Current Enzyme Inhibition (v.7, #4)

The simple sphingolipids ceramide, sphingosine, ceramide 1-phosphate and sphingosine 1-phosphate, and their metabolizing enzymes are implicated in the regulation of vital cellular functions, including angiogenesis, cell differentiation, migration, and cell growth and death. These sphingolipids are also involved in the establishment and progression of many illnesses including cardiovascular diseases such as atherosclerosis, or neurodegenerative diseases such as Parkinson's or Alzheimer's diseases, chronic inflammation, and cancer. Therefore, the development of inhibitors of the enzymes that are responsible for the synthesis and degradation of these metabolites may be essential tools for studying normal cell metabolism as well as for establishing new therapeutic strategies for treatment of disease.

Dipeptidyl peptidase-4 (DPP-4) is a widely expressed enzyme that functions to cleave the two N-terminal amino acids from certain bioactive peptides. The incretin hormone, glucagon-like peptide-1 (GLP-1) is a substrate for this cleavage and this results in inactivation of the hormone. Since GLP-1 is antidiabetic due to its combined actions to stimulate insulin secretion, increase beta-cell mass and inhibit glucagon secretion, inhibition of DPP-4 has been developed as a therapy of type 2 diabetes to target the islet dysfunction in the disease. This development has been successful: in 2006, the first DPP-4 inhibitor reached the market and today several DPP-4 inhibitors exist. They are all orally active and work through a two to three-fold increase in the circulating concentration of GLP-1. DPP-4 inhibitors are safe and highly tolerable with reported incidences of adverse events being similar as in study groups with placebo. Of special interest is that the risk for hypoglycemia is low and that the treatment is weight neutral rather than being associated with risk for hypoglycemia and/or weight gain which are commonly seen for other therapies. DPP-4 inhibition is most commonly used as add-on therapy to on-going metformin therapy when metformin alone is inadequate for a sufficient glycemic control. DPP-4 inhibition is also, however, used as monotherapy in patients in whom metformin is contraindicated or intolerable, and as add-on to on-going treatment with sulfonylurea, thiazolidinediones or insulin. HbA1c is lowered by approximately 0.6-1.1% by DPP-4 inhibition, with the outcome related to baseline HbA1c. Hence, DPP-4 inhibition is now established as an efficient and highly tolerable oral treatment of type 2 diabetes.

Orally administered drugs targeted against human diseases may have undesired side effects because of unforeseen interactions with enzyme activities encoded by the symbiotic microbiome in the gastrointestinal tract. A prime example is that of the common colon cancer chemotherapeutic CPT-11, a prodrug that is activated in the liver and becomes excreted as a glucuronidated end product; this inactivated product becomes reactivated in the intestine by the action of bacterial β-glucuronidases encoded by the microbiome, which remove the glucuronate moiety. Thus released, CPT-11 causes grave side effects in the intestinal epithelium leading to severe diarrhea and bloody diarrhea. A potential solution consists of a combined therapy where anticancer prodrugs as CPT-11 are supplied in conjunction with selective inhibitors against the bacterial enzymes that reactivate the liver-inactivated drug. Here we review efforts to design inhibitors against bacterial β-glucuronidases based on biochemical and structural analyses aimed at combination therapies with CPT-11 as a brilliant illustration of the complex interactions between the microbiota and current drug therapies, and discuss further examples of drugs that undergo microbiota-induced modifications that alter their pharmacological properties. Indeed, the realization that the microbiota's enzymatic repertoire has a greater than anticipated impact on therapeutic molecules given to human and animal patients has become a turning point in pharmacology and the medical sciences, and, therefore, a deeper and fuller understanding of the biotransformations of drugs catalyzed by the symbiotic flora can help the discovery of more effective treatments (and with far fewer side effects) than ever before.

A new electrofusion method has been developed with high efficiency for hybridoma formation. This method induces more positive clones than conventional fusion methods do. This has led to the successful establishment of functional monoclonal antibodies that can modulate the function of antigens. Such antibodies are difficult to obtain using existing methods. Monoclonal antibodies that inhibit the luminescent activity of the Gaussia luciferase (Gluc) have been prepared using this method. The affinities of antibodies for their antigens were assessed qualitatively by Biacore measurements. Epitope mapping experiments showed that these antibodies recognize the conformation of Gluc antigens rather than their specific sequences. The amino acid sequence of an antigen-recognition region of an antibody was also determined. The mechanism of luminescence inhibition by the monoclonal antibodies is discussed.

Two series A and B of 12 and 7 new carbamate derivates were tested in vitro as acetylcholinesterase inhibitors. The tests were performed in the batch stirred reactor at 25°C, pH 8, ionic strength 0.11 M and catalytic activity of the enzyme preparation 0.14 U mL-1 of the reaction mixture. The temporal dependences of actual concentrations of acetylcholine, choline and acetic acid were determined by two independent analytical methods, HPLC and pH-stat. For all used inhibitors, the model of competitive irreversible inhibition was valid. The inhibition rate constant k3 and qualified estimation of the absolute acetylcholinesterase concentration in the reaction mixture were calculated. The partition coefficients Kow between n-octanol and water of all used inhibitors were determined. The k3 and Kow values were correlated with the Hammett and Hansch substituent constants and with the calculated docking and binding energies of the reaction between the tested inhibitors and acetylcholinesterase.

Poly(ADP-ribosylation), a post-translational modification of proteins involved in DNA repair, replication, transcription and cell death, consists in the conversion of ß-NAD+ into ADP-ribose, and the further formation of polymers of variable length and structure bound to nuclear protein acceptors. Polymer synthesis and degradation are performed respectively by poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG) enzymes. Poly(ADP-ribosylation) represents an emergency reaction to DNA damage; however, PARP overactivation promotes NAD depletion and consequent necrosis, thus exerting a noxious function in many circumstances. The search for chemical compounds able to inhibit poly(ADP-ribosylation) allowed the discovery of new molecules and potent derivatives. Pharmacological inhibition of PARP enzymes is able to reverse the deleterious NAD consumption, thus having a protective role towards many pathological conditions. Of note, the combined treatment of tumors with PARP inhibitors and anticancer drugs has been shown to have a beneficial effect in anticancer therapy. On the whole, pharmacological inactivation of poly(ADP-ribosylation) represents a novel therapeutic strategy to limit cellular injury and to improve the prognosis of a variety of diseases.

Glycogen phosphorylase (GP) has been firmly proved as an important target for treatment of type 2 diabetes. With the rapid increase of type 2 diabetic patients recently, it is becoming an interesting field to discover GP inhibitor for potential antidiabetic drugs. As GP is a typical allosteric protein with several key inhibitor binding sites including the inhibitor, the catalytic, the allosteric and the new allosteric sites, the research works were mainly focused on compounds that can bind these sites and show selective inhibitory effect. This paper mainly reviewed the advances in the design of inhibitors for different binding sites of GP and aimed at providing readers with some useful hints towards more effective GP inhibitors.