Current Enzyme Inhibition - Volume 5, Issue 2, 2009

Phosphoinositide 3-kinases (PI3Ks) are a family of enzymes extensively involved in cell signaling, acting downstream of tyrosine kinase and G-protein coupled receptors. Based on structural and substrate specificity, mammalian PI3Ks include class I (A, B), class II and class III enzymes. Several lines of evidence have shown that PI3Ks participate in the regulation of fundamental cellular functions such as cell growth, apoptosis, motility and vesicular trafficking, as well as in the modulation of physiological processes such as immunity, metabolism and cardiovascular physiology. Moreover, a deregulation of the PI3K pathway has been shown in several disease states such as cancer, inflammation and cardiovascular diseases, in human and animal models. Hence, the pharmacological inhibition of PI3Ks has emerged as a novel potential therapeutic option. Older and broad-spectrum PI3Ks inhibitors such as wortmannin and LY294002 have been useful tools for the elucidation of signaling pathways. However, isoform selectivity and adequate pharmacokinetic properties are now necessary for a selective therapeutic approach. Recently, the understanding of the crystallographic structure of PI3Ks has allowed the development of a multitude of inhibitors. In this review, we will present a general outline of PI3K functions in cellular, physiological and disease processes and discuss the main pharmacological compounds available for isoform-specific inhibition and the related evidence regarding their use in disease models.

Proteases are a class of proteins that degrade other proteins. There are several classes of proteases and these are defined by the chemical nature of the catalytic sites. The process of proteolysis is a vital function that is exquisitely regulated. Many diseases involve deregulation of proteolysis due to over-expression of one or more proteases or endogenous protease inhibitors being faulty or missing. Engineered protease inhibitors that control proteolysis are a well recognized class of therapeutics. For certain classes of proteases, small molecule inhibitors have been very successful (Threonine, Cysteine, and Aspartate proteases). For Serine and Zinc proteases, there has been little success with small molecules. Kunitz domains are natural inhibitors of Serine proteases. Using phage display, Kunitz domains can be engineered rapidly to have very high affinity and specificity for a particular serine protease. One of these, DX-88, is a potent, specific inhibitor of human plasma kallikrein and has successfully completed two phase 3 trials for hereditary angioedema, for which a BLA has been filed to the FDA, and is currently in phase 2 for reduction of blood loss in thoracic surgery. For metalloproteases, potent, specific inhibitory antibodies against several MMPs have been discovered also by phage display.

Poly(ADP-ribosylation) is a post-translational modification of proteins which plays a crucial role in basic cellular processes, including DNA repair and replication, transcription and cell death. The biochemical reaction of poly(ADPribosylation) consists of 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. Although poly(ADPribosylation) is considered as an emergency reaction to DNA damage, high levels of PARP activation cause NAD depletion and consequent necrosis, thus having a pathogenetic role in many diseases. As for chemical compounds able to inhibit poly(ADP-ribosylation), since the use of nicotinamide and benzamide, potent derivatives have been developed and new molecules have been tested. Pharmacological inhibition of PARP enzymes proved to reverse the noxious effects of ROS, thus exerting a protective role towards a number of pathological conditions. Moreover, the combined treatment of tumors with PARP inhibitors and anticancer agents has been shown to have a beneficial effect in cancer therapy. Remarkably, pharmacological inactivation of poly(ADP-ribosylation) represents a novel therapeutical strategy to limit cellular injury and to improve the prognosis of a variety of diseases. For these reasons, at present a lot of Companies and research laboratories are actively involved in the modeling of new compounds able to modulate poly(ADP-ribosylation).

Serine protease inhibitor Kazal type 1 (SPINK1) was originally identified as a trypsin inhibitor in the pancreatic acinar cells in 1948. Recent studies showed an association of mutations in SPINK1 gene and hereditary chronic pancreatitis. Thus, a lack of SPINK1 may result in the premature conversion of trypsinogen into active trypsin in acinar cells, leading to pancreatitis. However, we found that mice deficient for Spink3, a mouse homologue of SPINK1, died after birth due to excessive autophagy (cellular self-digestion) in the pancreatic acinar cells, suggesting that Spink3 is involved in the regulation of autophagy. We further demonstrated that autophagy is involved in trypsinogen activation within the pancreatic acinar cells in experimentally induced pancreatitis. These results suggest that Spink3 has protective roles in pancreatitis by dual mechanisms, one as a trypsin inhibitor and a second as a suppressor of trypsinogen activation through negative regulation of autophagy. On the other hand, SPINK1 is structurally similar to epidermal growth factor (EGF), in terms of the number of amino acid residues and the presence of 3 intrachain disulfide bridges. In fact, Spink3 acts as a growth factor in various cell lines in vitro. To gain additional insight into the new function of Spink3 in vivo, we examined the expression pattern of Spink3 during development. We found that Spink3 was expressed in unexpected tissues such brain and mesonephric tubules. In this review, we summarize the old and new roles of SPINK1/Spink3 in trypsin inhibition, autophagy, and cell proliferation/differentiation.