This invention relates to a reagent and method for determining the activity of retroviral protease, e.g. human immunodeficiency virus (HIV) protease. More particularly, the invention relates to novel fluorogenic substrates and a fluorometric assay for HIV protease and other retroviral proteases.
Acquired immune deficiency syndrome, which only a few years ago was a medical curiosity, is now a serious disease. As a consequence, a great effort is being made to develop drugs and vaccines to combat AIDS. The AIDS virus, first identified in 1983, has been described by several names. It is the third known T-lymphocyte virus (HTLV-III) and has the capacity to replicate within cells of the immune system and thereby lead to a profound destruction of T4.sup.+ T-cells (or CD4.sup.30 cells). See, e.g., Gallo et al., Science 224, 500-503 (1984), and Popovic et al., Ibid., 497-500 (1984). This retrovirus has been known as lymphadenopathy-associated virus (LAV) or AIDS-related virus (ARV) and, most recently, as human immunodeficiency virus (HIV). Two distinct AIDS viruses, HIV-1 and HIV-2, have been described. HIV-1 is the virus originally identified in 1983 by Montagnier and co-workers at the Pasteur Institute in Paris [Ann. Virol. Inst. Pasteur 135 E, 119-134 (1984)], while HIV-2 was more recently isolated by Montagnier and his co-workers in 1986 [Nature 326, 662 (1987)]. As used herein HIV is meant to refer to these viruses in a generic sense.
A useful approach being investigated recently for potential use in the treatment of AIDS is the development of synthetic peptides as inhibitors of retroviral protease. Thus, it is known that retroviruses, including the human immunodeficiency virus (HIV), express their genetic content by directing the synthesis of a polyprotein by the host. This precursor is then processed by proteolysis to give essential viral enzymes and structural proteins. A virally encoded enzyme, the retroviral protease, is contained within the polyprotein and is responsible for the specific cleavages of the polyprotein yielding mature viral proteins.
Various novel peptide inhibitors of retroviral protease such as HIV protease are described in applicants' copending application Ser. No. 07/320,742, filed Mar. 8, 1989. Included among these are short peptides of about 4 to 8 amino acid residues derived from HIV cleavage sites such as, e.g. p24/p15, which have been modified to incorporate an internal CH.sub.2 NH bond isostere. An illustrative example of such peptides is
Ac--Thr--Ile--Nle--.psi.[CH.sub.2 NH]--Nle--Gln--Arg--NH.sub.2.
HIV protease thus is a logical therapeutic target in the search for AIDS drugs. In order to facilitate the exploration of potential inhibitors of HIV protease and to optomize lead compounds, a convenient assay to kinetically characterize the inhibitor which allows for automation and high throughput would be desirable. Initial assays used in the characterization of HIV protease were based on HPLC separation of products and substrate at fixed time intervals. See Moore et al., Biochem. Biophys. Res. Commun. 159, 420-425 (1989); Darke et al., Ibid. 156, 297-303 (1988); Schneider and Kent, Cell 54, 363-368 (1988). The HPLC assay quickly became the rate-limiting step in the development of HIV protease inhibitors with desirable therapeutic properties. A continuous assay which allows for quantitative kinetic characterization of the interaction of the inhibitors with HIV protease would be preferable. The strategy of developing either a chromogenic or fluorogenic substrate is proposed by the present inventors based on their work characterizing short peptide substrates of known HIV protease cleavage sites. Toth et al., Proc. 11th Am. Peptide Symp., Rivier and Marshall, Eds., ESCOM, Leiden, In Press 1990. Fluorogenic substrates for hydrolytic enzymes have been widely used in biochemistry because of their high sensitivity. See, e.g., Guilbault, Enzymatic Methods of Analysis, Pergamon Press, 1970, pp. 43-47.
Recently, Nashed et al., Biochem. Biophys. Res. Commun. 163, 1079-1085 (1989), and Hyland et al., Abstr. 2nd Int. Conf. Drugs Res. Immunologic and Infectious Disease. AIDS. New York Acad. Sci., I-20 (1989), have reported a spectrophotometric assay based on cleaved chromogenic substrates containing p--NO.sub.2 --Phe at position P1. In these cases, the substances were an octapeptide, a nonapeptide, and a decapeptide. A high-throughput, radiometric assay for screening for HIV protease inhibitors, which is not continuous, has also been reported by Hyland et al., supra.