Minocycline (7-dimethylamino-6-dimethyl-6-deoxytetracycline) is a second-generation tetracycline that can cross the blood-brain barrier and has anti-inflammatory and neuroprotective effects. The potential of minocycline as a drug for treating Huntington's disease has been studied; however, the molecular mechanism underlying the neuroprotective properties of minocycline remains elusive. In this study, we tested the hypothesis that a principal cellular target of minocycline is Apaf-1, a key protein in the formation of the apoptosome, a multiprotein complex involved in caspase activation. Minocycline binds to Apaf-1, as shown by nuclear magnetic resonance spectroscopy, and inhibits apoptosome activity in vitro and in ex vivo models. As a consequence, minocycline-treated cells as well as Apaf-1 knock-out cells are resistant to the development of mutant huntingtin-dependent protein aggregation.

ABSTRACT:The majority of snakebite envenomations in Central America are caused by the viperid species Bothrops asper, whose venom contains a high proportion of zinc-dependent metalloproteinases that play a relevant role in the pathogenesis of hemorrhage characteristic of these envenomations. Broad metalloproteinase inhibitors, such as the peptidomimetic hydroxamate Batimastat, have been shown to inhibit snake venom metalloproteinases (SVMP). However, the difficulty in having open public access to Batimastat and similar molecules highlights the need to design new inhibitors of SVMPs that could be applied in the treatment of snakebite envenomations. We have chosen the SVMP BaP1 as a model to search for new inhibitors using different strategies, that is, screening of the Prestwick Chemical Library and rational peptide design. Results from these approaches provide clues on the structural requirements for efficient BaP1 inhibition and pave the way for the design of new inhibitors of SVMP. KEYWORDS: BaP1, metalloproteinase inhibitors, protein docking, snake venom metalloproteinases T he treatment of snakebite envenomations is based on the parenteral administration of animal-derived antivenoms, 1 which have proved highly effective in the neutralization of systemic effects induced by snake venoms; however, they are only partially effective in abrogating the local pathological alterations induced by viperid snake venoms. 2 This is in part due to the very rapid onset of these effects, associated with the delay in reaching health centers where antivenoms are available. 3 Local pathological alterations induced by viperid snake venoms are predominantly due to the action of hemorrhagic zinc-dependent metalloproteinases (SVMP) and myotoxic phospholipases A 2 (PLA 2 ). 2 Bothrops asper metalloproteinase P1 (BaP1) is a representative member of the SVMP family. In the high resolution structure of BaP1, as well as in matrix metalloproteinases (MMP), a Zn 2+ ion is coordinated by a tri(histidine) motif, which is critical for substrate binding and cleavage. 4−9 Most MMP inhibitors to date developed consist of a zinc-binding group (ZBG), which binds the catalytic metal ion, 5,8,10 and a peptidomimetic backbone, which interacts noncovalently with the active site of the enzyme. 7,11 The peptidomimetic Batimastat (BB-94) is a first generation MMP inhibitor that contains the most common ZBG, that is, a hydroxamate moiety.Because of the difficulty in neutralizing locally acting SVMPs by antivenoms, the possibility has been raised that specific enzyme inhibitors may represent a new alternative for the treatment of these envenomations. 12 At the experimental level, it has been shown that chelating agents, such as EDTA salts, as well as Batimastat, are effective at inhibiting both the isolated SVMPs and the hemorrhagic activity of crude viperid venoms in animal models, 13,14 underscoring the potential therapeutic value of such inhibitors in this pathology. Nevertheless, the public access to metalloproteinase inhibitors designed by the pharma...

BackgroundApaf1 (apoptotic protease activating factor 1) is the central component of the apoptosome, a multiprotein complex that activates procaspase-9 after cytochrome c release from the mitochondria in the intrinsic pathway of apoptosis. Other cellular roles, including a pro-survival role, have also been described for Apaf1, while the relative contribution of each function to cell death, but also to cell homeostatic conditions, remain to be clarified.Methodology and Principal FindingsHere we examined the response to apoptosis induction of available embryonic fibroblasts from Apaf1 knockout mice (MEFS KO Apaf1). In the absence of Apaf1, cells showed mitochondria with an altered morphology that affects cytochrome c release and basal metabolic status.ConclusionsWe analysed mitochondrial features and cell death response to etoposide and ABT-737 in two different Apaf1-deficient MEFS, which differ in the immortalisation protocol. Unexpectedly, MEFS KO Apaf1 immortalised with the SV40 antigen (SV40IM-MEFS Apaf1) and those which spontaneously immortalised (SIM-MEFS Apaf1) respond differently to apoptotic stimuli, but both presented relevant differences at the mitochondria when compared to MEFS WT, indicating a role for Apaf1 at the mitochondria.

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