Assays for modulators of prolyl-4-hydroxylase

Details Assays for modulators of prolyl-4-hydroxylase Assays for modulators of prolyl-4-hydroxylase

2000-09-15

2004-12-21

Shukla, Ram R. (Department: 1632)

Multicellular living organisms and unmodified parts thereof and

Method of using a transgenic nonhuman animal in an in vivo...

C800S013000, C800S008000

Reexamination Certificate

active

06833489

ABSTRACT:

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
BACKGROUND OF THE INVENTION
Prolyl-4-hydroxylases (P4H) are enzymes that modify collagen in a manner that stabilizes the conformation of collagen. The synthesis of hydroxyproline residues by P4H is a critical step in intracellular collagen processing.
Reduced P4H enzyme activity leads to unstable collagen and disease symptoms such as those seen in patients suffering from scurvy. Increased activity creates less pliable tissue and is associated with fibrotic diseases. P4H is recognized as an ideal target for the pharmacological control of collagen biosynthesis (Bickel, et al.,
Hepatology
August:404-405, 1998).
BRIEF SUMMARY OF THE INVENTION
We have discovered an assay for modulators of P4H enzyme activity in the nematode
Caenorhabditis elegans
. Loss of one isoform of prolyl-4-hydroxylase causes the nematode to be short and fat, a morphology termed “dumpy” or “dpy”. (There are other nematode genes that can be mutated to the dpy phenotype, but there are methods known to one of skill in the art for determining which gene is responsible for the phenotype.) Loss of the second isoform of prolyl4-hydroxylase while retaining the first isoform of prolyl4-hydroxylase gives the nematode no apparent phenotype. Mutations in both prolyl-4-hydroxylase isoforms in the same animal result in embryonic lethality. The embryos develop to the pretzel stage and then retract into a mass of cells. These phenotypes provide an easy assay for detecting changes in prolyl4-hydroxylase activity.
In another embodiment of the present invention, one would introduce the human version of P4H-gene into a P4H-modified nematode and, thus, complement the P4H mutation. One would then expose the test chimeric nematode to a test compound and determine whether the test compound interferes with the P4H activity by examining whether the chimeric nematode or its progeny develop a phenotype that can be attributed to modified P4H activity. We predict that the P4H-modified nematode, which has been exposed to the test compound, will have a phenotype similar to the dpy-18 mutant or the phy-1 mutant or the combined dpy-18; phy-1 double mutant phenotype.
In another embodiment of the present invention, one would attempt to recover P4H activity, thus indicating that the test compound is a P4H activator. In that embodiment, one would introduce a test compound to a P4H-modified nematode and examine the nematode and its progeny for either recovered P4H activity or a phenotype demonstrating wild-type P4H activity.


REFERENCES:
Levy et al. Molecular and genetic analyses of the caenorhabditis elegans dpy-2 and dpy-10 collagen genes. A variety of molecular alterations affect organismal morphology pp. 803-817 vol. 4 1993.*
Hill et al. dpy-18 Encodes an a-subunit of proly-4-hydroxylase in caenorhabditis elegans pp 1139-1148 2000.*
M. Bickel, et al., “Selective Inhibition of Hepatic Collagen Accumulation in Experimental Liver Fibrosis in Rats by a New Prolyl 4-Hydroxylase Inhibitor,”Heptologypp. 403-404, 1998.
L. Friedman, et al., “Prolyl 4-Hydroxylase is Required for Viability and Morphogenesis inCaenorhabditis elegans,” Proc. Natl. Acad. Sci. USA97 (9):4736-4741, 2000.
V. Gunzler and K, Weidman, “Prolyl 4-Hydroxylase Inhibitors,”Prolyl Hydroxylase, Protein Disulfide Isomerase, and Other Structually, Related Proteins, pp. 65-95, 1997.
N.A. Guzman, “Prolyl 4-Hydroxylase: An Overview,”Prolyl Hydroxylase, Protein Disulfide Isomerase, and Other Structurally Related Proteins, pp. 1-64-1997.
J. Veijola, et al., “Cloning Baculovirus Expression , and Characterization of the &agr; Subunit of Prolyl 4-Hydroxylase from the NematodeCaenorhabditis elegans,” J. Biol. Chem. 269(43):26746-26753, 1994.

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