable role, however, its mechanism of action(MoA) is not fully understood.Methods: We used the principle of photo-affinity labeling and click chemistry-based functionalization in the design of a CQ probe and developed a combined deconvolution strategy of activity-based protein profiling(ABPP) and mass spectrometry-coupled cellular thermal shift assay(MS-CETSA) that identified the protein targets of CQ in an unbiased manner in this study. The interactions between CQ and these identified potential protein hits were confirmed by biophysical and enzymatic assays.Results: We developed a novel clickable, photo-affinity chloroquine analog probe(CQP) which retains the antimalarial activity in the nanomole range, and identified a total of 40 proteins that specifically interacted and photocrosslinked with CQP which was inhibited in the presence of excess CQ. Using MS-CETSA, we identified 83 candidate interacting proteins out of a total of 3375 measured parasite proteins. At the same time, we identified 8 proteins as the most potential hits which were commonly identified by both methods.Conclusions: We found that CQ could disrupt glycolysis and energy metabolism of malarial parasites through direct binding with some of the key enzymes, a new mechanism that is different from its well-known inhibitory effect of hemozoin formation. This is the first report of identifying CQ antimalarial targets by a parallel usage of labeled(ABPP)and label-free(MS-CETSA) methods.展开更多
Glioma is difficult to treat due to the unique tumor microenvironment and bloodebrain barrier.(13aS)-3-Hydroxyl-6,7-dimethoxyphenanthro[9,10-b]indolizidine(PF403),a phenanthroindolizidine alkaloid,has been identified ...Glioma is difficult to treat due to the unique tumor microenvironment and bloodebrain barrier.(13aS)-3-Hydroxyl-6,7-dimethoxyphenanthro[9,10-b]indolizidine(PF403),a phenanthroindolizidine alkaloid,has been identified as a promising therapeutic agent for the treatment of glioma.However,the anti-glioma mechanism of PF403 in vivo has not been conclusively verified and must be further elucidated.Hence,a strategy without chemical modification was applied to identify the target of PF403.In this study,we identified nicotinamide phosphoribosyl transferase(NAMPT)as the target of PF403 by using thermal proteome profiling(TPP).Moreover,microscale thermophoresis(MST),surface plasmon resonance(SPR),and isothermal titration calorimetry(ITC)experiments confirmed that NAMPT exhibits good affinity for PF403.Direct and indirect enzyme activity assays revealed that PF403 inhibited the catalytic activity of NAMPT,leading to a decrease in the concentration of nicotinamide adenine dinucleotide(NAD ^(+))in U87 cells.X-ray diffraction and amino acid spot mutation experiments revealed that PF403 primarily relies on the formation of piepi interactions with residue Tyr188 to maintain binding with NAMPT(PDB code 8Y55).After NAMPT was knocked down with lentivirus,PF403 lost or partially lost its antitumor activity at the cellular and animal levels.These findings suggest that PF403 exerts antitumor activity by directly targeting NAMPT.展开更多
基金suppor ted by the National Key Research and Development Program of China(2020YFA0908000)the Innovation Team and Talents Cultivation Program of National Administration of Traditional Chinese Medicine(ZYYCXTD-C-202002)+2 种基金the National Natural Science Foundation of China(82074098,82003814)the CACMS Innovation Fund(CI2021A05101)the Fundamental Research Funds for the Central public welfare research institutes(ZZ14-YQ-050,ZZ14-YQ-051,ZZ14-ND-010,ZZ15-ND-10 and ZZ14-FL-002)。
文摘Background: Malaria is a devastating infectious disease that disproportionally threatens hundreds of millions of people in developing countries. In the history of anti-malaria campaign, chloroquine(CQ) has played an indispensable role, however, its mechanism of action(MoA) is not fully understood.Methods: We used the principle of photo-affinity labeling and click chemistry-based functionalization in the design of a CQ probe and developed a combined deconvolution strategy of activity-based protein profiling(ABPP) and mass spectrometry-coupled cellular thermal shift assay(MS-CETSA) that identified the protein targets of CQ in an unbiased manner in this study. The interactions between CQ and these identified potential protein hits were confirmed by biophysical and enzymatic assays.Results: We developed a novel clickable, photo-affinity chloroquine analog probe(CQP) which retains the antimalarial activity in the nanomole range, and identified a total of 40 proteins that specifically interacted and photocrosslinked with CQP which was inhibited in the presence of excess CQ. Using MS-CETSA, we identified 83 candidate interacting proteins out of a total of 3375 measured parasite proteins. At the same time, we identified 8 proteins as the most potential hits which were commonly identified by both methods.Conclusions: We found that CQ could disrupt glycolysis and energy metabolism of malarial parasites through direct binding with some of the key enzymes, a new mechanism that is different from its well-known inhibitory effect of hemozoin formation. This is the first report of identifying CQ antimalarial targets by a parallel usage of labeled(ABPP)and label-free(MS-CETSA) methods.
基金supported financially by the National Natural Science Foundation of China(No.22337007).
文摘Glioma is difficult to treat due to the unique tumor microenvironment and bloodebrain barrier.(13aS)-3-Hydroxyl-6,7-dimethoxyphenanthro[9,10-b]indolizidine(PF403),a phenanthroindolizidine alkaloid,has been identified as a promising therapeutic agent for the treatment of glioma.However,the anti-glioma mechanism of PF403 in vivo has not been conclusively verified and must be further elucidated.Hence,a strategy without chemical modification was applied to identify the target of PF403.In this study,we identified nicotinamide phosphoribosyl transferase(NAMPT)as the target of PF403 by using thermal proteome profiling(TPP).Moreover,microscale thermophoresis(MST),surface plasmon resonance(SPR),and isothermal titration calorimetry(ITC)experiments confirmed that NAMPT exhibits good affinity for PF403.Direct and indirect enzyme activity assays revealed that PF403 inhibited the catalytic activity of NAMPT,leading to a decrease in the concentration of nicotinamide adenine dinucleotide(NAD ^(+))in U87 cells.X-ray diffraction and amino acid spot mutation experiments revealed that PF403 primarily relies on the formation of piepi interactions with residue Tyr188 to maintain binding with NAMPT(PDB code 8Y55).After NAMPT was knocked down with lentivirus,PF403 lost or partially lost its antitumor activity at the cellular and animal levels.These findings suggest that PF403 exerts antitumor activity by directly targeting NAMPT.
