Current Pharmaceutical Design - Volume 15, Issue 31, 2009

Zinc (Zn) metal ion along with other transition metals like copper and iron are of vital significance in living organisms with zinc being the second most abundant transition metal ion in living organism following iron. Zn(II) exhibits remarkable stability in redox processes due to its d10 electronic configuration and is known to be indispensable to growth and development, to metabolic pathways as well as to transmission of the genetic message. The coordination properties of the zinc that allows the metal to bind within a broad range of tetrahedral sites in proteins can discriminate the role of zinc in four classes, such as catalytic, cocatalytic, structural and protein interface. Zinc metal sites are encountered in a wide variety of enzymes implicated in synthesis of nucleic acid and proteins, catalysis, protein/peptide degradation, signaling etc. Recent studies propose that zinc proteins may comprise the 10% of the human proteome. In this issue of Current Pharmaceutical Design journal a variety of zinc enzymes are discussed and the latest achievements in the study of their structural and functional properties are highlighted along with the recent developments in the design and biological properties of new zinc chelators, which might act as modulators of enzymes’ function, with potential interest in pharmacology and medicine. I. Bertini and co-workers [1] at the Center of Magnetic Resonance and the University of Florence (Italy) provide a report on the recent advances in Matrix Metalloproteases genetics and function, highlighting the variability of the structural features among the family of these enzymes along with their intra- or inter-domain dynamics that are also taken into account in latest attempts for design and biological evaluation of new inhibitors.

Matrix metalloproteinases are involved in many biological processes and in a large set of diseases. In the last twenty years the genetics, functions, and the structural features of this family of proteolytic enzymes have been investigated and a large number of synthetic inhibitors designed and tested. A better knowledge of the dynamical features of these proteins can be relevant not only to reveal new biological activities but also to design more specific and selective inhibitors. Here, we report the common and the distinct structural features of these proteins, the most recent published information on protein dynamics in matrix metalloproteinases and the recent results on the catalytic mechanism. The implications of the observed intra- and interdomain flexibility in matrix metalloproteinases for drug design have been analyzed and discussed.

The fusion of therapeutics and diagnostic medicine in an effort to provide individualized pharmacotherapy frequently requires the manipulation of drugs that target different enzymes and receptors. To this end, and as a strategy to increase the efficiency of drug development pipelines, new chemical entities are often developed that interact with more than one target. Angiotensin-converting enzyme (ACE), its homologue ACE2, neutral endopeptidase (NEP) and endothelin-converting enzyme (ECE-1) are metallopeptidases that are involved in the metabolism of biologically active peptides that impact on the regulation of the cardiovascular system. The benefit of the ACE/NEP; NEP/ECE and ACE/NEP/ECE dual and triple inhibitors is not only their possible increased efficacy with respect to blood pressure control, but also their other activities, such as antiproliferative, anti-fibrotic and anti-inflammatory, mediated by angiotensin II and atrial natriuretic peptide. Over the last few years a number of three-dimensional structures of these metallopeptidases have advanced our understanding of the mode of interaction between various ligands and their target binding sites. This information is invaluable in the rational design of new and improved drugs. Here we review the structural basis for the design of single and multiple metallopeptidase inhibitors for the treatment of cardiovascular disease. Moreover, we present recent advances in the development of ACE/ECE-1 inhibitors that are likely to have high potency and improved side effect profiles.

In recent decades, the most successful strategy for controlling blood pressure has been inhibition of the angiotensin-converting enzyme (ACE). ACE inhibitors of chemical synthesis (captopril, enalapril, ramipril and trandolapril) have been widely used clinically to reduce mortality in patients with heart failure, and in patients with recent myocardial infarction and heart failure or marked left ventricular dysfunction. In addition to preventive and therapeutic drugs, increased attention has been paid to identifying dietary compounds that may contribute to cardiovascular treatment and prevention. ACE inhibitory peptides, derived from a multitude of plant and animal proteins such as milk, soy or fish, represent sources of health-enhancing components. These ACE inhibitory peptides can be enzymatically released from precursor proteins in vitro and in vivo, respectively during food processing and gastrointestinal digestion. They have shown the ability to lower blood pressure by limiting the vasoconstrictory effects of Angiotensin II and potentiating the vasodilatory effects of Bradykinin. By using specific procedures they may be generated in or incorporated into functional foods for the development of ‘natural’ beneficial health products. Several products containing peptides with ACE inhibitory properties are currently on the market or in development. This review focuses on the use, application and future perspective of bioactive peptides with properties relevant to cardiovascular health.

Insulin-degrading enzyme (IDE) or insulysin is a highly conserved Zn2+ -dependent endopeptidase with an “inverted” HxxEH motif. In vivo, IDE contributes to regulate the steady state levels of peripheral insulin and cerebral amyloid β peptide (Aβ) of Alzheimer's disease. In vitro, substrates of IDE include a broad spectrum of peptides with relevant physiological functions such as atrial natriuretic factor, insulin-like growth factor-II, transforming growth factor- α, β-endorphin, amylin or glucagon. The recently solved crystal structures of an inactive IDE mutant bound to four different substrates indicate, in accordance with previous compelling biochemical data, that peptide backbone conformation and size are major determinants of IDE recognition and substrate selectivity. IDE-N and IDE-C halves contribute to substrate binding and may rotate away from each other leading to open and closed conformers that permit or preclude the entry of substrates. Noteworthy, stabilization of substrate β strands in their IDE-bound form may explain the preference of IDE for peptides with a high tendency to self-assembly as amyloid fibrils. These structural requirements may underlie the capability of some amyloid peptides of forming extremely stable complexes with IDE and raise the possibility of a dead-end chaperone-like function of IDE independent of catalysis. Furthermore, the recent recognition of IDE as a varicella zoster virus receptor and its putative involvement in muscle cell differentiation, steroid receptor signaling or proteasome modulation suggest that IDE is a multi-functional protein with broad and relevant roles in several basic cellular processes. Accordingly, IDE functions, regulation or trafficking may partake in the molecular pathogenesis of major human diseases and become potential targets for therapeutic intervention.

During the last few years a novel role for previously known Zn(II) aminopeptidases has emerged, attracting a great deal of scientific interest to these molecules. Aminopeptidases appear now to play a key role in the last, yet crucial, proteolytic steps that generate small peptides for presentation onto MHC class I molecules so that the mature MHCpeptide complexes can be recognized by cytotoxic T-lymphocytes. In that context, ER aminopeptidases have been shown to strongly affect the adaptive immune response. ER aminopeptidase 1 (ERAP1) has been demonstrated to be a critical determinant of the immune response by generating mature antigenic epitopes from peptide precursors that arrive into the ER originating primarily from intracellular proteins degraded by the proteasome. At least one more related aminopeptidase, renamed ERAP2, appears to have important yet distinct roles in antigenic peptide generation. This review discusses recent findings that help to unravel the role of ER aminopeptidases in the immune response as well as the molecular properties that underlie this role. Determining the exact role and mechanism of action of these aminopeptidases will potentially provide tools for the pharmaceutical manipulation of the immune response on a subtle and qualitative level leading to novel therapeutic opportunities for the treatments of diseases ranging from autoimmunity to cancer.

Botulinum toxin has long been known for its paralytic effects on the human voluntary musculature via inhibition of acetylcholine release at neuromuscular junctions. Its original clinical use for the treatment of strabismus has expanded significantly to include neurological conditions related to muscle hyperactivity and/or spasticity (e.g., dystonia, spasticity, tics, tremor, dysphonia). Recently, botulinum toxin has been shown to impact autonomic disorders by acting at acceptors on glands and smooth muscle, and consequently it has been used in the management of a number of other conditions including hypersecretory disorders, pain syndromes, detrusor sphinchter dyssenergia or overactivity and gastointestinal smooth muscle/sphincter spasm; it may also reduce pain in patients for whom it is used to treat these and other primary conditions. This article will review the pharmacology and formulations of botulinum toxins as well as data from clinical trials demonstrating their efficacy for numerous conditions based on their effects on cholinergic synapses outside the motor nervous system.

LIM (Lin-11, Isl-1, Mec-3), RING (Really interesting new gene), PHD (Plant homology domain) and MYND (myeloid, Nervy, DEAF-1) domains are all zinc-binding domains that ligate two zinc ions. Unlike the better known classical zinc fingers, these domains do not bind DNA, but instead mediate interactions with other proteins. LIM-domain containing proteins have diverse functions as regulators of gene expression, cell adhesion and motility and signal transduction. RING finger proteins are generally associated with ubiquitination; the presence of such a domain is the defining feature of a class of E3 ubiquitin protein ligases. PHD proteins have been associated with SUMOylation but most recently have emerged as a chromatin recognition motif that reads the methylation state of histones. The function of the MYND domain is less clear, but MYND domains are also found in proteins that have ubiquitin ligase and/or histone methyltransferase activity. Here we review the structure-function relationships for these domains and discuss strategies to modulate their activity.

The ubiquitin proteasome system (UPS) plays a fundamental role in maintaining the correct balance of protein levels inside all living cells. Degradation of proteins by this pathway is essential for most cellular processes including cell signalling, DNA repair, apoptosis and gene transcription. Any disruption to the system is likely to have severe consequences which may lead to disorders including neurodegeneration and cancer. Ubiquitin protein ligases are a group of UPS proteins of particular importance because these proteins determine targeting specificity via recognition of a ‘target’ protein and its' subsequent ‘tagging’ with ubiquitin. The 26S proteasome recognises these mutli-ubiquitylated proteins, allowing the correct protein to be degraded at the correct time and place within each cell. Several types of ubiquitin protein ligase have now been identified, however, the largest group by far are those proteins containing a ‘RING’ motif. In this review, examples will be given whereby abnormal protein ubiquitylation due to absence or inefficiency of a RING protein ligase is proposed to be a key regulator of the disease process. Ways in which we may be able to reverse these effects or manipulate these proteins to restore function will be discussed.

The RING (Really Interesting New Gene) family is the largest type of E3 ubiquitin ligases. RING finger domains bind two zinc ions in a unique “cross-brace” arrangement through a defined motif of cysteine and histidine residues. This arrangement endows the RING domain with a globular conformation, characterized by a central α-helix and variable-length loops separated by several small β-strands. RING E3 ubiquitin ligases, play an essential role in the regulation of many biologic processes and defects in some of them are involved in cancer development. Furthermore, some RING E3 ligases are frequently overexpressed in human cancers. Today, RING ligases represent potentially molecular targets for disease intervention and could act as prognostic biomarkers. Targeting specific RING E3 ligases could lead to the development of a novel class of anticancer drugs. However RING fingers exhibit remarkable variations in their sequence and their topology characteristics. Structure determination of new RING finger domain is in the core of the design of new pharmaceuticals and what is presented in this article is a thorough review of achievements on the NMR or Xray structure determinations. Protein preparation protocols along with analysis of the structural features of known RING finger are also presented.