Structure–activity relationships by interligand NOE-based design and synthesis of antiapoptotic compounds targeting Bid

Abstract

Bcl-2 family proteins play a crucial role in tissue homeostasis and apoptosis (programmed cell death). Bid is a proapoptotic member of the Bcl-2 family, promoting cell death when activated by caspase-8. Following an NMR-based approach (structure-activity relationships by interligand NOE) we were able to identify two chemical fragments that bind on the surface of Bid. Covalent linkage of the two fragments led to high-affinity bidentate derivatives. In vitro and in-cell assays demonstrate that the compounds prevent tBid translocation to the mitochondrial membrane and the subsequent release of proapoptotic stimuli and inhibit neuronal apoptosis in the low micromolar range. Therefore, by using a rational chemical-biology approach, we derived antiapoptotic compounds that may have a therapeutic potential for disorders associated with Bid activation, e.g., neurodegenerative diseases, cerebral ischemia, or brain trauma.apoptosis ͉ drug discovery ͉ neurodegeneration P rogrammed cell death (1-3) is a process associated with several pathologies such as neurodegenerative diseases, spinal cord injury, amyotrophic lateral sclerosis, and brain ischemia (4-6). Bid (7, 8) is a proapoptotic member of the Bcl-2 family proteins, and its altered expression accounts for onset and propagation of these pathologies (4-6). The sequence of events leading to programmed cell death has been well characterized in nonneuronal cells (1-3), where Bid provides one mechanism by which TNF-Fas family death receptor activation is linked to downstream events (9). These death receptors activate caspase-8, which cleaves Bid to its truncated active form, tBid (8). tBID targets the outer mitochondrial membrane and induces conformational changes in Bak and Bax (10), which results in the release of proapoptotic stimuli such as Smac and cytochrome c (11). Cytochrome c, together with APAF-1 and caspase-9, form the apoptosome complex, which results in the activation of caspase-3 and other effector caspases, which ultimately cause cell death (12).Recent studies clearly point to Bid as a mediator upstream of mitochondria in neuronal death after cerebral ischemia (13). By using an in vivo model of mild focal cerebral ischemia and an in vitro neuronal oxygen glucose deprivation (OGD) that favors apoptotic cell death, we showed that Bid is a critical mediator of ischemic cell death within the CNS (13). Reduced cell death was observed in highly enriched neuronal cultures from BidϪ͞Ϫ mice after OGD and reduced ischemic brain injury in mutant mice with a deletion in Bid. It was also found that both Bid and caspase-3 are activated in an ischemic brain and in cultured neurons after OGD.The results generated by using BidϪ͞Ϫ mice demonstrate convincingly that Bid plays a prominent role in acute CNS injury. Phenotypically, these mutant mice do not have any CNS developmental defect (11) or any difference in microscopic or macroscopic CNS morphologies, as compared with WT mice. It can be concluded that Bid promotes death in neurons after OGD in vitro and in the brain after f...

Inhibition of Drp1 provides neuroprotection in vitro and in vivo

“…5,19 The pivotal role for Bid upstream of such mitochondrial dysfunction is substantiated by experiments demonstrating pronounced neuroprotective effects of the Bid inhibitor BI-6c9 in models of glutamate-induced excitotoxicity and OGD in primary cultured neurons, and similar studies in HT-22 neurons using Bid siRNA. 19,20,30,31 Further, reduced Bid expression attenuated neuronal death in a model of OGD in vitro and reduced brain damage in models of cerebral ischemia and brain trauma in vivo. 21 Recent studies showed that tBid and full-length Bid exert similar effects on mitochondria and AIF-dependent cell death in neurons.…”

Bid-mediated mitochondrial damage is a key mechanism in glutamate-induced oxidative stress and AIF-dependent cell death in immortalized HT-22 hippocampal neurons

“…26 Similar neuroprotective results were obtained in cultured neurons from Bid-deficient mice when exposed to oxygen glucose deprivation (OGD). 24 Further, small-molecule inhibitors of Bid provided protective effects against glutamateinduced excitotoxicity or OGD in cultured primary neurons 10,27 and prevented mitochondrial demise, AIF release and cell death in HT-22 cells exposed to oxidative stress induced by glutamate or amyloid-beta peptide. 13 These different paradigms of lethal stress induce activation of Bid, which translocates to the mitochondria, where it mediates mitochondrial membrane permeabilization and release of death proteins such as cytochrome c or AIF.…”

Proapoptotic BH3-Only Protein Bid Is Essential For Death Receptor–Induced Apoptosis of Pancreatic β-Cells

“…Collectively, the observations that loss of Bid protects ␤-cells against FasL, TNF-␣, inflammatory cytokines (albeit only partially), and (at least in vitro) perforin plus granzyme B indicate that pharmacological inhibition of Bid may be able to prevent or limit apoptosis induced by multiple pathways without interfering with normal physiological ␤-cell function. Inhibitors targeted at Bid have been recently described (49), making this a feasible approach for preserving ␤-cell survival and prevention of diabetes.…”