Profiling the enzymatic properties and inhibition of human complement factor B.

Human complement factor B is the crucial catalytic component of the C3 convertase enzyme that activates the alternative pathway of complement-mediated immunity. Although a serine protease in its own right, factor B circulates in human serum as an inactive zymogen and there is a crystal structure only for the inactive state of factor B and various fragments. To provide greater insight to the catalytic function and properties of factor B, we have used short para-nitroanilide derivatives of 4- to 15-residue peptides as substrates to profile the catalytic properties of factor B. Among factors found to influence catalytic activity of factor B was an unusual dependence on pH. Non-physiological alkaline conditions strongly promoted substrate cleavage by factor B, consistent with a pH-accessible conformation of the enzyme that may be critical for catalytic function. Small N-terminal extensions to conventional hexapeptide para-nitroanilide substrates significantly increased catalytic activity of factor B, which was more selective for its cleavage site than trypsin. The new chromogenic assay enabled optimization of catalysis conditions, the profiling of different substrate sequences, and the development of the first reversible and competitive substrate-based inhibitor of factor B. The inhibitor was also shown to prevent in vitro formation of C3a from C3 by factor B, by synthetic and by natural C3 convertase of the alternative complement activation pathway, and to block formation of membrane attack complex. The availability of a reversible substrate-based inhibitor that could stabilize the active conformation of factor B, in conjunction with a pH-promoted higher processing activity, may offer a new avenue to obtain crystal structures of factor B and C3 convertase in an active conformation.

Carbohydrate-based scaffolds in drug discovery.

Carbohydrates have been proven as valuable scaffolds to display pharmocophores and the resulting molecules have demonstrated useful biological activity towards various targets including the somatostatin receptors (SSTR), integrins, HIV-1 protease, matrix metalloproteinases (MMP), multidrug resistance-associated protein (MRP), and as RNA binders. Carbohydrate-based compounds have also shown antibacterial and herbicidal activity.

Carbohydrates as scaffolds in drug discovery.

Drug discovery has long suffered from the difficulty of having to place pharmacophoric groups in just the right spatial arrangement to elicit the desired biological response. Although some molecule classes have been discovered that seem to be privileged structures for at least some drug-receptor interactions, there remains the challenge to design and synthesize molecules with high specific affinity to pharmacologically important targets. With their high density of stereochemical information and their relative rigidity, carbohydrates provide excellent platforms upon which to display a number of substituents in a sterically defined way, hence offering the opportunity to harness their unique features for the drug-discovery process. This review highlights the progress that has been made in the development of carbohydrate scaffolds for drug discovery.

Organic azide inhibitors of cysteine proteases.

Cysteine proteases are crucial regulatory enzymes in human physiology and disease. Inhibitors are usually designed with reactive electrophiles to covalently bond to the catalytic cysteinyl sulfur, and consequently they also indiscriminately interact with biological thiolates and other nucleophiles, leading to toxic side effects in vivo. Here we describe an alternative to using reactive electrophiles, demonstrating the use of a much less reactive azidomethylene substituent (-CH2-N3) that confers potent inhibition of cysteine proteases. This new approach resulted in potent, reversible, competitive inhibitors of caspase-1 (IC50 < 10 nM), with significant advantages over aldehydes such as high stability in vitro to thiols (10 mM dithiothreitol (pH 7.2), 20 mM glutathione (pH 7.2, 9, 11)) and aqueous media, as well as some highly desirable druglike features. It was also demonstrated that azides can be incorporated into inhibitors of other caspases (e.g. 3, 8) and cathepsins (e.g. K, S, B), indicating the versatility of this valuable new approach to cysteine protease inhibition.

Molecular diversity through sugar scaffolds.

Monosaccharides provide an excellent platform to tailor molecular diversity by appending desired substituents at selected positions around the sugar scaffold. The presence of five functionalized and stereo-controlled centres on the sugar scaffolds gives the chemist plenty of scope to custom design molecules to a pharmacophore model. This review focuses on the peptidomimetic developments in this area, as well as the concept of tailoring structural and functional diversity in a library using carbohydrate scaffolds and how this can lead to increased hit rates and rapid identification of leads, which has promising prospects for drug development.