PPIases (Peptidyl-prolyl cis-trans isomerases)

Peptidyl-prolyl cis-trans isomerases (PPIases) are ubiquitous enzymes that participate in protein folding. They have been shown to accelerate the cis-trans isomerization of the X-Pro peptide bond in polypeptide chains in vitro and in vivo (where X is any amino acid). They can be divided into structurally distinct families based on amino acid sequence homology and their ability to bind certain immunosuppressive drugs. PPIases that bind cyclosporin A (CsA) are termed cyclophilins, while those which bind FK506 or rapamycin are called FK506-binding Proteins (FKBP’s). The binding of drugs to their respective receptors can result in inhibition of PPIase activity to varying degrees. Cyclophilins and FKBP’s are of interest as drug targets because of their likely involvement in the broad spectrum, anti-infective activities of CsA (1,2) and FK506 (3), and non-immunosuppressive derivatives thereof (4).

References:
(1) Chappell, L.H. and Wastling, J.M. (1992) Parasitology 105, S25–40.
(2) Page, A.P. et al. (1995) Parasitology Today 11, 385–388.
(3) Moro, A. et al. (1995) EMBO J. 14, 2483–90.
(4) Bell, A. et al. (1994) Biochem. Pharmacol. 48, 495–503.

Chitinases

Chitinases hydrolyze the b1-4 linkages of chitin, an unbranched polymer of b1-4 linked N-acetyl-D-glucosamine (GlcNAc). Since chitin is the second most abundant polymer in nature, many organisms including prokaryotes, vertebrates, plants, fungi and insects produce chitinases with roles in nutrition, morphogen-esis, aggression and defense (1,2).

Chitinases are separated into two of the families of glycohydrolases, family 18 and family 19, based on their hydrolytic mechanisms (3). The four human parasite chitinases available from NEB, Brugia malayi chitinase (BmCHT1), Onchocerca volvulus chitinase (OvCHT1), Entamoeba histolytica chitinase (EhCHT1), and Plasmodium falciparum chitinase (PfCHT1) are all family 18 glycohydrolases.

The activities of chitinases on various available substrates differ. To illustrate, we have used the same amount of enzyme activity for each chitinase as assayed on the standard substrate, 4-methylumbelliferyl-N,N’,N’’-triacetyl-b-chitotrioside, and performed an activity assay using 3H-chitin as a substrate. The results are shown in figure1.

Fig1: 160 units of each chitinase were incubated with 10,000 cpm 3H-chitin in 20 mM NaPO4 (pH 6.0), 0.2 M NaCl, 1 mM EDTA, 500 µg/ml BSA in 200 µl at 37°C. Aliquots were removed at various time points, mixed with unlabeled chitin and after centrifugation, the total soluble cpm for each was determined.

References:
(1) Cohen-Kupiec, R. and Chet, I. (1998) Current Opinion in Biotechnology 9, 270–277.
(2) Gooday, G.W. (1999) EXS 87, 157–169.
(3) Henrissat, B. and Bairoch, A. (1996) Biochem. J. 316, 695–696.

Chitin Synthase

Chitin synthases (EC 2.4.1.16) are membrane bound enzymes that produce chitin, an extracellular polysaccharide composed of an unbranched chain of b 1,4 linked N-acetyl-D-glucosamine (GlcNAc). These enzymes transfer GlcNAc to the growing chitin chain from uridine diphospho-GlcNAc (UDP-GlcNAc) (1,2). The critical role of chitin synthases in the life cycle of many pathogens especially fungal, protozoan and nematode species that cause disease in humans, makes them an extremely important drug target. Chitin serves as an essential structural component of these pathogens. For example, disruption of these enzymes in fungal species results in a loss in cell wall integrity (3), in protozoan species (ie., Entamoeba), the infectious cyst formation stage is affected (4) and in filarial parasites, the synthesis of egg shell is inhibited (5).

Naturally occurring, antibiotic, UDP-GlcNAc analogues (nikkomycins and polyoxins) made by different Streptomyces have been found to be competitive inhibitors of chitin synthases. These inhibitors, though, are effective (Ki values in µM range) in systems only when tested in vitro and have weak and variable effects when tested in vivo. This is presumably due to cell permeability issues as well as the fact that numerous chitin synthases with differing susceptibilities are present within the same organism (3). More potent and universal chitin synthase inhibitors are needed.

In Saccharomyces cerevisiae, there are three chitin synthases: Chs1p, Chs2p and Chs3p encoded respectively by the genes CHS1, CHS2, CHS3. Of these three, Chs1p is the most active when assayed in vitro. It is activated in vitro by a controlled proteolysis with trypsin and requires Mg²⁺ for activity (6,7). A partial purification of chitin synthase activity from S. cerevisiae cells is now available from NEB.

References:
(1) Glaser, L. and Brown, D.H. (1957) J. Biol. Chem. 228, 729–742.
(2) Choi, W-J. and Cabib, E. (1994) Anal. Biochem. 219, 368–372.
(3) Ruiz-Herrera, J. and San-Blas, (2003) G. Curr. Drug Targets Infect. Disord. 3, 77–91.
(4) Das, S. and Gillin, F.D. (1991) Biochem. J. 280, 641–647.
(5) Zhang, Y. et al., New England Biolabs, unpublished observations.
(6) Valdivieso, M.H. et al. (1999) EXS 87, 55–69.
(7) Cabib, E. et al. (1989) J. Cell Biol. 138, 97–102.

Subtilisin-like Proprotein Convertases

Subtilisin-like proprotein convertases (SPCs) are processing enzymes in the secretory pathway of eukaryotic cells. Endoproteolytic cleavage by these enzymes is an essential step in the post-translational processing of many peptide hormones, membrane receptors, viral proteins and bacterial toxins (1). SPCs are interesting drug targets because they may process proteins required for parasite survival in the vertebrate host (2). These calcium dependent serine proteases cleave on the carboxy side of basic amino acid clusters such as (Lys/Arg)-Arg and Arg-X-(Lys/Arg)-Arg (where X is any amino acid). Inhibitors of SPCs include EGTA, polyarginine compounds (3) and a1-Antitrypsin Portland (4).

References:
(1) Steiner, D.F. (1998) Curr. Opin. Chem. Biol. 2, 31–39.
(2) Jin, J. et al (1999) Gene 237, 161–175.
(3) Cameron, A. et al (2000) J. Biol. Chem. 275, 36741–36749.
(4) Jean, F. et al (1998) Proc. Natl. Acad. Sci. USA 95, 7293–7298.

Cytochrome P450s

The cytochrome P450 enzymes, abbreviated CYP450s, are a family of heme containing proteins that are known to modify a wide variety of compounds. First discovered in 1958, these proteins display an unusual absorbance maximum at 450 nm upon carbon monoxide binding to a reduced form of the heme; this led to the initial P450 designation. CYP450s are now known to be distributed over all five kingdoms of life and are classified based upon their respective redox partner. In addition to the general importance of these enzymes, interest has been fueled by their role in the metabolism of xenobiotics, including pharmaceutical agents, in humans. Specifically, focus has centered on the type II isozymes abundant in liver. These liver enzymes are membrane-bound proteins that require the presence of another protein, NADPH-cytochrome P450 reductase, for effective enzymatic activity.

Over 90% of currently available pharmaceutical agents are metabolized by the cytochrome P450 enzymes. Incredibly, from the large human CYP450 family a preponderance of the drugs, approximately 50%, are acted upon by a single isoform, CYP3A4. These enzymes can alter the pharmacological activity of a compound in a number of ways. For example, the CYP450 can simply modify the compound so that it is cleared from the system; alternatively, these enzymes may convert a compound or prodrug from a biologically inactive form into an active one. Some researchers hope to use the fact that many types of cancers express CYP450s in order to administer a relatively harmless prodrug that is metabolized at the tumor site into a potent chemotherapeutic agent. Due to these effects and the role of CYP450s in drug-drug interaction, it is important to determine whether a drug or drug target inhibits or is metabolized by one of these intriguing enzymes.

Fig2: NADPH-cytochrome P450 Reductase interacts with CYP3A4 and mediates electron transfer from NADPH to the cytochrome P450 heme iron. Conversion of testosterone to 6-b-hydroxy testosterone is shown above.