Australian researchers have developed the first potent new small-molecule inhibitors capable of blocking the activation of apoptotic cell death before it causes damage to mitochondria, they reported in a study published in the Oct. 7, 2019, issue of Nature Chemical Biology.
Those first-in-class inhibitors will be useful tools for evaluating the mechanisms underlying apoptosis, assessing the impact of the pharmacological blockade of apoptosis in experimental models and potentially have multiple clinical indications.
"Such inhibitors could potentially be applied to indications where excessive or undesired apoptosis contributes to the pathology of disease," said study first author Mark van Delft, a senior postdoctoral scientist in the Blood Cells and Blood Cancer Division at The Walter and Eliza Hall Institute of Medical Research (WEHI) in Melbourne, Australia.
"Examples include ischemia reperfusion and acute traumatic tissue injuries, neurodegenerative conditions including Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis, and ocular diseases such as retinal detachment and glaucoma," said van Delft.
"At this stage, however, it remains unclear where the potential is greatest, because such inhibitors have been unavailable, so our new inhibitors will allow us to begin exploring this important question in mouse models."
Apoptosis is recognized as being essential for tissue homeostasis, whereas its deregulation is known to underlie many diseases, including cancer, which has spurred numerous drug development efforts.
Those efforts have resulted in progress in inducing apoptosis in tumor cells. For example, the anti-apoptotic B-cell lymphoma-2 (Bcl-2) protein inhibitor Venclexta (venetoclax, Abbvie Inc.) has high efficacy in chronic lymphocytic leukemia patients and has been approved by the U.S. FDA for that indication.
Unfortunately, attempts to prevent loss of healthy cells by blocking apoptosis, for example following ischemic stroke or organ transplantation, have been less successful.
Much research in that regard has been focused on targeting the caspase protease enzymes mediating many aspects of apoptotic cell death. However, pro-apoptotic caspases are now known mainly to promote rapid clearance of dying cells and prevent damage-associated molecular signaling; i.e. they accelerate, but are not mandatory for, cell death.
Consequently, clinical trials with caspase inhibitors have been disappointing, prompting researchers to investigate whether a more effective strategy would be to block apoptosis pharmacologically prior to caspase activation.
The two essential mediators of intrinsic mitochondrial apoptosis are the Bcl-2-associated X (BAX) and Bcl-2 antagonist killer (BAK) nuclear encoded proteins.
BAX and BAK are normally restrained by other Bcl-2 proteins, but stress signals activate yet another Bcl-2 family member, which initiates apoptosis by inhibiting pro-survival proteins and activating BAX/BAK, resulting in mitochondrial outer membrane permeabilization.
The effects of BAK inhibition on apoptosis were investigated in the new study, to which professors Benjamin Kile, head of Anatomy and Developmental Biology at Monash University, and Guillaume Lessene and David Huang, respective laboratory heads at WEHI and at the Department of Pharmacology and Therapeutics and Department of Medical Biology at the University of Melbourne, contributed equally.
The researchers demonstrated that WEHI-9625, a novel tricyclic sulfone small molecule, bound to voltage-dependent anion-selective channel protein 2 (VDAC2), which allows ions to transit between mitochondria and the cytoplasm, and showed it could inhibit BAK-driven apoptosis.
"We identified the starting point small molecule for this research via high-throughput screening at the WEHI National Drug Discovery Centre, with WEHI-9625 being developed from this starting point through extensive medicinal chemistry over seven years," explained Lessene.
"VDAC2 is known to regulate apoptosis through interactions with BAX and BAK, but its exact role has been controversial. Our study provides strong evidence that stabilizing the interaction between VDAC2 and BAK blocks BAK activation and therefore the apoptosis mediated by this protein," he said.
"WEHI-9625 potently inhibited the mouse form of BAK, but not the human form of the protein, making it a potentially useful tool for proof-of-concept studies in mouse models and a starting point for developing inhibitors for use in humans."
Importantly, unlike caspase inhibitors, WEHI-9625 was shown to block apoptosis before mitochondrial damage occurred, preserving cell function and long-term cloning potential.
"Ours and other recent studies have found that once mitochondria are damaged, cell death is inevitable, whereas if caspases are blocked, cell death is delayed but not prevented," noted Lessene.
"We show that inhibiting apoptosis before mitochondrial damage enables cells to remain viable and to grow and divide normally," he told BioWorld.
Those findings improve understanding of the key role played by VDAC2 in regulating apoptosis and demonstrate that blocking apoptosis at an early stage is both advantageous and pharmacologically controllable.
Regarding drug development, said Lessene, "WEHI-9625 is a proof-of-concept molecule that works specifically in mouse cells and, as such, will not be progressed to the clinic for the treatment of human diseases.
"Although we are currently developing apoptosis inhibitors that work in human cells, applications aimed at preservation of healthy tissue following injury in cardiovascular, ocular or neurodegenerative conditions in humans remain years ahead," he said.
In the meantime, "the next phase of our research will be to develop small molecules that are potent inhibitors of apoptosis mediated by human BAK and BAX. Our work will also contribute to determining which diseases are best managed by such new medicines."