Current Enzyme Inhibition - Volume 5, Issue 3, 2009

Disulfide reductases are involved in many functions in the cell, in particular, maintaining the intracellular redox balance. Many classes of these enzymes exist, and their inhibition has served as an effective therapy against different types of human diseases. The involvement of disulfide reductases in various cellular processes is reviewed, and therapeutic strategies against pathogenic bacteria, parasitic infections, and human diseases based on their inhibition are discussed.

Somatic angiotensin-converting enzyme (ACE) - well known for its role in cardiovascular pathophysiology - has an unusual, two-domain, double active-site structure. The two domains (designated N and C) are 55% identical and each contains a similar active site with overlapping but distinct substrate preferences. While both convert angiotensin I to angiotensin II in vitro, current evidence suggests the C domain site predominates in this role in vivo. The N domain site inactivates a hemoregulatory and antifibrotic peptide, AcSDKP, in vivo, although the significance of this remains unclear. However, differences in the characteristics of the two domains may result in different context-dependent activities, as is the case with other enzymes containing tandem repeats. The N domain may also have a role in modulating C domain activity, through a combination of inter-domain cooperativity and structural stabilization. Comparison of ACE with its structural homologues reveals conservation of peptidase activity and a tendency to hinge about the active-site cleft. Recent work on ACE active-site mutants containing one or more key residues replaced by their cognate residues from the other domain, synthesis of domain-selective inhibitors, and co-crystal structures of each domain with such inhibitors, has led to a better resolution of the basis for domain selectivity and should enable the design of next-generation, domain-selective inhibitors with distinct pharmacological profiles.

Diacylglycerol kinases (DGKs), a family of lipid kinases, convert diacylglycerol (DG) to phosphatidic acid (PA). Acting as a second messenger, DG activates protein kinase C (PKC). PA, a signaling lipid, regulates diverse functions involved in physiological responses. Since DGK modulates two lipid second messengers, DG and PA, regulation of DGK could induce related cellular responses. Currently, there are 10 mammalian isoforms of DGK that are categorized into five groups based on their structural features. These diverse isoforms of DGK are considered to activate distinct cellular functions according to extracellular stimuli. Each DGK isoform is thought to play various roles inside the cell, depending on its subcellular localization (nuclear, ER, Golgi complex or cytoplasm). In vascular smooth muscle, vasoconstrictors such as angiotensin II, endothelin-1 and norepinephrine stimulate contraction by increasing inositol trisphosphate (IP3), calcium, DG and PKC activity. Inhibition of DGK could increase DG availability and decrease PA levels, as well as alter intracellular responses, including calcium-mediated and PKC-mediated vascular contraction. The purpose of this review is to demonstrate a role of DGK in vascular function. Selective inhibition of DGK isoforms may represent a novel therapeutic approach in vascular dysfunction.

SHIP (SH2 domain containing inositol phosphatases) was identified as a 145kDa multi-domain cytosolic protein expressed specifically in hematopoietic cells that negatively regulates cell growth, survival and proliferation. Aberrant SHIP function is associated with many disease pathologies. There are two aspects to SHIP function: the catalytic function mediated by the central catalytic domain and the non-catalytic function mediated by the protein-protein interaction domains of SHIP. Much is known about the catalytic function of SHIP and its role in regulation of growth factor and immune receptor signaling. However, not much is known about the mechanistic details of the non-catalytic functions of SHIP and this is an active area of research. In this review, we discuss the role of SHIP in different cell types of immune system, its role in various diseases and its potential as a therapeutic target.

Hydrolysis of D-valyl-L-leucyl-L-arginine 4-nitroanilide (120-640 μM) by rat tissue kallikrein (rK1) (3.15 nM) was studied in both the absence and the presence of increasing concentrations of aprotinin (10.4-34.6 nM), a serine protease inhibitor also known as basic pancreatic trypsin inhibitor, which inhibits trypsin, chymotrypsin, plasmin and kallikrein. The data indicate that the inhibition of rK1 by aprotinin is a parabolic competitive inhibition, with two inhibitor molecules binding to one enzyme molecule. Statistical analysis of the data supports the kinetic model proposed. The calculated values of the constants Ki and Kii (mean + SE) were 26.4 ± 5.4 nM and 16.9 ± 11.4 nM, respectively. Ki ≈ Kii, suggests that the aprotinin molecules bind to two enzyme sites with approximately the same affinity. Parabolic competitive inhibition was also reported for the inhibition of human tissue kallikrein (hK1) by aprotinin with Ki and Kii values of 16.20 nM and 1.10 nM, respectively. It is noteworthy that as the Kii < Ki, the second aprotinin molecule binds to the enzyme with a larger affinity suggesting that the second binding site was probably created or positively modulated as a consequence of the binding of the first aprotinin molecule. These results suggest different kinetic properties for the two tissue kallikreins. The second binding site for aprotinin in rK1 and also in hK1 may have important implications for the physiological activity of these enzymes. This study also presents a review of the literature on parabolic competitive inhibition.

Free radicals are causative factor for aminoglycoside induced tissue injury. Fixed dose combination therapy of ceftazidime plus tobramycin reduces liver toxicity than single therapy of tobramycin. The objective of present study was designed to determine the activity of antioxidant enzymes as well as some biochemical parameters along with MDA level by the administration of tobramycin and its fixed dose combination (FDC) with ceftazidime in liver of Mus musculus mice. Eighteen mice were selected and divided into three groups of six mice each. Control group was treated with normal saline and other groups were treated with tobramycin sulphate (4.0 mg/Kg body weight/ day) and FDC of ceftazidime plus tobramycin group (34.1 mg/Kg body weight/ day) for seven days treatment. Overnight fasting animals were sacrificed on 8th day and liver was taken out after perfusion and the Serum glutamic oxaloacetic transaminase (SGOT), Serum glutamic pyruvic transaminase (SGPT) and alkaline phosphatase as well as antioxidant enzymes along with MDA level in mice were determined. Our results showed that the antioxidant enzyme activities and alkaline phosphatase level were significantly decreased along with significant increase in the SGOT, SGPT and MDA levels in tobramycin sulphate treated group as compared to control group. But in case of fixed dose combination of ceftazidime and tobramycin (tobarcef) treated group, all enzymes activities and levels were found to be significantly improved as compared to tobramycin alone treated group. These findings indicate that a fixed dose combination of ceftazidime + tobramycin prevents liver toxicity by improving the level of MDA, liver enzymes and antioxidant activities, due to induction of tobramycin drug.