Dissecting Programmed Cell Death with Small Molecules
Yingjie Bai, Hiu C. Lam, and Xiaoguang Lei*
CONSPECTUS:
Programmed cell death (PCD) is fundamentally an indispensable process in all cellular activities, including cell develop- ment, wound healing, and immune surveillance of tumors (Galluzzi, L. et al. Cell Death Differ. 2018, 25, 486−541). Malfunctioning of PCD has been shown to be closely related to human diseases such as acute pancreatitis, neurodegenerative diseases, and diverse types of cancers. To date, multiple PCD processes have been discovered and the corresponding regulatory pathways have been elucidated. For example, apoptosis and autophagy are two PCD mechanisms that have been well studied by sophisticated models and probe toolkits. However, limited genetic and chemical tools for other types of PCD hamper the elucidation of their molecular mechanisms. Our group has been studying PCD using both function-oriented synthesis and chemical biology strategies, including the development of diverse chemical probes based on novel PCD modulators. For instance, in the development of downstream programmed necrosis (or necroptosis) inhibitor necrosulfonamide, we used a chemical probe to unveil a functional protein that was not previously implicated in necroptosis, miXed lineage kinase domain-like protein (MLKL). In addition, high throughput screening and medicinal chemistry enabled the discovery of bioymifi, a small molecule agonist which selectively causes oligomerization of the death receptor 5 (DR5), to induce extrinsic apoptosis. Furthermore, we developed a biomimetic synthetic strategy based on diverse Diels−Alder reactions in the total syntheses of ainsliadimers A and B, ainsliatrimers A and B, and gonchnatiolides A−C, which are natural product inhibitors or activators for PCD. Using synthetic ainsliadimer A probe, we elucidated that ainsliadimer A inhibits the NF-κB pathway by covalently binding to Cys46 of IKKβ and triggers apoptosis of cancer cells. We have also revealed that IKKβ is allosterically inhibited by ainsliadimer A. In addition to total synthesis, we have developed a bioorthogonal click hetero-Diels−Alder cycloaddition of vinyl thioether and o-quinolinone quinone methide (TQ-ligation) to facilitate small molecule target identification. The combination of total synthesis and TQ-ligation enables subcellular imaging and identification of the cellular target of ainsliatrimer A to be PPARγ. In addition, TQ-ligation has been applied in the discovery of heat shock protein 90 (HSP90) as one of the functional target proteins for kongensin A. We also confirmed that kongensin A covalently attaches to Cys420 within HSP90 and demonstrated that kongensin A blocks the interaction between HSP90 and CDC37 and subsequently inhibits necroptosis. Our development of these diverse PCD modulators provides not only effective chemical tools for fundamental biomedical research, but also the foundation for drug discovery targeting important human diseases such as cancers and inflammation caused by malfunction of PCD.
■ INTRODUCTION
Programmed cell death (PCD) can be classified into apoptosis and nonapoptotic PCDs based on the nature of ubiquitous causes and outcome of cell death. Apoptosis, the prototype PCD, was first described by Kerr et al. in 1972,1 characterized by its morphological changes, unique biochemistry, and lack of inflammation.1−4 Besides apoptosis, typical nonapoptotic PCDs including autophagy,5 necroptosis,6 ferroptosis,7 and pyroptosis,8 have been discovered recently.9,10 In addition to PCDs, regulated cell death (RCD) has emerged to extend the definition of PCDs to include the promiscuous cell death pathways.9 In 2018, the Nomenclature Committee on Cell Death accepted MPT-driven necrosis, NETotic cell deaths,11 parthanatos, entotic cell death, and mitotic catastrophe as RCD pathways.9 Since the characterization of novel PCD-related signaling pathways has not yet been completed, space remains for component characterizations and development of small molecule modulators for PCDs.
Indeed, recent discoveries of PCDs (e.g., ferroptosis9 and oncosis12) were initiated from their corresponding inhibitors or PCD pathway components via chemical screenings using cellular phenotypes as readouts. However, the target identification of these small molecule inhibitors posed a challenge. Bioorthogonal reactions have been broadly applied in bioimaging and target identifications.13,14 This methodology might be applied in the study of PCDs with the corresponding modulators by attaching chemical handles for various purposes (e.g., attachment of biotin for affinity purification or fluorescence for visualization). The rapid reaction time, high spatiotemporal resolution, unique reactivity, and high specific- ity features of these chemical biological tools are comple- mentary to conventional studies in biochemistry and genetics. Therefore, despite the fact that the reagents might be somewhat toXic,15 bioorthogonal reactions have been currently applied in many research areas including microbiology, oncology, developmental biology, and neurobiology.14,16−19 Herein, we summarize our efforts in revealing the mechanisms of PCDs using chemical biology approaches with various small molecule probes and bioorthogonal reactions which were developed in our laboratory (Figure 1). First, we will discuss our chemical biology studies which led to the discovery of PCD modulators necrosulfonamide and bioymifi. Next, we present our endeavors using natural product total synthesis and subsequent target identification of PCD- inducers ainsliadimer A and ainsliatrimer A. Moreover, we introduce the combination of biomimetic synthesis and TQ- ligation that facilitates the target identifications of these PCD modulators. Ultimately, the research summarized in this Account alongside other studies of PCDs from different research groups5,7,20 has collectively contributed to stimulate fundamental biological discoveries and develop potential therapeutics for PCD-related human diseases such as cancers and autoimmune diseases.
DISSECTING NECROPTOSIS WITH NECROSULFONAMIDE
Necroptosis was first introduced by Yuan and co-workers in 2006, along with an antinecroptosis compound named as necrostatin-1 (Nec-1) which targets receptor-interacting protein 1 (RIPK1).6 Subsequently, multiple antinecroptosis compounds targeting RIPK1 or RIPK3 have been identified.21 It has been shown that RIPK3 can phosphorylate itself and its partner RIPK1, within the necrosome, an alternative name for the RIPK1/RIPK3 complex.21 However, the biological consequences of the phosphorylation related to necroptosis and the necroptotic downstream substrate(s) of RIPK1/ RIPK3 complex remained elusive. Therefore, we initiated a program in further deciphering the molecular pathway of necroptosis. We used a cell-based assay of necroptosis for high- throughput screening to discover antinecroptosis small molecules. Two previously established cell lines, Jurkat and HT-29 cells, were each treated with a combination of TNF-α to initiate programmed cell death, a cell-permeable small molecule that mimics Smac function (Smac mimetic) to enhance the cell death signal, and the pan-caspase inhibitor z- VAD-fmk to block apoptosis and initiate necroposis.22,23 After screening ∼200 000 compounds using this cell-based necroptosis assay, we identified a sulfonylaminopyrimidine hit as a necroptosis inhibitor with an IC50 of less than 1 μM. Shortly after structure−activity relationship (SAR) study, we discov- ered a more potent derivative, with an IC50 of 124 nM, which we named as necrosulfonamide. Subsequent cell-based bio- logical evaluations of how necrosulfonamide inhibits necrop- tosis were performed, and the results showed that the formation of RIPK3 punctae in the necrosulfonamide treated cells was significantly different from the one of Nec-1 treated cells. These results suggested that the cellular target(s) and mode of action for necrosulfonamide might be different from Nec-1 (Figure 2A).24
We then conducted co-immunoprecipitation experiments with necrosulfonamide to examine the possible RIPK3- interacting proteins. Indeed, pull down experiments and subsequent mass spectrometry analysis revealed that the miXed lineage kinase-domain like protein (MLKL) is an interacting partner of RIPK3 (Figure 2B). MLKL was later confirmed to be a crucial new component of necrosome, the RIPK1/RIPK3-containing complex that promotes necroptosis. The inhibition of necroptosis mediated by siRNA knockdown of MLKL further demonstrated that MLKL is indispensable for the necroptosis pathway. Therefore, our search for RIPK3 downstream inhibitors and the subsequent discovery of necrosulfonamide have revealed that MLKL as one of the central and essential components in the necroptosis process (Figure 2D).24
After the functional study of MLKL, we then proposed a hypothesis that MLKL might be the direct cellular target of necrosulfonamide. Therefore, we designed and synthesized a necrosulfonamide probe for the pull-down assay. From our SAR studies, we have gained crucial information about the heterocyclic moiety of necrosulfonamide could tolerate modifications without compromising its biological activity. Then a small molecule probe was prepared which contained a biotin tag, and a rigid polyproline linker between biotin and necrosulfonamide to enhance the binding activity of the probe to the potential target proteins (Figure 2C). Upon the treatment of RIP3-overexpressing HeLa cell lysates with this probe, a subsequent pull-down assay by streptavidin yielded endogenous MLKL, which could be competed off by the addition of excess necrosulfonamide as a binding competitor. Mutagenesis experiments also revealed that the C86S mutant of MLKL still mediated necroptosis despite the addition of necrosulfonamide, suggesting that the Cys86 residue of MLKL is α,β-unsaturated amide group might act as a Michael acceptor that covalently attaches to MLKL. Collectively, these data revealed that MLKL plays a significant role in initiating necroptosis and necrosulfonamide is a potent and selective small molecule inhibitor of necroptosis by covalently targeting MLKL.24
DISCOVERY OF THE FIRST DR5 AGONIST BIOYMIFI AS A POTENTIAL ANTICANCER AGENT
Because selective activation of specific apoptotic pathway has been proven to be an effective anticancer strategy, we were interested in searching for a compound to activate extrinsic apoptosis, which is an apoptotic pathway initiated by death receptors.25,26 By screening over 200 000 compounds combined with Smac mimetic in T98G cells, we discovered an apoptosis-stimulating hit called A1 (Figure 3A). However, our synthetic A1 was unfortunately not biologically active. After an extensive investigation, we revealed that the structure of “A1” compound from the commercial chemical library was wrongly assigned. The real composition of “A1” was a 1:9 miXture of A2 and C2 (Figure 3A). To our surprise, apoptosis was not induced when cells were treated with either A2 or C2 alone. Our attention then turned to de novo syntheses of many other analogues to further explore the SAR. Ultimately, we developed a bromine-substituted thiazolidin-4-one derivative, namely, bioymifi, which showed an improved activity with an EC50 at 2 μM for inducing apoptosis (Figure 3A).27
We then determined which apoptotic pathway was targeted by bioymifi.24 Our initial study showed that the pan-caspase inhibitor z-VAD-FMK rescued bioymifi-treated cells; therefore, we proposed that a caspase-dependent pathway would be involved in apoptosis. We excluded the intrinsic apoptotic pathway, as RNAi targeting caspase-9 did not improve cell survival after treatment with bioymifi. Moreover, biochemical studies showed that caspase-3, caspase-8, and PARP are cleaved consecutively after the treatment of bioymifi, suggesting bioymifi kills cancer cells via the extrinsic apoptotic pathway. We then hypothesized the target of bioymifi could be a death receptor. After screening all known members of death receptors by RNAi, we found that death receptor 5 (DR5) is a possible cellular target of bioymifi (Figure 3B). Further isothermal titration calorimetry (ITC) experiments illustrated that bioymifi reversibly binds to the ECD domain of DR5 with a Kd of 1.2 μM similar to its LC50 to cancer cells. From our studies, DR5 was found to be a functional cellular target of bioymifi to inducing extrinsic apoptosis.27
Since death receptors have been shown to oligomerize to activate the downstream apoptosis executors by the activation of TNFα,28 we wondered if bioymifi triggers a similar apoptosis-inducing mechanism. Therefore, full-length Flag- tagged DR5 from cells was treated with bioymifi, assayed with semi-denaturing detergent agarose gel electrophoresis (SDD- AGE), and followed by immunoblot analysis. The high molecule weight of DR5-Flag polymers indicated the aggregation of DR5 was induced by bioymifi or A2C2. In addition, aggregation of the purified DR5-ECD was observed after the treatment with bioymifi or A2C2, which concluded that ECD is required for aggregation. Subsequent immuno- fluorescence studies on DR5-overexpressed HCC115 tumor cells treated with bioymifi supported that bioymifi induces oligomerization of DR5 on the cellular level (Figure 3C). In conclusion, we have developed bioymifi as the first small molecule agonist for DR5 to trigger extrinsic apoptosis of cancer cells (Figure 3D).27
■ COLLECTIVE AND BIOMIMETIC SYNTHESES OF SESQUITERPENOID NATURAL PRODUCTS
A family of sesquiterpenoid oligomer natural products were isolated from Gochnatia and Ainslia plants. They have shown potent biological activity to induce apoptosis of various tumor cells, but the modes of action were unknown (Figure 4).29−32 We aimed to develop an efficient approach to access these complex natural products to facilitate the subsequent mode of action studies. Retrosynthetic analysis suggested that ainslia- dimer A might be derived from two molecules of guaianolide- type sesquiterpenoid dehydrozaluzanin C. Starting from the commercially available santonin, dehydrozaluzanin C was synthesized in 12 steps. After screening conditions for the hetero-Diels−Alder dimerization of dehydrozaluzanin C, BINOL was found to be the optimal catalyst in promoting the biomimetic dimerization. Hydrolysis of the alkenyl ether of the dimeric product, followed by cyclization via an intra- molecular aldol reaction furnished the total synthesis of ainsliadimer A.33
We then extended the synthesis and demonstrated that the similar biomimetic strategy could be applied for the syntheses of other sesquiterpenoid oligomers, including ainsliatrimers A and B, gochnatiolides A−C, and ainsliadimer B (Figure 4).34,35 The collective total syntheses of gochnatiolides A−C and ainsliadimer B were achieved in two steps and three steps, respectively, from dehydrozaluzanin C used as the common precursor. The biomimetic Diels−Alder reaction was applied as a key step under our optimized conditions to construct the polycyclic ring systems. The sesquiterpenoid trimers, ainslia- trimers A and B, were constructed in four steps via two sequential biomimetic Diels−Alder reactions from dehydroza- luzanin C. The first enantioselective syntheses also allowed the unambiguous confirmation of the chemical structure and absolute and relative configurations of these sesquiterpenoid oligomers such as gochnatiolide B and ainsliatrimers A and B. More importantly, this biomimetic strategy has accelerated the development of effective small molecule probes for further target identification of ainsliadimer A and ainsliatrimer A.33−36
■ MODE OF ACTION STUDY FOR AINSLIADIMER A
The biomimetic synthesis of ainsliadimer A has laid the foundation for our mechanistic study of the apoptosis-inducing activity of this natural product.33 First, we investigated which apoptotic pathways were triggered by ainsliadimer A. We found that the pan-caspase inhibitor z-VAD-FMK can rescue BCG-823 cells treated with ainsliadimer A, indicating the apoptosis pathway is involved. In addition, through real-time PCR analysis, we found that multiple apoptosis inhibitory A inhibits the phosphorylation of IκB to block the NF-κB pathway, which shares a comparable mechanism to the known IκB kinase (IKK) inhibitor BMS-345541 in both LPS and poly(I:C) induced models, suggesting ainsliadimer A might directly block the NF-κB signaling pathway.37
We further designed and synthesized an ainsliadimer A-downregulated, suggesting the regulatory proteins correspond- ing to their transcription factor pathway could be targeted.37 Subsequent investigation of the components in the nuclear factor-κB (NF-κB) signaling pathway showed that ainsliadimer derived chemical probe based on our SAR studies. This probe contains an ainsliadimer A warhead, a PEG linker, and a biotin tag. Given the fact that saturation of the α,β unsaturated lactones led to the loss of function for ainsliadimer A, we also constructed a negative control (NC) probe through selective reduction of the two conjugated alkene moieties for pull-down assay (Figure 5A). We then used these two probes in the pull- down experiments with cell lysates which were previously activated by TNFα treatments. Two specific protein bands, IKKα and IKKβ, were identified by mass spectrometry analysis (Figure 5B). To reveal the binding site of ainsliadimer A (1), we individually mutated the nine conserved cysteine residues of IKKβ into alanine residues (Figure 5C). Among these mutants, C46A was the sole mutant that lost the interaction with ainsliadimer A (1). Further LC-MS/MS analysis also confirmed that Cys46 residue of IKKβ is covalently attached by ainsliadimer A.37 Taken together, we have elucidated the detailed mode of action for ainsliadimer A. This remarkable natural product selectively and covalently targets IKKβ to activate the NF-κB pathway and trigger apoptosis of cancer cells.37
TQ-LIGATION FACILITATES NATURAL PRODUCT TARGET IDENTIFICATION
In order to facilitate natural product target identification, we have developed an efficient and robust bioorthogonal ligation (TQ-ligation) by “click” hetero-Diels−Alder cycloaddition of o-quinolinone quinone methide and vinyl thioether under physiological conditions (Figure 6A).38 The compatibility of TQ-ligation has been demonstrated in the imaging of taxol derivative in live cells.38 The second generation TQ-ligation was subsequently developed to improve the kinetic rate up to 18-fold compared to the prototype (Figure 6B).39 Moreover, the second generation TQ ligation is mutually orthogonal to the traditional strain-promoted azide−alkyne cyclization (SPAAC) both in vitro and in vivo.39,40 We also found that the second generation TQ ligation has a comparable rate constant to that of the widely used SPAAC (k2 = 7.6 × 10−2 M−1 s−1 in CH3CN).39 We further demonstrated that the TQ-ligation is an effective means to facilitate our target identifications of the apoptosis inducers such as ainsliatrimer A and kongensin A (Figure 6C).41,42 We will discuss these endeavors in details in the following sections.
■ AINSLIATRIMER A INDUCES APOPTOSIS BY ACTIVATING PPARγ
In the mode of action study of the apoptosis inducer ainsliatrimer A,41 we first synthesized a bioactive natural product probe in which a vinyl thioether was attached onto the 3″-O position of ainsliatrimer A (TV-ainsliatrimer A) (Figure 7A). The HeLa cells were treated with TV-ainsliatrimer A, followed by incubation with fluorescein-modified oQQM precursor which would undergo “click” hetero-Diels−Alder reaction with the vinyl thioether of TV-ainsliatrimer A, resulting a distribution of the fluorescent small molecule probes in cells. Pretarget imaging showed that fluorescence was visualized in the nucleus exclusively, suggesting the cellular targets of ainsliatrimer A should be located in the nucleus (Figure 7A, B). Next, we investigated which nuclear protein(s) might bind to ainsliatrimer A. We therefore synthesized the biotinylated ainsliatrimer A probe using the TQ-ligation strategy as well as the negative control probe from the saturated TV-ainsliatrimer A derivative. Treatment of HeLa cell lysates with these two probes, followed by pull-down assay using the extracted cell nuclear proteins and LC-MS/MS analysis, revealed that histone deacetylase 2 (HDAC2) and peroXisome proliferator activated receptor γ (PPARγ) as two candidate cellular targets (Figure 7D). siRNA knockdown at the protein level demonstrated that PPARγ knockdown could rescue cells treated with ainsliatrimer A while HDAC2 knockdown could not. Further validation experiments elucidated that ainsliatrimer A is a selective PPARγ agonist to trigger apoptosis.41 Along with our discovery, other independent studies also demonstrated that the activation of PPARγ could induce apoptosis.43,44 Therefore, the PPARγ agonists such as ainsliatrimer A could serve as a promising lead for anticancer therapy.
■ KONGENSIN A IS AN EFFECTIVE INHIBITOR OF NECROPTOSIS BY TARGETING HSP90
To continue to search for novel small molecules modulating programmed cell death such as necroptosis, we screened our in-house ∼300 000 small molecule library and discovered kongensin A (KA), a diterpenoid natural product isolated from Croton kongensis as a hit to block necroptosis.45 Initial validation assays showed that KA blocks necroptosis efficiently at 2 μM. To identify the cellular target of kongensin A, we first prepared a positive probe by attaching vinyl thioether onto KA, and a negative control probe from KA derivative (Figure 8A). HT-29 cells were incubated with the KA probe and NC probe, followed by the treatment of biotin-modified oQQM precursor for TQ-ligation. Pull-down assay followed by LC-MS/MS analysis revealed that one of the cellular targets for KA is heat shock protein 90 (HSP90). In addition, Cys420 residue was confirmed to be the binding site by LC-MS/MS analysis and site-directed mutagenesis (Figure 8B−D).42
Moreover, coimmunoprecipitation of HSP90 in kongensin A-treated HeLa lysates revealed that KA inhibits the protein− protein interaction between HSP90 and its co-chaperone cell division cycle protein 37 (CDC37), a protein which can stabilize multiple kinases such as CDK4.46 In addition, we further demonstrated that the HSP90−CDC37 complex is crucial for RIPK3-dependent necroptosis.47 In summary, we have elucidated the mode of action of KA: the covalent interaction of KA to Cys420 of HSP90 disrupts the formation of the HSP90−CDC37 complex and subsequently inhibits RIPK3-dependent necroptosis.42 This study provided a new means to develop a HSP90 inhibitor for the treatment of necroptosis-related diseases such as inflammation and atherosclerosis.48
■ CONCLUSION
Our laboratory conducts research at the interface between chemistry and biology. We systematically use bioactive small molecules including complex natural products to dissect fundamental biological process and to develop novel therapeutic agents for the currently intractable human diseases. Over the past decade, we have extensively utilized various small molecule probes to dissect the extrinsic programmed cell death pathways, including both apoptosis and programmed necrosis (necroptosis), and illuminated a number of new molecular mechanisms underlying these fundamental cellular processes. These discoveries not only significantly enriched our current understanding of programmed cell death mechanisms, but also led to the discovery of a number of potential drug leads for the treatment of cancers, ischemia-reperfusion injury, and auto- immune disorders, which are caused by dysfunction of the regulated cell death machinery.
Forward chemical genetics has been proven to be a very powerful approach to the discovery of new targets. However, the most significant challenge associated with this approach is identification of the functional targets of small-molecule probes. The great potential of chemical genetics cannot be realized without the establishment of efficient means for target identification. In this regard, synthetic chemistry plays an important role in the development of effective chemical probes, especially for the complex natural product derived probes. Many issues such as thorough SAR studies, the choice of labeling, and target ID methods should be carefully considered for the chemical probe design and development. Several leading chemical biologists have recently published a commentary describing the key properties of successful chemical probes.49 These include high potency, good selectivity, and the availability of appropriate control compounds. In our studies, we have inevitably applied many different approaches, including direct labeling with biotin tags, utilization of bioorthogonal ligation methods, or application of pretarget imaging. In our opinion, no unified method could be successfully applied in all cases. EXtensive evaluations of different approaches should be performed in order to obtain the most conclusive results. Overall, our work to date provides a model workflow for the transformation of bioactive small molecules including complex natural products into effective chemical probes and the identification of their functional protein targets.
In conclusion, we can certainly envision that the combined efforts of function-oriented synthesis and forward chemical genetics will continue to uncover the unknown molecular mechanisms of fundamental cellular process such as pro- grammed cell death to advance our understanding of human biology and provide new meaningful approaches to combat human diseases.
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