Investigation into structural and functional relationships of short-chain dehydrogenases and reductases (SDRs) using a compound library
The short-chain dehydrogenases/reductases (SDR) protein family is one of the largest and most evolutionarily conserved protein superfamilies known to date. SDRs show a remarkable ability to accommodate hundreds of reactions/substrates by using the versatile nucleotide binding domain as their central...
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| Format: | Thesis |
| Language: | English |
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2018
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| _version_ | 1797078479755280384 |
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| author | Bhatia, C |
| author2 | Oppermann, U |
| author_facet | Oppermann, U Bhatia, C |
| author_sort | Bhatia, C |
| collection | OXFORD |
| description | The short-chain dehydrogenases/reductases (SDR) protein family is one of the largest and most evolutionarily conserved protein superfamilies known to date. SDRs show a remarkable ability to accommodate hundreds of reactions/substrates by using the versatile nucleotide binding domain as their central scaffold. SDRs are implicated in many different disease pathways, and represent a ‘druggable’ enzyme class, which has generated extensive biotechnological and pharmaceutical interest. For 28 human SDR members, the three- dimensional structures have been determined. Despite this, almost 25 % of human SDR members still have unknown functions, and many members lack any chemical descriptors. To address the dearth of ligand knowledge, 28 SDRs were cloned, expressed and purified to further characterise members by the application of differential scanning fluorimetry (DSF). A family annotation through cofactor preference, substrate accommodation and subcellular localisation has shown a few distinctive traits within the SDR family. This in silico approach has led to the creation of a systematic flow chart, which provides a starting point to identify substrate classes for orphan SDRs. Cofactor and inhibitor screening of SDRs by DSF has shown Tm shifts that are in-line with previous experimental data. DSF screening showed the decryption of 6 cofactor preferences for orphan SDRs, five of which have been confirmed in crystal structures. In contrast, screening of substrates in DSF produce little thermal stability (15 % detection rate), rendering it difficult to distinguish specific substrates for orphan SDRs. However, over 70 % of SDRs screened in DSF with known enzyme activity produce thermal shifts with compounds that resemble their substrate structures. This can therefore infer substrate classes for orphan SDRs that compare well with those from computational predictions. DSF screening against a ligand library has led to the discovery of the first small molecule binders for Nmr-like family domain-conatining protein 1 (NMRAL1) and selective inhibitors for Hydroxyprostaglandin dehydrogenase (HPGD). The use of small molecule ligands identified from DSF has also contributed to the success of crystal formation in the case of Hydroxysteroid dehydrogenase-like protein 2 (HSDL2) and Dehydrogenase/reductase SDR family member 2 (DHRS2). Overall this study has provided a platform for ligand screening to further structurally characterise the SDR family. |
| first_indexed | 2024-03-07T00:32:27Z |
| format | Thesis |
| id | oxford-uuid:804b39f0-8a25-4561-afeb-3c11e5ad6c8c |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T00:32:27Z |
| publishDate | 2018 |
| record_format | dspace |
| spelling | oxford-uuid:804b39f0-8a25-4561-afeb-3c11e5ad6c8c2022-03-26T21:22:17ZInvestigation into structural and functional relationships of short-chain dehydrogenases and reductases (SDRs) using a compound libraryThesishttp://purl.org/coar/resource_type/c_db06uuid:804b39f0-8a25-4561-afeb-3c11e5ad6c8cStructural biologyProtein putificationThermal shiftsProtein expressionCrystallographyFamily AnnotationEnglishHyrax Deposit2018Bhatia, COppermann, UThe short-chain dehydrogenases/reductases (SDR) protein family is one of the largest and most evolutionarily conserved protein superfamilies known to date. SDRs show a remarkable ability to accommodate hundreds of reactions/substrates by using the versatile nucleotide binding domain as their central scaffold. SDRs are implicated in many different disease pathways, and represent a ‘druggable’ enzyme class, which has generated extensive biotechnological and pharmaceutical interest. For 28 human SDR members, the three- dimensional structures have been determined. Despite this, almost 25 % of human SDR members still have unknown functions, and many members lack any chemical descriptors. To address the dearth of ligand knowledge, 28 SDRs were cloned, expressed and purified to further characterise members by the application of differential scanning fluorimetry (DSF). A family annotation through cofactor preference, substrate accommodation and subcellular localisation has shown a few distinctive traits within the SDR family. This in silico approach has led to the creation of a systematic flow chart, which provides a starting point to identify substrate classes for orphan SDRs. Cofactor and inhibitor screening of SDRs by DSF has shown Tm shifts that are in-line with previous experimental data. DSF screening showed the decryption of 6 cofactor preferences for orphan SDRs, five of which have been confirmed in crystal structures. In contrast, screening of substrates in DSF produce little thermal stability (15 % detection rate), rendering it difficult to distinguish specific substrates for orphan SDRs. However, over 70 % of SDRs screened in DSF with known enzyme activity produce thermal shifts with compounds that resemble their substrate structures. This can therefore infer substrate classes for orphan SDRs that compare well with those from computational predictions. DSF screening against a ligand library has led to the discovery of the first small molecule binders for Nmr-like family domain-conatining protein 1 (NMRAL1) and selective inhibitors for Hydroxyprostaglandin dehydrogenase (HPGD). The use of small molecule ligands identified from DSF has also contributed to the success of crystal formation in the case of Hydroxysteroid dehydrogenase-like protein 2 (HSDL2) and Dehydrogenase/reductase SDR family member 2 (DHRS2). Overall this study has provided a platform for ligand screening to further structurally characterise the SDR family. |
| spellingShingle | Structural biology Protein putification Thermal shifts Protein expression Crystallography Family Annotation Bhatia, C Investigation into structural and functional relationships of short-chain dehydrogenases and reductases (SDRs) using a compound library |
| title | Investigation into structural and functional relationships of short-chain dehydrogenases and reductases (SDRs) using a compound library |
| title_full | Investigation into structural and functional relationships of short-chain dehydrogenases and reductases (SDRs) using a compound library |
| title_fullStr | Investigation into structural and functional relationships of short-chain dehydrogenases and reductases (SDRs) using a compound library |
| title_full_unstemmed | Investigation into structural and functional relationships of short-chain dehydrogenases and reductases (SDRs) using a compound library |
| title_short | Investigation into structural and functional relationships of short-chain dehydrogenases and reductases (SDRs) using a compound library |
| title_sort | investigation into structural and functional relationships of short chain dehydrogenases and reductases sdrs using a compound library |
| topic | Structural biology Protein putification Thermal shifts Protein expression Crystallography Family Annotation |
| work_keys_str_mv | AT bhatiac investigationintostructuralandfunctionalrelationshipsofshortchaindehydrogenasesandreductasessdrsusingacompoundlibrary |