The studies on the mechanism and inhibition of Isocitrate Dehydrogenases
<p>Isocitrate dehydrogenases (IDHs) are human metabolic enzymes producing NADPH (IDH1/2) or NADH (IDH3). IDHs are involved in cancer metabolism and progress; upregulation and mutations to human IDH1/2 that catalyse reduction of 2-oxoglutarate (2OG) to 2-hydroxyglutarate have been identified in...
| Main Author: | |
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| Other Authors: | |
| Format: | Thesis |
| Language: | English |
| Published: |
2022
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| _version_ | 1826308605079453696 |
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| author | Liu, X |
| author2 | Schofield, C |
| author_facet | Schofield, C Liu, X |
| author_sort | Liu, X |
| collection | OXFORD |
| description | <p>Isocitrate dehydrogenases (IDHs) are human metabolic enzymes producing NADPH (IDH1/2) or NADH
(IDH3). IDHs are involved in cancer metabolism and progress; upregulation and mutations to human
IDH1/2 that catalyse reduction of 2-oxoglutarate (2OG) to 2-hydroxyglutarate have been identified in
multiple cancers. The work described in this thesis attempted to gain a better understanding of IDH1
and IDH2 biochemistry by studying the inhibition and kinetics of wild type (wt) IDH1/2 and mutant
IDH1/2 (mIDH1/2), with the aim of enabling improved IDH1/2 targeting treatments.</p>
<p>By comparison with IDH1, there are only limited inhibition studies reported on IDH2. The work
described in this thesis began with profiling of reported mIDH1/2 inhibitors with IDH2 using
biochemical and biophysical assays. Allosterically binding mIDH2 targeting inhibitors were found to be
Mg<sup>2+</sup> or Mg<sup>2+</sup>-2OG complex competitive, as is the case for IDH1. NaCl was observed to affect the
kinetics of both Mg<sup>2+</sup> and 2OG (lower their affinities) with mIDH1/2 in a manner that increases the
inhibition potencies of mIDH1/2 inhibitors, particularly with R172K IDH2.</p>
<p>In the assay optimisation of the inhibition assays for wt IDH1/2, the results support the proposal that
the substrate for wt IDH2 is the isocitrate-Mg<sup>2+</sup> complex as recently shown for IDH1. Screening for wt
IDH1/2 inhibitors was performed. As a result, several wt IDH1 and wt IDH2 selective inhibitors and a
pan-wt IDH1/2 inhibitor were identified. Mechanism of action studies suggested the pan-wt IDH1/2
inhibitors are likely to be Mg<sup>2+</sup> or Mg<sup>2+</sup>-isocitrate competitive.</p>
<p>To date, all potent mIDH1/2 inhibitors and the wt IDH1/2 inhibitors identified here are likely to be
allosteric binders; it is of interest to find an active site interacting inhibitor competing with
isocitrate/2OG binding. TCA cycle intermediates and their analogues, and synthetic 2OG derivatives
were studied with wt and mutant IDH1/2 to identify inhibitors and competitive substrates. A 2OG
derivative and N-oxalylglycine were identified as IDH1/2 variant inhibitors. Some synthetic 2OG
derivatives bearing C3-substitution manifested substantial activity as substrates with IDH1/2 variants.
Mechanistic studies with synthetic 2OG derivatives and R132H IDH1 are consistent with half-site
activity and positive cooperativity processes involving the IDH dimers.</p>
<p>Overall, the work described in this thesis reveals that the allosteric inhibition of both wt and variant
IDH1/2s leads to Mg<sup>2+</sup> or Mg<sup>2+</sup>-substrate competition. In addition, 2OG derivatives can act as both
inhibitors and alternative substrates for IDH1/2 variants; they will be useful as biochemical probes to
study IDH catalysis. </p> |
| first_indexed | 2024-03-07T07:21:50Z |
| format | Thesis |
| id | oxford-uuid:c7bf342a-75fa-4d7a-bcb9-c2ad4724ac25 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T07:21:50Z |
| publishDate | 2022 |
| record_format | dspace |
| spelling | oxford-uuid:c7bf342a-75fa-4d7a-bcb9-c2ad4724ac252022-10-19T15:14:44ZThe studies on the mechanism and inhibition of Isocitrate Dehydrogenases Thesishttp://purl.org/coar/resource_type/c_db06uuid:c7bf342a-75fa-4d7a-bcb9-c2ad4724ac25EnglishHyrax Deposit2022Liu, XSchofield, C<p>Isocitrate dehydrogenases (IDHs) are human metabolic enzymes producing NADPH (IDH1/2) or NADH (IDH3). IDHs are involved in cancer metabolism and progress; upregulation and mutations to human IDH1/2 that catalyse reduction of 2-oxoglutarate (2OG) to 2-hydroxyglutarate have been identified in multiple cancers. The work described in this thesis attempted to gain a better understanding of IDH1 and IDH2 biochemistry by studying the inhibition and kinetics of wild type (wt) IDH1/2 and mutant IDH1/2 (mIDH1/2), with the aim of enabling improved IDH1/2 targeting treatments.</p> <p>By comparison with IDH1, there are only limited inhibition studies reported on IDH2. The work described in this thesis began with profiling of reported mIDH1/2 inhibitors with IDH2 using biochemical and biophysical assays. Allosterically binding mIDH2 targeting inhibitors were found to be Mg<sup>2+</sup> or Mg<sup>2+</sup>-2OG complex competitive, as is the case for IDH1. NaCl was observed to affect the kinetics of both Mg<sup>2+</sup> and 2OG (lower their affinities) with mIDH1/2 in a manner that increases the inhibition potencies of mIDH1/2 inhibitors, particularly with R172K IDH2.</p> <p>In the assay optimisation of the inhibition assays for wt IDH1/2, the results support the proposal that the substrate for wt IDH2 is the isocitrate-Mg<sup>2+</sup> complex as recently shown for IDH1. Screening for wt IDH1/2 inhibitors was performed. As a result, several wt IDH1 and wt IDH2 selective inhibitors and a pan-wt IDH1/2 inhibitor were identified. Mechanism of action studies suggested the pan-wt IDH1/2 inhibitors are likely to be Mg<sup>2+</sup> or Mg<sup>2+</sup>-isocitrate competitive.</p> <p>To date, all potent mIDH1/2 inhibitors and the wt IDH1/2 inhibitors identified here are likely to be allosteric binders; it is of interest to find an active site interacting inhibitor competing with isocitrate/2OG binding. TCA cycle intermediates and their analogues, and synthetic 2OG derivatives were studied with wt and mutant IDH1/2 to identify inhibitors and competitive substrates. A 2OG derivative and N-oxalylglycine were identified as IDH1/2 variant inhibitors. Some synthetic 2OG derivatives bearing C3-substitution manifested substantial activity as substrates with IDH1/2 variants. Mechanistic studies with synthetic 2OG derivatives and R132H IDH1 are consistent with half-site activity and positive cooperativity processes involving the IDH dimers.</p> <p>Overall, the work described in this thesis reveals that the allosteric inhibition of both wt and variant IDH1/2s leads to Mg<sup>2+</sup> or Mg<sup>2+</sup>-substrate competition. In addition, 2OG derivatives can act as both inhibitors and alternative substrates for IDH1/2 variants; they will be useful as biochemical probes to study IDH catalysis. </p> |
| spellingShingle | Liu, X The studies on the mechanism and inhibition of Isocitrate Dehydrogenases |
| title | The studies on the mechanism and inhibition of Isocitrate Dehydrogenases |
| title_full | The studies on the mechanism and inhibition of Isocitrate Dehydrogenases |
| title_fullStr | The studies on the mechanism and inhibition of Isocitrate Dehydrogenases |
| title_full_unstemmed | The studies on the mechanism and inhibition of Isocitrate Dehydrogenases |
| title_short | The studies on the mechanism and inhibition of Isocitrate Dehydrogenases |
| title_sort | studies on the mechanism and inhibition of isocitrate dehydrogenases |
| work_keys_str_mv | AT liux thestudiesonthemechanismandinhibitionofisocitratedehydrogenases AT liux studiesonthemechanismandinhibitionofisocitratedehydrogenases |