Related Experiment Video
Updated: Jan 28, 2026
An In Ovo Model for Testing Insulin-mimetic Compounds
Published on: April 23, 2018
Confounding off-target effects of BH3 mimetics at commonly used concentrations: MIM1, UMI-77, and A-1210477
David J Mallick1, Ryan S Soderquist2, Darcy Bates3
1Department of Molecular and Systems Biology, Geisel School of Medicine at Dartmouth, Lebanon, NH, 03756, USA.
Abstract:
Targeting anti-apoptotic BCL2 family proteins has become an attractive therapeutic strategy for many cancers, and the BCL2-selective inhibitor ABT-199 (venetoclax) has obtained clinical success. However, MCL1 can promote drug resistance and overall cancer cell survival. Thus, there is a critical need to develop an effective drug that antagonizes MCL1. However, most putative MCL1 inhibitors have been misclassified as they fail to directly inhibit MCL1 in cells, but rather induce the pro-apoptotic protein NOXA. We have investigated three putative MCL1 inhibitors: MIM1, UMI-77, and A-1210477. All three compounds were developed in cell-free assays and then found to be cytotoxic, and hence assumed to directly target MCL1 in cells. Here, we investigated whether these compounds directly inhibit MCL1 or inhibit MCL1 indirectly through the induction of NOXA. Both MIM1- and UMI-77-induced NOXA through the unfolded protein response pathway, and sensitized leukemia cells to ABT-199; this cytotoxicity was dependent on NOXA suggesting that these compounds do not directly target MCL1. A-1210477 was the only compound that did not induce NOXA, but it still sensitized cells to ABT-199. A-1210477 induced accumulation of MCL1 protein consistent with it binding and preventing MCL1 degradation. However, at concentrations used in several prior studies, A-1210477 also induced cytochrome c release, caspase activation, and apoptosis in a BAX/BAK-independent manner. Furthermore, the release of cytochrome c occurred without loss of mitochondrial membrane potential. This apoptosis was extremely rapid, sometimes occurring within 0.5-1 h. Hence, we have identified a novel mechanism of apoptosis that circumvents the known mechanisms of cytochrome c release. It remains to be determined whether these unexpected mechanisms of action of putative BH3 mimetics will have therapeutic potential.
Insights
Researchers investigated MCL1 inhibitors, finding MIM1 and UMI-77 indirectly kill cancer cells by inducing NOXA. A-1210477 showed a novel, rapid apoptosis pathway independent of BAX/BAK, offering potential therapeutic avenues.
Area of Science:
- Molecular Biology
- Cancer Therapeutics
- Drug Discovery
Background:
- Targeting anti-apoptotic BCL2 family proteins is a key cancer therapy strategy, with BCL2-selective inhibitor ABT-199 (venetoclax) showing clinical success.
- MCL1 is crucial for cancer cell survival and can drive drug resistance, necessitating the development of effective MCL1 inhibitors.
- Many purported MCL1 inhibitors have been misclassified, acting indirectly by inducing the pro-apoptotic protein NOXA rather than directly inhibiting MCL1.
Purpose of the Study:
- To investigate whether three putative MCL1 inhibitors (MIM1, UMI-77, and A-1210477) directly inhibit MCL1 or indirectly induce NOXA.
- To characterize the mechanism of action for compounds that sensitize cancer cells to ABT-199.
- To identify novel mechanisms of apoptosis induction for potential therapeutic development.
Main Methods:
- Assessed the effect of MIM1, UMI-77, and A-1210477 on NOXA induction and MCL1 inhibition in cancer cells.
- Utilized cell-free assays and cytotoxicity assessments to evaluate compound activity.
- Investigated apoptosis pathways, including cytochrome c release, caspase activation, and mitochondrial membrane potential, in response to A-1210477.
Main Results:
- MIM1 and UMI-77 induced NOXA via the unfolded protein response pathway, sensitizing leukemia cells to ABT-199 in a NOXA-dependent manner, indicating indirect MCL1 antagonism.
- A-1210477 did not induce NOXA but sensitized cells to ABT-199 by preventing MCL1 degradation.
- At higher concentrations, A-1210477 triggered rapid, BAX/BAK-independent apoptosis with cytochrome c release and caspase activation, bypassing canonical mitochondrial pathways.
Conclusions:
- MIM1 and UMI-77 function indirectly by upregulating NOXA, not by directly targeting MCL1.
- A-1210477 exhibits a novel, rapid apoptotic mechanism independent of BAX/BAK and mitochondrial membrane potential.
- The unique apoptotic pathway induced by A-1210477 warrants further investigation for its therapeutic potential in cancer treatment.
Related Concept Videos
01:20Radical Reactivity: Concentration Effects
01:27Confounding in Epidemiological Studies
03:24Common Ion Effect
01:25Strategies for Assessing and Addressing Confounding
Confounding can be addressed at both the design phase of a study and through analytical methods after data...
03:03Concentration and Rate Law
For example, in a generic reaction aA + bB ⟶ products, where a and b are stoichiometric coefficients, the rate law can be written as:
02:48Expressing Solution Concentration
Concentrations may be quantitatively assessed using a wide variety of measurement units, each convenient for particular applications. Molarity (M) is a useful concentration unit for many applications in chemistry.