Targeting the mevalonate pathway for pharmacological intervention
Farnesyl pyrophosphate synthase (FPPS) is a key branch point enzyme in the mevalonate pathway and the main molecular target of nitrogen-containing bisphosphonates (N-BPs), potent inhibitors of osteoclastic activity and the leading drug of choice for conditions characterized by excessive bone resorpt...
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| Natura: | Tesi |
| Lingua: | English |
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2011
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| _version_ | 1826307063379132416 |
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| author | Tsoumpra, M |
| author2 | Russell, R |
| author_facet | Russell, R Tsoumpra, M |
| author_sort | Tsoumpra, M |
| collection | OXFORD |
| description | Farnesyl pyrophosphate synthase (FPPS) is a key branch point enzyme in the mevalonate pathway and the main molecular target of nitrogen-containing bisphosphonates (N-BPs), potent inhibitors of osteoclastic activity and the leading drug of choice for conditions characterized by excessive bone resorption. The main aim of this thesis is to investigate the interaction of N-BPs with FPPS in order to gain further insights into the mechanism of drug inhibition. Kinetic and crystallographic studies following site-directed mutagenesis of FPPS reveal key residues involved in stabilization of carbocation intermediate, substrate binding and formation of a tight enzyme-inhibitor complex. The aromatic ring of Tyr204 is involved in N-BP binding but not in the catalytic mechanism, where the hydroxyl moiety plays an important role. Lys200 is implicated in regulation of substrate binding, product specificity and enzyme isomerization which leads to a tight binding inhibition. Phe239 is considered important for the FPPS C-terminal switch which stabilizes substrate binding and promotes the inhibitor induced isomerized state. The highly conserved Arg112, Asp103 and Asp107 are pivotal for catalysis. Successful purification of the full length of Rab geranylgeranyl transferase (RGGT) complex downstream of the FPPS in the mevalonate pathway was achieved and may lead to co-crystallization with BP analogues and identification of the putative site of drug binding. Investigation of the in vitro effect of N-BPs on osteoclastogenesis suggest a correlation with FPPS inhibition kinetics for the most potent N-BPs but indicate an alternative mechanism of the disruption of bone resorption by alendronate. Together these results highlight the importance of the multiple interactions of N-BPs with side-chain residues of FPPS which dictate their strength of binding and advance the understanding of their pharmacophore effect. |
| first_indexed | 2024-03-07T06:57:22Z |
| format | Thesis |
| id | oxford-uuid:fe945074-e645-4c1d-9598-e28b51a43cca |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T06:57:22Z |
| publishDate | 2011 |
| record_format | dspace |
| spelling | oxford-uuid:fe945074-e645-4c1d-9598-e28b51a43cca2022-03-27T13:37:45ZTargeting the mevalonate pathway for pharmacological interventionThesishttp://purl.org/coar/resource_type/c_db06uuid:fe945074-e645-4c1d-9598-e28b51a43ccaCrystallographyChemical kineticsPharmacologyEnzymesStructural chemistryBiochemistryEnglishOxford University Research Archive - Valet2011Tsoumpra, MRussell, ROppermann, UDunford, JFarnesyl pyrophosphate synthase (FPPS) is a key branch point enzyme in the mevalonate pathway and the main molecular target of nitrogen-containing bisphosphonates (N-BPs), potent inhibitors of osteoclastic activity and the leading drug of choice for conditions characterized by excessive bone resorption. The main aim of this thesis is to investigate the interaction of N-BPs with FPPS in order to gain further insights into the mechanism of drug inhibition. Kinetic and crystallographic studies following site-directed mutagenesis of FPPS reveal key residues involved in stabilization of carbocation intermediate, substrate binding and formation of a tight enzyme-inhibitor complex. The aromatic ring of Tyr204 is involved in N-BP binding but not in the catalytic mechanism, where the hydroxyl moiety plays an important role. Lys200 is implicated in regulation of substrate binding, product specificity and enzyme isomerization which leads to a tight binding inhibition. Phe239 is considered important for the FPPS C-terminal switch which stabilizes substrate binding and promotes the inhibitor induced isomerized state. The highly conserved Arg112, Asp103 and Asp107 are pivotal for catalysis. Successful purification of the full length of Rab geranylgeranyl transferase (RGGT) complex downstream of the FPPS in the mevalonate pathway was achieved and may lead to co-crystallization with BP analogues and identification of the putative site of drug binding. Investigation of the in vitro effect of N-BPs on osteoclastogenesis suggest a correlation with FPPS inhibition kinetics for the most potent N-BPs but indicate an alternative mechanism of the disruption of bone resorption by alendronate. Together these results highlight the importance of the multiple interactions of N-BPs with side-chain residues of FPPS which dictate their strength of binding and advance the understanding of their pharmacophore effect. |
| spellingShingle | Crystallography Chemical kinetics Pharmacology Enzymes Structural chemistry Biochemistry Tsoumpra, M Targeting the mevalonate pathway for pharmacological intervention |
| title | Targeting the mevalonate pathway for pharmacological intervention |
| title_full | Targeting the mevalonate pathway for pharmacological intervention |
| title_fullStr | Targeting the mevalonate pathway for pharmacological intervention |
| title_full_unstemmed | Targeting the mevalonate pathway for pharmacological intervention |
| title_short | Targeting the mevalonate pathway for pharmacological intervention |
| title_sort | targeting the mevalonate pathway for pharmacological intervention |
| topic | Crystallography Chemical kinetics Pharmacology Enzymes Structural chemistry Biochemistry |
| work_keys_str_mv | AT tsoumpram targetingthemevalonatepathwayforpharmacologicalintervention |