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Assays, methods and means

a technology of transcription factor and hypoxia, applied in the field of transcription factor hif (hypoxia inducible factor), can solve the problem that the peptides derived from them may not be well suited as active agents for oral compositions

Inactive Publication Date: 2004-07-29
ISIS INNOVATION LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention relates to hydroxylases that regulate the cellular turnover of HIF (hypoxia inducible factor) alpha. These enzymes play a key role in the body's response to hypoxia and are involved in various physiological and pathological processes. The invention provides methods for identifying and modulating the interaction of HIF hydroxylases with their substrates, which can have therapeutic applications. The invention also includes the HIF hydroxylases themselves.

Problems solved by technology

This might be desirable where the active compound is difficult or expensive to synthesise or where it is unsuitable for a particular method of administration, for instance, HIF-.alpha. or HIF hydroxylase and in particular peptides derived from them may not be well suited as active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal.

Method used

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Examples

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example 1

Oxygen Regulated Modification by Enzymatic Prolyl Hydroxylation Targets HIF-.alpha. to the Von Hippel-Lindau Ubiquitylation Complex

[0504] In this example it is shown that the interaction between pVHL and a specific domain of the HIF-1.alpha. subunit is regulated by enzymatic hydroxylation of a proline residue (HIF-1.alpha. P564) in a manner that is dependent on oxygen and iron. An absolute requirement of the enzyme for dioxygen as a co-substrate and iron as a co-factor suggests a direct mechanism of cellular oxygen sensing.

[0505] In previous studies of the HIF-.alpha. / pVHL interaction we found that treatment of cells with cobaltous ions and iron chelators prevented the HIF-.alpha. / pVHL association suggesting that the oxygen sensing mechanism might impinge directly on this protein interaction (8). Surprisingly, these studies indicated that the HIF-.alpha. / pVHL complex could be retrieved intact from hypoxic cells. Given the rapidity of pVHL dependent proteolysis of HIF-.alpha. in oxyg...

example 2

Identification of Hypoxia Inducible Factor and Von Hippel-Lindau Tumour Suppressor Homologues in C. elegans

[0576] In this Example we define a HIF homologue in c. elegans and demonstrate that both the transcriptional response to hypoxia, and an important mode of regulation through interaction with the von Hippel-Lindau tumour suppressor are conserved.

[0577] 2.1 Identification of a Homologue.

[0578] We sought homologues to HIF-.alpha. subunits in the c. elegans EST database using an tBLASTn enquiry with the human sequence. Prior to completion of the c. elegans sequencing programme an EST was found with significant homology to HIF-.alpha. in the basic-helix-loop-helix region, and we assembled a contig of ESTs covering the putative homologue. Complete determination of the c. elegans sequence revealed a further six predicted PAS proteins but no closer matches to mammalian HIF-.alpha.. The EST contig we had identified corresponds to a predicted open reading frame (ORF) on chromosome V (F38...

example 3

The VHL E3 Ligase Complex Interacts with Two Independent Regions of HIF-1.alpha.

[0606] In this Example we show that two independent regions of the HIF-1.alpha. a ODDD are targeted for ubiquitylation by VHL E3 in a manner dependent upon proline hydroxylation. However these two VHL E3 target sites differ in their overall sequence, their ability to bind VHL directly and their requirement for other cellular factors. These data reinforce the critical role for pVHL in HIF-.alpha. regulation, but implicate a more complex model for pVHL / HIF-.alpha. interactions.

[0607] Immunoprecipitation and band shift assays show that VHL and HIF-.alpha. subunits are physically associated in a wide range of cell types, consistent with a general role for VHL in oxygen-dependent regulation of HIF-.alpha. subunits. At the same time biochemical studies show that VHL exists as a multiprotein complex with elongins B and C, CUL-2 and RBX1. This complex is homologous to the SCF (Skp-1-Cdc53 / Cullin-F-box) class of ...

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Abstract

A novel class of hydroxylases is described having the amino acid sequence of SEQ ID NO: 2, 4, 6 and 8, and variants and fragments thereof having HIF hydroxylation activity. The polypeptides of the invention have in particular prolyl hydroxylase activity. An assay method monitors the interaction of the HIF hydroxylase with a substrate. Modulators of HIF hydroxylase are provided for use in the treatment of a condition associated with increased or decreased HIF levels or activity or for the treatment of a condition where it is desirable to modulate HIF levels or activity.

Description

[0001] The present invention relates to hydroxylases which act on hypoxia inducible factor alpha (HIF-.alpha.) and which are involved in the regulation of the cellular turnover of HIF. Compounds, methods and means for the modulation of the activity of these enzymes are provided.BACKGROUND OF INVENTION[0002] The transcription factor HIF (hypoxia inducible factor) system is a key regulator of responses to hypoxia, occupying a central position in oxygen homeostasis in a wide range of organisms (1). A large number of transcriptional targets have been identified, with critical roles in angiogenesis, erythropoiesis, energy metabolism, inflammation, vasomotor function, and apoptotic / proliferative responses (1). The system is essential for normal development (2, 3), and plays a key role in pathophysiological responses to ischaemia / hypoxia (1). HIF is also important in cancer, in which it is commonly upregulated, and has major effects on tumour growth and angiogenesis (1). The WAIF DNA bindi...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K31/7088C12N15/09A61K38/00A61K38/16A61K38/55A61K39/395A61K45/00A61K48/00A61P9/10A61P9/12A61P17/02A61P29/00A61P35/00A61P37/06C07K7/08C07K14/47C07K16/40C12N9/02C12N9/06C12Q1/26
CPCA01K2217/05A61K38/00C07K14/4702C07K16/40C12Q1/26G01N2500/20C12N9/0071A61K31/14A61K31/194A61K31/221A61K31/455C07D213/80C07D213/81C07D213/82A61K31/137A61K31/166A61K31/185A61K31/198A61K31/21A61K31/225A61K31/44A61K31/192A61K31/195A61K31/197A61K31/24A61K31/327A61K31/4412A61K31/165A61K31/223A61K31/235A61K31/265A61P17/02A61P29/00A61P35/00A61P37/06A61P43/00A61P9/10A61P9/12G01N33/573G01N2333/90245C07K2317/30A61K39/3955C07K14/475A61K38/005C07C235/80C07C323/60C07C327/32G01N2500/04C12Q1/34
Inventor MAXWELL, PATRICK HENRYPUGH, CHRISTOPHER WILLIAMRATCLIFFE, PETER JOHNSCHOFIELD, CHRISTOPHER JOSEPH
Owner ISIS INNOVATION LTD
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