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The protease fibroblast activation protein (FAP) is a specific marker of activated mesenchymal cells in tumour stroma and fibrotic liver. A specific, reliable FAP enzyme assay has been lacking. FAP's unique and restricted cleavage of the post proline bond was exploited to generate a new specific substrate to quantify FAP enzyme activity. This sensitive assay detected no FAP activity in any tissue or fluid of FAP gene knockout mice, thus confirming assay specificity. Circulating FAP activity was ∼20- and 1.3-fold less in baboon than in mouse and human plasma, respectively. Serum and plasma contained comparable FAP activity. In mice, the highest levels of FAP activity were in uterus, pancreas, submaxillary gland and skin, whereas the lowest levels were in brain, prostate, leukocytes and testis. Baboon organs high in FAP activity included skin, epididymis, bladder, colon, adipose tissue, nerve and tongue. FAP activity was greatly elevated in tumours and associated lymph nodes and in fungal-infected skin of unhealthy baboons. FAP activity was 14- to 18-fold greater in cirrhotic than in non-diseased human liver, and circulating FAP activity was almost doubled in alcoholic cirrhosis. Parallel DPP4 measurements concorded with the literature, except for the novel finding of high DPP4 activity in bile. The new FAP enzyme assay is the first to be thoroughly characterised and shows that FAP activity is measurable in most organs and at high levels in some. This new assay is a robust tool for specific quantitation of FAP enzyme activity in both preclinical and clinical samples, particularly liver fibrosis.

FGF-21 is a key regulator of metabolism and potential drug candidate for the treatment of type II diabetes and other metabolic disorders. However, the half-life of active, circulating, human FGF-21 has recently been shown to be limited in mice and monkeys by a proteolytic cleavage between P171 and S172. Here, we show that fibroblast activation protein is the enzyme responsible for this proteolysis by demonstrating that purified FAP cleaves human FGF-21 at this site in vitro, and that an FAP-specific inhibitor, ARI-3099, blocks the activity in mouse, monkey and human plasma and prolongs the half-life of circulating human FGF-21 in mice. Mouse FGF-21, however, lacks the FAP cleavage site and is not cleaved by FAP. These findings indicate FAP may function in the regulation of metabolism and that FAP inhibitors may prove useful in the treatment of diabetes and metabolic disorders in humans, but pre-clinical proof of concept studies in rodents will be problematic.

Six new N-arly-boroGly derivatives, along with their N-acyl-boroSar analogues, are synthesized by modification of conventional procedures and (super 11)B and (super 1)H NMR spectroscopy are used for the structural characterization of alpha-amidoboronic acids. The results show that the formation of such intramolecular B-O dative bonds depends on the nature of the substituents at both the acyl carbon and the nitrogen atoms.

Dipeptide boronic acids of the type H2N-X-Y-B(OH)2 are potent inhibitors of the enzyme protease. When X, the N-terminal amino acid, contains a side chain having a functional group with boron affinity and suitable geometry, additional cyclization in the form of bidentate intramolecular chelation or 'autochelation' is likely to occur, predominantly at mid pH.

We describe here the epimerization-free synthesis and characterization of a new class of conformationally constrained lactam aminoboronic acid inhibitors of dipeptidyl peptidase IV (DPP IV; E.C. 3.4.14.5). These compounds have the advantage that they cannot undergo the pH-dependent cyclization prevalent in most dipeptidyl boronic acids that attenuates their potency at physiological pH. For example, d-3-amino-1-l-1-boronic-ethyl-pyrrolidine-2-one (amino-d-lactam-l-boroAla), one of the best lactam inhibitors of DPP IV, is several orders of magnitude less potent than l-Ala-l-boroPro, as measured by Kivalues (2.3 nM vs 30 pM, respectively). At physiological pH, however, it is actually more potent than l-Ala-l-boroPro, as measured by IC50values (4.2 nM vs 1400 nM), owing to the absence of the potency-attenuating cyclization. In an interesting and at first sight surprising reversal of the relationship between stereochemistry and potency observed with the conformationally unrestrained Xaa-boroPro class of inhibitors, the l−ldiastereomers of the lactams are orders of magnitude less effective than the d−llactams. However, this interesting reversal and the unexpected potency of the d−llactams as DPP IV inhibitors can be understood in structural terms, which is explained and discussed here. [ABSTRACT FROM AUTHOR]

Presents a letter to the editor about the backbone resonance assignments of Matrilysin Metallo-Proteinase-7 complexed with a sulfonamide hydroxamate-type inhibitor.

The ClpP1P2 protease complex is essential for viability in Mycobacteria tuberculosis and is an attractive drug target. Using a fluorogenic tripeptide library (Ac-X3X2X1-aminomethylcoumarin) and by determining specificity constants (kcat/Km), we show that ClpP1P2 prefers Met >> Leu > Phe > Ala in the X1 position, basic residues or Trp in the X2 position, and Pro >> Ala > Trp in the X3 position. We identified peptide substrates that are hydrolyzed up to 1000 times faster than the standard ClpP substrate. These positional preferences were consistent with cleavage sites in the protein GFPssrA by ClpXP1P2. Studies of ClpP1P2 with inactive ClpP1 or ClpP2 indicated that ClpP1 was responsible for nearly all the peptidase activity, whereas both ClpP1 and ClpP2 contributed to protein degradation. Substrate-based peptide boronates were synthesized that inhibit ClpP1P2 peptidase activity in the submicromolar range. Some of them inhibited the growth of Mtb cells in the low micromolar range indicating that cleavage specificity of Mtb ClpP1P2 can be used to design novel anti-bacterial agents. [ABSTRACT FROM AUTHOR]