Structural Plasticity of Malaria Dihydroorotate Dehydrogenase Allows Selective Binding of Diverse Chemical Scaffolds

Malaria remains a major global health burden and current drug therapies are compromised by resistance. Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) was validated as a new drug target through the identification of potent and selective triazolopyrimidine-based DHODH inhibitors with anti-malarial activity in vivo. Here we report x-ray structure determination of PfDHODH bound to three inhibitors from this series, representing the first of the enzyme bound to malaria specific inhibitors. We demonstrate that conformational flexibility results in an unexpected binding mode identifying a new hydrophobic pocket on the enzyme. Importantly this plasticity allows PfDHODH to bind inhibitors from different chemical classes and to accommodate inhibitor modifications during lead optimization, increasing the value of PfDHODH as a drug target. A second discovery, based on small molecule crystallography, is that the triazolopyrimidines populate a resonance form that promotes charge separation. These intrinsic dipoles allow formation of energetically favorable H-bond interactions with the enzyme. The importance of delocalization to binding affinity was supported by site-directed mutagenesis and the demonstration that triazolopyrimidine analogs that lack this intrinsic dipole are inactive. Finally, the PfDHODH-triazolopyrimidine bound structures provide considerable new insight into species-selective inhibitor binding in this enzyme family. Together, these studies will directly impact efforts to exploit PfDHODH for the development of anti-malarial chemotherapy. Malaria remains a major global health burden and current drug therapies are compromised by resistance. Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) was validated as a new drug target through the identification of potent and selective triazolopyrimidine-based DHODH inhibitors with anti-malarial activity in vivo. Here we report x-ray structure determination of PfDHODH bound to three inhibitors from this series, representing the first of the enzyme bound to malaria specific inhibitors. We demonstrate that conformational flexibility results in an unexpected binding mode identifying a new hydrophobic pocket on the enzyme. Importantly this plasticity allows PfDHODH to bind inhibitors from different chemical classes and to accommodate inhibitor modifications during lead optimization, increasing the value of PfDHODH as a drug target. A second discovery, based on small molecule crystallography, is that the triazolopyrimidines populate a resonance form that promotes charge separation. These intrinsic dipoles allow formation of energetically favorable H-bond interactions with the enzyme. The importance of delocalization to binding affinity was supported by site-directed mutagenesis and the demonstration that triazolopyrimidine analogs that lack this intrinsic dipole are inactive. Finally, the PfDHODH-triazolopyrimidine bound structures provide considerable new insight into species-selective inhibitor binding in this enzyme family. Together, these studies will directly impact efforts to exploit PfDHODH for the development of anti-malarial chemotherapy. The human malaria parasite is endemic in 87 countries putting 2.5 billion people in the poorest nations of the tropics at risk for the disease (1Greenwood B.M. Fidock D.A. Kyle D.E. Kappe S.H. Alonso P.L. Collins F.H. Duffy P.E. J. Clin. Invest. 2008; 118: 1266-1276Crossref PubMed Scopus (479) Google Scholar, 2Guerra C.A. Gikandi P.W. Tatem A.J. Noor A.M. Smith D.L. Hay S.I. Snow R.W. PLoS Med. 2008; 5: e38Crossref PubMed Scopus (318) Google Scholar). Despite intensive efforts to control malaria through combination drug therapy and insect control programs, malaria remains one of the largest global health problems. The most severe form of the disease is caused by Plasmodium falciparum, which kills 1–2 million people yearly, primarily children and pregnant woman. Effective vaccines have not been developed, and chemotherapy remains the mainstay of both treatment and prevention of the disease. Unfortunately widespread drug resistance to almost every known anti-malarial agent has compromised the effectiveness of malaria control programs (3White N.J. J. Clin. Invest. 2004; 113: 1084-1092Crossref PubMed Scopus (830) Google Scholar). The introduction of artemisinin combination chemotherapy has provided new treatment options to combat drug-resistant parasites (4Rosenthal P.J. N. Engl. J. Med. 2008; 358: 1829-1836Crossref PubMed Scopus (139) Google Scholar). However, recent reports by the World Health Organization suggest that resistance to artemisinin is developing along the Thai-Cambodian border, underscoring the need for a continual pipeline of new drug development to combat this disease.The malaria parasite relies exclusively on de novo pyrimidine biosynthesis to supply precursors for DNA and RNA biosynthesis (5Gutteridge W.E. Trigg P.I. J. Protozool. 1970; 17: 89-96Crossref PubMed Scopus (103) Google Scholar, 6Reyes P. Rathod 5: PubMed Scopus Google Scholar). the human the for both de novo pyrimidine biosynthesis and for of pyrimidine and The lack of a to in malaria has in this as a for new dehydrogenase x-ray structure of bound to x-ray structure of bound to x-ray structure of bound to x-ray structure of PfDHODH bound to x-ray structure of bound to x-ray structure of bound to activity x-ray structure of bound to x-ray structure of bound to x-ray structure of bound to x-ray structure of PfDHODH bound to x-ray structure of bound to x-ray structure of bound to activity is a enzyme that the of dihydroorotate to the in de novo pyrimidine biosynthesis PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). is to the of the and recent studies suggest that the of in the parasite is to supply for this PubMed Scopus Google Scholar). These studies provide that PfDHODH is an enzyme to the malaria inhibitor of human DHODH the of is for the treatment of that DHODH is a target Clin. PubMed Scopus Google Scholar, N.J. N. Engl. J. Med. 2004; PubMed Scopus Google Scholar). Finally, J. A.M. Rathod J. PubMed Scopus Google Scholar, J. A.M. J. PubMed Scopus Google and studies D.E. J. J. PubMed Scopus Google Scholar, J. PubMed Scopus Google that the identification of species-selective inhibitors this target was recent studies have directly to the of PfDHODH as a new target for the of We a to classes of potent and selective inhibitors of PfDHODH J. A.M. Rathod J. PubMed Scopus Google Scholar, J. J. Rathod J. PubMed Scopus Google Scholar, J. J. Rathod J. Med. PubMed Scopus Google Scholar, J. J. Rathod J. Med. 2008; PubMed Scopus Google Scholar). These are with and the of not the of mutagenesis supported a the not with the inhibitor inhibitors and a that binding J. J. Rathod J. Med. 2008; PubMed Scopus Google Scholar, Rathod 2008; PubMed Scopus Google Scholar). the one has based on a triazolopyrimidine structure and PfDHODH inhibitors in this potent activity P. falciparum in with of PfDHODH and activity the parasite J. J. Rathod J. Med. PubMed Scopus Google Scholar, J. J. Rathod J. Med. 2008; PubMed Scopus Google Scholar). We a of this that is to Plasmodium in the malaria the first that PfDHODH inhibitors have anti-malarial activity in J. J. Rathod J. Med. PubMed Scopus Google of and from from from from from from from J. J. Rathod J. Med. 2008; PubMed Scopus Google from J. J. Rathod J. Med. PubMed Scopus Google in a new a of PfDHODH one of a new for the development of anti-malarial the that the in the PubMed Scopus Google Scholar). has to a to target PfDHODH for drug programs and to the identification of species-selective of the enzyme J. J. Rathod J. Med. PubMed Scopus Google Scholar, J. J. Rathod J. Med. 2008; PubMed Scopus Google Scholar, A.M. Med. PubMed Scopus Google Scholar, A.M. J. Med. PubMed Scopus Google Scholar, Med. PubMed Scopus Google Scholar, C.A. P. J. J. 2008; PubMed Scopus Google Scholar). The structure of PfDHODH to D.E. J. J. PubMed Scopus Google a inhibitor with affinity for PfDHODH Rathod 2008; PubMed Scopus Google the of PfDHODH to bind the of inhibitors an of the developing for the triazolopyrimidine-based inhibitor we report the x-ray structures of PfDHODH bound to three triazolopyrimidine-based inhibitors with different bound to the triazolopyrimidine we the small molecule x-ray structures of these inhibitors and with the Finally, the PfDHODH structures with the structures of bound to and to a potent inhibitor of the human enzyme PubMed Scopus Google Scholar, The Scholar). these studies both the affinity binding and of this of PfDHODH the for lead programs for the anti-malarial this unexpected insight into PfDHODH is a drug target for a of chemical with for lead remains one of the most global health of drug resistance current of the in developing new anti-malarial is the identification of that will allow the of chemical of the triazolopyrimidine-based inhibitors of PfDHODH has to the of PfDHODH as a target for the development of new and to the identification of a chemical with in anti-malarial activity J. J. Rathod J. Med. PubMed Scopus Google Scholar, J. J. Rathod J. Med. 2008; PubMed Scopus Google Scholar). Here we report the x-ray structures of PfDHODH bound to three triazolopyrimidine-based inhibitors. these that plasticity allows the triazolopyrimidine-based inhibitors to an unexpected hydrophobic binding pocket that was not in flexibility allows the target to accommodate a of inhibitors from different A second unexpected insight was by of the small molecule x-ray structures of the inhibitors with the The intrinsic of the triazolopyrimidine charge delocalization from to insight into the of the triazolopyrimidine-based Finally, the in the human and malaria DHODH to species-selective inhibitor binding that the of the triazolopyrimidine-based malaria enzyme has the flexibility to form different pocket and the flexibility is to the that inhibitor have been for PfDHODH that chemical J. J. Rathod J. Med. PubMed Scopus Google Scholar, J. J. Rathod J. Med. 2008; PubMed Scopus Google Scholar, A.M. Med. PubMed Scopus Google Scholar, A.M. J. Med. PubMed Scopus Google Scholar, Med. PubMed Scopus Google Scholar, C.A. P. J. J. 2008; PubMed Scopus Google Scholar). the triazolopyrimidine with the inhibitors are to bind to the the enzyme accommodate these flexibility the pocket to the of this pocket to bind of in inhibitor binding has been in with conformational in the of and J. J. J. Med. PubMed Scopus Google the N.J. N. Engl. J. Med. 2004; PubMed Scopus Google and a to a 2008; PubMed Scopus Google Scholar). plasticity to an of the DHODH enzyme the of this enzyme as a drug target by options and the that with a chemical the of the enzyme to to different analogs a for during lead to flexibility has been in a of known drug PubMed Scopus Google Scholar, PubMed Scopus (103) Google Scholar, PubMed Scopus Google and is into drug J. 2008; PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, P. 2008; PubMed Scopus Google Scholar, 2008; 5: PubMed Scopus Google Scholar). plasticity to binding of inhibitor classes as an of value drug target for drug of drug interactions that are in conformational in the target that hydrophobic inhibitors of of the binding pocket to affinity interactions PubMed Scopus Google Scholar). that the triazolopyrimidine-based inhibitors a of the by and the to into an that through the the inhibitors bind and a of the that is of the that has not been interactions provide binding a PubMed Scopus Google as has been for potent binding of inhibitors PubMed Scopus Google Scholar). a for these interactions in inhibitor binding to PfDHODH of in this and in a in binding intrinsic of the triazolopyrimidine allow to populate a resonance form that promotes charge which is to the formation of H-bond interactions with the enzyme. The importance of these interactions to inhibitor is supported both by the site-directed mutagenesis and by the chemical of the is with that are to into the triazolopyrimidine the by of the binding affinity by that the of these are the in binding by the with is to both x-ray structures of PfDHODH bound to the triazolopyrimidine-based inhibitors the first studies of the enzyme bound to and as provide considerable new insight into selective binding in this enzyme family. The pocket into A H-bond which interactions with the bound and which is hydrophobic malaria and inhibitors with of inhibitor binding from in the of to A the These have in the of the the triazolopyrimidine for PfDHODH from the of an from the into the which is in the human for the of the on the to of allows with the this by the pocket on The for binding of is The that that form with the triazolopyrimidine and are in the human enzyme inhibitors of based on this x-ray structures of PfDHODH bound to the inhibitors provide insight into the for this to the of activity the with of the J. J. Rathod J. Med. PubMed Scopus Google Scholar, J. J. Rathod J. Med. 2008; PubMed Scopus Google we have that the is a binding are inactive. The structure that the are from the of on one and from the of on the based on the current structures is not in the pocket to accommodate is this of the binding pocket of the conformational flexibility that to accommodate this the hydrophobic of the pocket the and the a is at this and the of a on the Finally, the for interactions and in to the hydrophobic of the are x-ray structure determination of PfDHODH in to three triazolopyrimidine-based inhibitors has provided insight into the for potent and species-selective binding of this of inhibitors. has of the for this lead efforts to the and in of the will by these the results have provided insight into the for species-selective inhibitors of human DHODH that to the triazolopyrimidine Finally, the of in lead for interactions for The human malaria parasite is endemic in 87 countries putting 2.5 billion people in the poorest nations of the tropics at risk for the disease (1Greenwood B.M. Fidock D.A. Kyle D.E. Kappe S.H. Alonso P.L. Collins F.H. Duffy P.E. J. Clin. Invest. 2008; 118: 1266-1276Crossref PubMed Scopus (479) Google Scholar, 2Guerra C.A. Gikandi P.W. Tatem A.J. Noor A.M. Smith D.L. Hay S.I. Snow R.W. PLoS Med. 2008; 5: e38Crossref PubMed Scopus (318) Google Scholar). Despite intensive efforts to control malaria through combination drug therapy and insect control programs, malaria remains one of the largest global health problems. The most severe form of the disease is caused by Plasmodium falciparum, which kills 1–2 million people yearly, primarily children and pregnant woman. Effective vaccines have not been developed, and chemotherapy remains the mainstay of both treatment and prevention of the disease. Unfortunately widespread drug resistance to almost every known anti-malarial agent has compromised the effectiveness of malaria control programs (3White N.J. J. Clin. Invest. 2004; 113: 1084-1092Crossref PubMed Scopus (830) Google Scholar). The introduction of artemisinin combination chemotherapy has provided new treatment options to combat drug-resistant parasites (4Rosenthal P.J. N. Engl. J. Med. 2008; 358: 1829-1836Crossref PubMed Scopus (139) Google Scholar). However, recent reports by the World Health Organization suggest that resistance to artemisinin is developing along the Thai-Cambodian border, underscoring the need for a continual pipeline of new drug development to combat this disease. The malaria parasite relies exclusively on de novo pyrimidine biosynthesis to supply precursors for DNA and RNA biosynthesis (5Gutteridge W.E. Trigg P.I. J. Protozool. 1970; 17: 89-96Crossref PubMed Scopus (103) Google Scholar, 6Reyes P. Rathod 5: PubMed Scopus Google Scholar). the human the for both de novo pyrimidine biosynthesis and for of pyrimidine and The lack of a to in malaria has in this as a for new dehydrogenase x-ray structure of bound to x-ray structure of bound to x-ray structure of bound to x-ray structure of PfDHODH bound to x-ray structure of bound to x-ray structure of bound to activity x-ray structure of bound to x-ray structure of bound to x-ray structure of bound to x-ray structure of PfDHODH bound to x-ray structure of bound to x-ray structure of bound to activity is a enzyme that the of dihydroorotate to the in de novo pyrimidine biosynthesis PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). is to the of the and recent studies suggest that the of in the parasite is to supply for this PubMed Scopus Google Scholar). These studies provide that PfDHODH is an enzyme to the malaria inhibitor of human DHODH the of is for the treatment of that DHODH is a target Clin. PubMed Scopus Google Scholar, N.J. N. Engl. J. Med. 2004; PubMed Scopus Google Scholar). Finally, J. A.M. Rathod J. PubMed Scopus Google Scholar, J. A.M. J. PubMed Scopus Google and studies D.E. J. J. PubMed Scopus Google Scholar, J. PubMed Scopus Google that the identification of species-selective inhibitors this target was dihydroorotate dehydrogenase P. falciparum human dihydroorotate the x-ray structure of bound to the x-ray structure of bound to the x-ray structure of bound to the x-ray structure of PfDHODH bound to the x-ray structure of bound to the x-ray structure of bound to structure activity dihydroorotate dehydrogenase P. falciparum human dihydroorotate the x-ray structure of bound to the x-ray structure of bound to the x-ray structure of bound to the x-ray structure of PfDHODH bound to the x-ray structure of bound to the x-ray structure of bound to structure activity recent studies have directly to the of PfDHODH as a new target for the of We a to classes of potent and selective inhibitors of PfDHODH J. A.M. Rathod J. PubMed Scopus Google Scholar, J. J. Rathod J. PubMed Scopus Google Scholar, J. J. Rathod J. Med. PubMed Scopus Google Scholar, J. J. Rathod J. Med. 2008; PubMed Scopus Google Scholar). These are with and the of not the of mutagenesis supported a the not with the inhibitor inhibitors and a that binding J. J. Rathod J. Med. 2008; PubMed Scopus Google Scholar, Rathod 2008; PubMed Scopus Google Scholar). the one has based on a triazolopyrimidine structure and PfDHODH inhibitors in this potent activity P. falciparum in with of PfDHODH and activity the parasite J. J. Rathod J. Med. PubMed Scopus Google Scholar, J. J. Rathod J. Med. 2008; PubMed Scopus Google Scholar). We a of this that is to Plasmodium in the malaria the first that PfDHODH inhibitors have anti-malarial activity in J. J. Rathod J. Med. PubMed Scopus Google Scholar). a of PfDHODH one of a new for the development of anti-malarial the that the in the PubMed Scopus Google Scholar). has to a to target PfDHODH for drug programs and to the identification of species-selective of the enzyme J. J. Rathod J. Med. PubMed Scopus Google Scholar, J. J. Rathod J. Med. 2008; PubMed Scopus Google Scholar, A.M. Med. PubMed Scopus Google Scholar, A.M. J. Med. PubMed Scopus Google Scholar, Med. PubMed Scopus Google Scholar, C.A. P. J. J. 2008; PubMed Scopus Google Scholar). The structure of PfDHODH to D.E. J. J. PubMed Scopus Google a inhibitor with affinity for PfDHODH Rathod 2008; PubMed Scopus Google the of PfDHODH to bind the of inhibitors an of the developing for the triazolopyrimidine-based inhibitor Here we report the x-ray structures of PfDHODH bound to three triazolopyrimidine-based inhibitors with different bound to the triazolopyrimidine we the small molecule x-ray structures of these inhibitors and with the Finally, the PfDHODH structures with the structures of bound to and to a potent inhibitor of the human enzyme PubMed Scopus Google Scholar, The Scholar). these studies both the affinity binding and of this of PfDHODH the for lead programs for the anti-malarial this unexpected insight into PfDHODH is a drug target for a of chemical with for lead remains one of the most global health of drug resistance current of the in developing new anti-malarial is the identification of that will allow the of chemical of the triazolopyrimidine-based inhibitors of PfDHODH has to the of PfDHODH as a target for the development of new and to the identification of a chemical with in anti-malarial activity J. J. Rathod J. Med. PubMed Scopus Google Scholar, J. J. Rathod J. Med. 2008; PubMed Scopus Google Scholar). Here we report the x-ray structures of PfDHODH bound to three triazolopyrimidine-based inhibitors. these that plasticity allows the triazolopyrimidine-based inhibitors to an unexpected hydrophobic binding pocket that was not in flexibility allows the target to accommodate a of inhibitors from different A second unexpected insight was by of the small molecule x-ray structures of the inhibitors with the The intrinsic of the triazolopyrimidine charge delocalization from to insight into the of the triazolopyrimidine-based Finally, the in the human and malaria DHODH to species-selective inhibitor binding that the of the triazolopyrimidine-based malaria enzyme has the flexibility to form different pocket and the flexibility is to the that inhibitor have been for PfDHODH that chemical J. J. Rathod J. Med. PubMed Scopus Google Scholar, J. J. Rathod J. Med. 2008; PubMed Scopus Google Scholar, A.M. Med. PubMed Scopus Google Scholar, A.M. J. Med. PubMed Scopus Google Scholar, Med. PubMed Scopus Google Scholar, C.A. P. J. J. 2008; PubMed Scopus Google Scholar). the triazolopyrimidine with the inhibitors are to bind to the the enzyme accommodate these flexibility the pocket to the of this pocket to bind of in inhibitor binding has been in with conformational in the of and J. J. J. Med. PubMed Scopus Google the N.J. N. Engl. J. Med. 2004; PubMed Scopus Google and a to a 2008; PubMed Scopus Google Scholar). plasticity to an of the DHODH enzyme the of this enzyme as a drug target by options and the that with a chemical the of the enzyme to to different analogs a for during lead to flexibility has been in a of known drug PubMed Scopus Google Scholar, PubMed Scopus (103) Google Scholar, PubMed Scopus Google and is into drug J. 2008; PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, P. 2008; PubMed Scopus Google Scholar, 2008; 5: PubMed Scopus Google Scholar). plasticity to binding of inhibitor classes as an of value drug target for drug of drug interactions that are in conformational in the target that hydrophobic inhibitors of of the binding pocket to affinity interactions PubMed Scopus Google Scholar). that the triazolopyrimidine-based inhibitors a of the by and the to into an that through the the inhibitors bind and a of the that is of the that has not been interactions provide binding a PubMed Scopus Google as has been for potent binding of inhibitors PubMed Scopus Google Scholar). a for these interactions in inhibitor binding to PfDHODH of in this and in a in binding intrinsic of the triazolopyrimidine allow to populate a resonance form that promotes charge which is to the formation of H-bond interactions with the enzyme. The importance of these interactions to inhibitor is supported both by the site-directed mutagenesis and by the chemical of the is with that are to into the triazolopyrimidine the by of the binding affinity by that the of these are the in binding by the with is to both x-ray structures of PfDHODH bound to the triazolopyrimidine-based inhibitors the first studies of the enzyme bound to and as provide considerable new insight into selective binding in this enzyme family. The pocket into A H-bond which interactions with the bound and which is hydrophobic malaria and inhibitors with of inhibitor binding from in the of to A the These have in the of the the triazolopyrimidine for PfDHODH from the of an from the into the which is in the human for the of the on the to of allows with the this by the pocket on The for binding of is The that that form with the triazolopyrimidine and are in the human enzyme inhibitors of based on this x-ray structures of PfDHODH bound to the inhibitors provide insight into the for this to the of activity the with of the J. J. Rathod J. Med. PubMed Scopus Google Scholar, J. J. Rathod J. Med. 2008; PubMed Scopus Google we have that the is a binding are inactive. The structure that the are from the of on one and from the of on the based on the current structures is not in the pocket to accommodate is this of the binding pocket of the conformational flexibility that to accommodate this the hydrophobic of the pocket the and the a is at this and the of a on the Finally, the for interactions and in to the hydrophobic of the are x-ray structure determination of PfDHODH in to three triazolopyrimidine-based inhibitors has provided insight into the for potent and species-selective binding of this of inhibitors. has of the for this lead efforts to the and in of the will by these the results have provided insight into the for species-selective inhibitors of human DHODH that to the triazolopyrimidine Finally, the of in lead for interactions for Malaria remains one of the most global health of drug resistance current of the in developing new anti-malarial is the identification of that will allow the of chemical of the triazolopyrimidine-based inhibitors of PfDHODH has to the of PfDHODH as a target for the development of new and to the identification of a chemical with in anti-malarial activity J. J. Rathod J. Med. PubMed Scopus Google Scholar, J. J. Rathod J. Med. 2008; PubMed Scopus Google Scholar). Here we report the x-ray structures of PfDHODH bound to three triazolopyrimidine-based inhibitors. these that plasticity allows the triazolopyrimidine-based inhibitors to an unexpected hydrophobic binding pocket that was not in flexibility allows the target to accommodate a of inhibitors from different A second unexpected insight was by of the small molecule x-ray structures of the inhibitors with the The intrinsic of the triazolopyrimidine charge delocalization from to insight into the of the triazolopyrimidine-based Finally, the in the human and malaria DHODH to species-selective inhibitor binding that the of the triazolopyrimidine-based inhibitors. The malaria enzyme has the flexibility to form different pocket and the flexibility is to the that inhibitor have been for PfDHODH that chemical J. J. Rathod J. Med. PubMed Scopus Google Scholar, J. J. Rathod J. Med. 2008; PubMed Scopus Google Scholar, A.M. Med. PubMed Scopus Google Scholar, A.M. J. Med. PubMed Scopus Google Scholar, Med. PubMed Scopus Google Scholar, C.A. P. J. J. 2008; PubMed Scopus Google Scholar). the triazolopyrimidine with the inhibitors are to bind to the the enzyme accommodate these flexibility the pocket to the of this pocket to bind of in inhibitor binding has been in with conformational in the of and J. J. J. Med. PubMed Scopus Google the N.J. N. Engl. J. Med. 2004; PubMed Scopus Google and a to a 2008; PubMed Scopus Google Scholar). plasticity to an of the DHODH enzyme the of this enzyme as a drug target by options and the that with a chemical the of the enzyme to to different analogs a for during lead to flexibility has been in a of known drug PubMed Scopus Google Scholar, PubMed Scopus (103) Google Scholar, PubMed Scopus Google and is into drug J. 2008; PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, P. 2008; PubMed Scopus Google Scholar, 2008; 5: PubMed Scopus Google Scholar). plasticity to binding of inhibitor classes as an of value drug target for drug of drug interactions that are in conformational in the target that hydrophobic inhibitors of of the binding pocket to affinity interactions PubMed Scopus Google Scholar). that the triazolopyrimidine-based inhibitors a of the by and the to into an that through the the inhibitors bind and a of the that is of the that has not been interactions provide binding a PubMed Scopus Google as has been for potent binding of inhibitors PubMed Scopus Google Scholar). a for these interactions in inhibitor binding to PfDHODH of in this and in a in binding The intrinsic of the triazolopyrimidine allow to populate a resonance form that promotes charge which is to the formation of H-bond interactions with the enzyme. The importance of these interactions to inhibitor is supported both by the site-directed mutagenesis and by the chemical of the is with that are to into the triazolopyrimidine the by of the binding affinity by that the of these are the in binding by the with is to both The x-ray structures of PfDHODH bound to the triazolopyrimidine-based inhibitors the first studies of the enzyme bound to and as provide considerable new insight into selective binding in this enzyme family. The pocket into A H-bond which interactions with the bound and which is hydrophobic malaria and inhibitors with of inhibitor binding from in the of to A the These have in the of the the triazolopyrimidine for PfDHODH from the of an from the into the which is in the human for the of the on the to of allows with the this by the pocket on The for binding of is The that that form with the triazolopyrimidine and are in the human enzyme inhibitors of based on this The x-ray structures of PfDHODH bound to the inhibitors provide insight into the for this to the of activity the with of the J. J. Rathod J. Med. PubMed Scopus Google Scholar, J. J. Rathod J. Med. 2008; PubMed Scopus Google we have that the is a binding are inactive. The structure that the are from the of on one and from the of on the based on the current structures is not in the pocket to accommodate is this of the binding pocket of the conformational flexibility that to accommodate this the hydrophobic of the pocket the and the a is at this and the of a on the Finally, the for interactions and in to the hydrophobic of the are x-ray structure determination of PfDHODH in to three triazolopyrimidine-based inhibitors has provided insight into the for potent and species-selective binding of this of inhibitors. has of the for this lead efforts to the and in of the will by these the results have provided insight into the for species-selective inhibitors of human DHODH that to the triazolopyrimidine Finally, the of in lead for interactions for We and of the of for with and and for and with with