Methods for identifying agents modulating reverse cholesterol transport or metabolism of steroid for treatment of cardiovascular disease, ovarian follicle dysfunction and other metabolic diseases.

The present invention relates to methods for identifying agents modulating reverse cholesterol transport or metab. of steroid mols. for treatment of cardiovascular disease, ovarian follicle dysfunction and other metabolic diseases. Haptoglobin (Hpt) was previously found to bind the high d. lipoprotein (HDL) apolipoprotein A-I (ApoA-I) and able to inhibit the ApoA-I-dependent activity of the enzyme lecithin:cholesterol acyltransferase (LCAT), which plays a major role in the reverse cholesterol transport. The ApoA-I sequence from Glu113 to Asn184 harbored the binding site for Hpt. Biotinylated peptides were synthesized overlapping such a sequence, and their Hpt binding activity was detd. by avidin-linked peroxidase. The highest activity was exhibited by the peptide P2a, contg. the ApoA-I sequence from Leu141 to Ala164. Such a sequence contains an ApoA-I domain required for binding cells, promoting cholesterol efflux, and stimulating LCAT. The peptide P2a effectively prevented both binding of Hpt to HDL-coated plastic wells and Hpt-dependent inhibition of LCAT, measured by anti-Hpt antibodies and cholesterol esterification activity, resp. The enzyme activity was not influenced, in the absence of Hpt, by P2a. Differently from ApoA-I or HDL, the peptide did not compete with Hb for Hpt binding in ELISA expts. The results suggest that Hpt might mask the ApoA-I domain required for LCAT stimulation, thus impairing the HDL function. Synthetic peptides, able to displace Hpt from ApoA-I without altering its property of binding Hb, might be used for treatment of diseases assocd. with defective LCAT function.