J. David Becherer

The enzymatic activity of ADAM8 and ADAM9 is not regulated by TIMPs

The ADAM family of proteases are type I transmembrane proteins with both metalloproteinase and disintegrin containing extracellular domains. ADAMs are implicated in the proteolytic processing of membrane‐bound precursors and involved in modulating cell–cell and cell–matrix interactions. ADAM8 (MS2, CD156) has been identified in myeloid and B cells. In this report we demonstrate that soluble ADAM8 is an active metalloprotease in vitro and is able to hydrolyse myelin basic protein and a variety of peptide substrates based on the cleavage sites of membrane‐bound cytokines, growth factors and receptors which are known to be processed by metalloproteinases. Interestingly, although ADAM8 was inhibited by a number of peptide analogue hydroxamate inhibitors, it was not inhibited by the tissue inhibitors of metalloproteinases (TIMPs). We also demonstrate that the activity of recombinant soluble ADAM9 (meltrin‐γ, MDC9) lacks inhibition by the TIMPs, but can be inhibited by hydroxamate inhibitors. The lack of TIMP inhibition of ADAM8 and 9 contrasts with other membrane‐associated metalloproteinases characterised to date in this respect (ADAM10, 12, 17, and the membrane‐type metalloproteinases) which have been implicated in protein processing at the cell surface.

Biochemical properties and functions of membrane-anchored metalloprotease-disintegrin proteins (ADAMs)

Publisher Summary Since their discovery about a decade ago, some very intriguing insights into the function of a few Anchored Metalloprotease-Disintegrin Proteins (ADAMs) have emerged. They have been linked to a role in cell–cell interactions as potential integrin ligands and in the cleavage and release and, in several cases, activation of membrane-anchored substrates. Yet much still remains to be discovered about the role of other ADAMs in cell–cell interaction, in protein ectodomain shedding, in processing of other molecules on the cell surface or in the extracellular matrix, or in signaling. From a medical point of view, it will be interesting to learn more about the role of ADAMs in diseases such as cancer and autoimmune disorders. Other potential functions in cell adhesion and signaling must also be taken into consideration. Given the insights that have already emerged from studies of TNFα converting enzyme (TACE) as a prototypical ADAM, similar approaches to characterizing other less well defined ADAMs are expected to yield interesting information about the roles of these proteins in normal homeostasis and in pathophysiology.

Discovery, Synthesis, and Biological Evaluation of Thiazoloquin(az)olin(on)es as Potent CD38 Inhibitors

A series of thiazoloquin(az)olinones were synthesized and found to have potent inhibitory activity against CD38. Several of these compounds were also shown to have good pharmacokinetic properties and demonstrated the ability to elevate NAD levels in plasma, liver, and muscle tissue. In particular, compound 78c was given to diet induced obese (DIO) C57Bl6 mice, elevating NAD > 5-fold in liver and >1.2-fold in muscle versus control animals at a 2 h time point. The compounds described herein possess the most potent CD38 inhibitory activity of any small molecules described in the literature to date. The inhibitors should allow for a more detailed assessment of how NAD elevation via CD38 inhibition affects physiology in NAD deficient states.

The C-terminal domains of TACE weaken the inhibitory action of N-TIMP-3

Tumor necrosis factor‐α converting enzyme (TACE) is an ADAM ( isintegrin nd etalloproteinases) that comprises an active catalytic domain and several C‐terminal domains. We compare the binding affinity and association rate constants of the N‐terminal domain form of wild‐type tissue inhibitor of metalloproteinase (TIMP‐3; N‐TIMP‐3) and its mutants against full‐length recombinant TACE and the truncated form of its catalytic domain. We show that the C‐terminal domains of TACE substantially weaken the inhibitory action of N‐TIMP‐3. Further probing with hydroxamate inhibitors indicates that both forms of TACE have similar active site configurations. Our findings highlight the potential role of the C‐terminal domains of ADAM proteinases in influencing TIMP interactions.

N-hydroxyformamide peptidomimetics as TACE/matrix metalloprotease inhibitors: oral activity via P1' isobutyl substitution.

N-Hydroxyformamide-class metalloprotease inhibitors were designed and synthesized, which have potent broad-spectrum activity versus matrix metalloproteases and TNF-alpha converting enzyme (TACE). Compound 13c possesses good oral and intravenous pharmacokinetics in the rat and dog.

Pyrrolidinyl pyridone and pyrazinone analogues as potent inhibitors of prolyl oligopeptidase (POP)

We report the synthesis and in vitro activity of a series of novel pyrrolidinyl pyridones and pyrazinones as potent inhibitors of prolyl oligopeptidase (POP). Within this series, compound 39 was co-crystallized within the catalytic site of a human chimeric POP protein which provided a more detailed understanding of how these inhibitors interacted with the key residues within the catalytic pocket.

Genetic Ablation of CD38 Protects against Western Diet-Induced Exercise Intolerance and Metabolic Inflexibility

Nicotinamide adenine dinucleotide (NAD+) is a key cofactor required for essential metabolic oxidation-reduction reactions. It also regulates various cellular activities, including gene expression, signaling, DNA repair and calcium homeostasis. Intracellular NAD+ levels are tightly regulated and often respond rapidly to nutritional and environmental changes. Numerous studies indicate that elevating NAD+ may be therapeutically beneficial in the context of numerous diseases. However, the role of NAD+ on skeletal muscle exercise performance is poorly understood. CD38, a multi-functional membrane receptor and enzyme, consumes NAD+ to generate products such as cyclic-ADP-ribose. CD38 knockout mice show elevated tissue and blood NAD+ level. Chronic feeding of high-fat, high-sucrose diet to wild type mice leads to exercise intolerance and reduced metabolic flexibility. Loss of CD38 by genetic mutation protects mice from diet-induced metabolic deficit. These animal model results suggest that elevation of tissue NAD+ through genetic ablation of CD38 can profoundly alter energy homeostasis in animals that are maintained on a calorically-excessive Western diet.

TNFα converting enzyme

Tumor necrosis factor a (TNFα) is a pleiotropic cytokine that mediates inflammatory and apoptotic processes by binding to two different receptors and thereby initiating complex signaling transduction pathways [1]. While TNFa has been studied since the earlier part of this century, the purification and cloning of this protein in 1985 [2 – 5] precipitated a decade of intensive research. These efforts demonstrated the cross-disciplinary significance of this molecule and gave insight into the signaling pathways that initiated TNFα transcription, the unique control of its translation and the TNF receptors that bind the secreted, 17 kDa molecule. These receptors reside on cells of nearly every tissue and, in turn, transduce the signals that result in changes in cell behavior.

A high throughput assay for the TNF converting enzyme

Tumor necrosis factor-α (TNFα) has been implicated in cancer and inflammatory diseases since it was first characterized and eventually identified by researchers in several laboratories in the mid-1980s [1-5]. However, only recently has the patho-logical role of TNF in arthritis and Crohn’s disease been demonstrated in the clinic with the FDA’s approval of Enbrel and Remicade [6-8]. The recent success of these biological agents that neutralize TNF has led to intense efforts to find small molecule TNF antagonists that will mimic the consequences, if not the mechanism, of these agents. Because TNF interacts at multiple contact points with either of its two receptors, researchers have struggled to find small molecular weight inhibitors that antagonize this interaction. Therefore, most efforts have focused on targets upstream of TNF synthesis or secretion and downstream of TNF receptor engagement since these targets appear more amenable to modulation by small molecular weight inhibitors [9].