Enzyme inhibition

Details Enzyme inhibition Enzyme inhibition

2000-05-11

2004-12-14

Carlson, Karen Cochrane (Department: 1653)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Having -c-, wherein x is chalcogen, bonded directly to...

C514S539000, C514S588000, C514S209000, C514S541000, C514S546000, C514S520000, C514S461000, C514S002600, C514S018700, C514S021800, C530S350000

Reexamination Certificate

active

06831099

ABSTRACT:

TECHNICAL FIELD
This invention relates to compounds and methods for enzyme inhibition. In particular, the invention relates to therapeutic methods deriving from enzyme inhibition.
BACKGROUND
In eukaryotes, protein degradation is predominately mediated through the ubiquitin pathway in which proteins targeted for destruction are ligated to the 76 amino acid polypeptide ubiquitin. Once targeted, ubiquitinated proteins then serve as substrates for the 26S proteasome, a multicatalytic protease, which cleaves proteins into short peptides through the action of its three major proteolytic activities. While having a general function in intracellular protein turnover, proteasome-mediated degradation also plays a key role in many processes such as major histocompatibility complex (MHC) class I presentation, apoptosis, cell division, and NF-&kgr;B activation.
The 20S proteasome is a 700 kDa cylindrical-shaped multicatalytic protease complex comprised of 28 subunits organized into four rings. In yeast and other eukaryotes, 7 different &agr; subunits form the outer rings and 7 different &bgr; subunits comprise the inner rings. The &agr; subunits serve as binding sites for the 19S (PA700) and 11S (PA28) regulatory complexes, as well as a physical barrier for the inner proteolytic chamber formed by the two &bgr; subunit rings. Thus, in vivo, the proteasome is believed to exist as a 26S particle (“the 26S proteasome”). In vivo experiments have shown that inhibition of the 20S form of the proteasome can be readily correlated to inhibition of 26S proteasome. Cleavage of amino-terminal prosequences of &bgr; subunits during particle formation expose amino-terminal threonine residues, which serve as the catalytic nucleophiles. The subunits responsible for catalytic activity in proteaseome thus possess an amino terminal nucleophilic residue, and these subunits belong to the family of N-terminal nucleophile (Ntn) hydrolases (where the nucleophilic N-terminal residue is, for example, Cys, Ser, Thr, and other nucleophilic moieties). This family includes, for example, penicillin G acylase (PGA), penicillin V acylase (PVA), glutamine PRPP amidotransferase (GAT), and bacterial glycosylasparaginase. In addition to the ubiquitously expressed &bgr; subunits, higher vertebrates also possess three &ggr;-interferon-inducible &bgr; subunits (LMP7, LMP2 and MECL1), which replace their normal counterparts, X, Y and Z respectively, thus altering the catalytic activities of the proteasome. Through the use of different peptide substrates, three major proteolytic activities have been defined for the eukaryote 20S proteasome: chymotrypsin-like activity (CT-L), which cleaves after large hydrophobic residues; trypsin-like activity (T-L), which cleaves after basic residues; and peptidylglutamyl peptide hydrolyzing activity (PGPH), which cleaves after acidic residues. Two additional less characterized activities have also been ascribed to the proteasome: BrAAP activity, which cleaves after branched-chain amino acids; and SNAAP activity, which cleaves after small neutral amino acids. The major proteasome proteolytic activities appear to be contributed by different catalytic sites, since inhibitors, point mutations in &bgr; subunits and the exchange of &ggr; interferon-inducing &bgr; subunits alter these activities to various degrees.
The 20S proteasome plays important roles in cell growth regulation, major histocompatibility complex class I presentation, apoptosis, antigen processing, NF-&kgr;B activation, and transduction of pro-inflammatory signals.
Small molecules which have been used to inhibit proteasome activity include lactacystin, and short peptides including aldehyde, vinyl sulfone, boronic acid and glyoxal functional groups. These compounds generally lack the specificity, stability, or potency necessary to explore the roles of the proteasome at the cellular and molecular level. For example, peptide aldehydes also inhibit lysosomal and Ca
+2
-activated proteases, thus complicating a precise dissection of their effects on cells. Vinyl sulfone-based inhibitors have been reported to bind and inhibit intracellular cysteine proteases (for example, cathepsin S), in addition to their actions against the proteasome. Lactacystin has a rate of proteasome inactivation which is significantly slower than that of vinyl sulfone peptide inhibitors. Lactacystin is also non-specific for 20S proteasome, as it has been found to significantly decrease the hydrolysis rate of human platelet lysosomal cathepsin A-like enzyme at pH 5.5.
SUMMARY
Enzyme inhibitors are valuable tools that enable the elucidation of details in cellular events that are regulated by these enzymes. Additionally, enzyme inhibitors have therapeutic applications and can be used to carry out mechanistic studies of the machinery of enzymatic processes. The invention relates to the discovery that classes of molecules known as peptide &agr;′,&bgr;′-epoxides and peptide &agr;′,&bgr;′-aziridines can bind efficiently, irreversibly and selectively to N-terminal nucleophile (Ntn) hydrolases, and can specifically inhibit particular activities of enzymes having multiple catalytic activity.
Once thought merely to dispose of denatured and misfolded proteins, the proteasome is now recognized as constituting proteolytic machinery that regulates the levels of diverse intracellular proteins through their degradation in a signal-dependent manner. Hence, there is great interest in identifying reagents that can specifically perturb the activities of the proteasome and other Ntn hydrolases and thereby be used as probes to study the role of these enzymes in biological processes. Compounds that target the Ntn hydrolases are herein described, synthesized and investigated. Peptide epoxides and peptide aziridines that can potently, selectively, and irreversibly inhibit particular proteasome activities are disclosed and claimed.
Particular peptide epoxides and peptide aziridines modify three catalytic subunits of the 20S proteasome resulting in inhibition primarily of the chymotrypsin-like activity; the trypsin-like and PGPH activities were also inhibited at approximately 100-fold and 1000-fold slower rates, respectively. Furthermore, in comparison with other potent irreversible proteasome inhibitors, peptide epoxides and peptide aziridines inhibit the chymotrypsin-like activity at least about 80-fold faster than lactacystin and at least about four-fold faster than clasto-lactacystin &bgr;-lactone. Even higher rates are obtainable.
Other particular peptide epoxides and peptide aziridines primarily inhibit PGPH activity, while having far less inhibitory effect on chymotrypsin-like activity, and virtually no effect on trypsin-like activity. In contrast to the enzyme inhibitors described above, which are highly specific for chymotrypsin-like activity of the proteasome, these other particular PGPH-specific peptide epoxides and peptide aziridines inhibit a catalytic step which is believe to be a rate-limiting step in protein degradation. Their use in elucidating the role(s) of other proteasomal subunits is thus limited. The PGPH-specific inhibitors allow separation of contributions of this particular catalytic activity in biological processes mediated by the proteasome.
Unlike several other peptide-based inhibitors, the peptide epoxides and peptide aziridines described herein do not substantially inhibit non-proteasomal proteases such trypsin, chymonypsin, cathepsin B, papain, and calpain at concentrations up to 50 &mgr;M. At higher concentrations, inhibition is observed, but is competitive and not irreversible, since the inhibitor merely competes with the substrate. The novel peptide epoxides and peptide aziridines are also shown to inhibit NF-&kgr;B activation and to stabilize p53 levels in cell culture. Moreover, we have demonstrated the potent anti-inflammatory activity of peptide epoxides and peptide aziridines in a mouse model of cutaneous inflammation. Thus, these compounds can be unique molecular probes, which have the versatility to explore Ntn enzyme function

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