Modular synthesis of functional libraries by accelerated SuFEx click chemistry.

Accelerated SuFEx Click Chemistry (ASCC) is a powerful method for coupling aryl and alkyl alcohols with SuFEx-compatible functional groups. With its hallmark favorable kinetics and exceptional product yields, ASCC streamlines the synthetic workflow, simplifies the purification process, and is ideally suited for discovering functional molecules. We showcase the versatility and practicality of the ASCC reaction as a tool for the late-stage derivatization of bioactive molecules and in the array synthesis of sulfonate-linked, high-potency, microtubule targeting agents (MTAs) that exhibit nanomolar anticancer activity against multidrug-resistant cancer cell lines. These findings underscore ASCC's promise as a robust platform for drug discovery.

BCL-XL inhibitors enhance the apoptotic efficacy of BRAF inhibitors in BRAFV600E colorectal cancer.

Metastatic BRAFV600E colorectal cancer (CRC) carries an extremely poor prognosis and is in urgent need of effective new treatments. While the BRAFV600E inhibitor encorafenib in combination with the EGFR inhibitor cetuximab (Enc+Cet) was recently approved for this indication, overall survival is only increased by 3.6 months and objective responses are observed in only 20% of patients. We have found that a limitation of Enc+Cet treatment is the failure to efficiently induce apoptosis in BRAFV600E CRCs, despite inducing expression of the pro-apoptotic protein BIM and repressing expression of the pro-survival protein MCL-1. Here, we show that BRAFV600E CRCs express high basal levels of the pro-survival proteins MCL-1 and BCL-XL, and that combining encorafenib with a BCL-XL inhibitor significantly enhances apoptosis in BRAFV600E CRC cell lines. This effect was partially dependent on the induction of BIM, as BIM deletion markedly attenuated BRAF plus BCL-XL inhibitor-induced apoptosis. As thrombocytopenia is an established on-target toxicity of BCL-XL inhibition, we also examined the effect of combining encorafenib with the BCL-XL -targeting PROTAC DT2216, and the novel BCL-2/BCL-XL inhibitor dendrimer conjugate AZD0466. Combining encorafenib with DT2216 significantly increased apoptosis induction in vitro, while combining encorafenib with AZD0466 was well tolerated in mice and further reduced growth of BRAFV600E CRC xenografts compared to either agent alone. Collectively, these findings demonstrate that combined BRAF and BCL-XL inhibition significantly enhances apoptosis in pre-clinical models of BRAFV600E CRC and is a combination regimen worthy of clinical investigation to improve outcomes for these patients.

BECLIN1 is essential for intestinal homeostasis involving autophagy-independent mechanisms through its function in endocytic trafficking.

Autophagy-related genes have been closely associated with intestinal homeostasis. BECLIN1 is a component of Class III phosphatidylinositol 3-kinase complexes that orchestrate autophagy initiation and endocytic trafficking. Here we show intestinal epithelium-specific BECLIN1 deletion in adult mice leads to rapid fatal enteritis with compromised gut barrier integrity, highlighting its intrinsic critical role in gut maintenance. BECLIN1-deficient intestinal epithelial cells exhibit extensive apoptosis, impaired autophagy, and stressed endoplasmic reticulum and mitochondria. Remaining absorptive enterocytes and secretory cells display morphological abnormalities. Deletion of the autophagy regulator, ATG7, fails to elicit similar effects, suggesting additional novel autophagy-independent functions of BECLIN1 distinct from ATG7. Indeed, organoids derived from BECLIN1 KO mice show E-CADHERIN mislocalisation associated with abnormalities in the endocytic trafficking pathway. This provides a mechanism linking endocytic trafficking mediated by BECLIN1 and loss of intestinal barrier integrity. Our findings establish an indispensable role of BECLIN1 in maintaining mammalian intestinal homeostasis and uncover its involvement in endocytic trafficking in this process. Hence, this study has important implications for our understanding of intestinal pathophysiology.

Differential membrane binding of α/ß-peptide foldamers: implications for cellular delivery and mitochondrial targeting.

The intrinsic pathway of apoptosis is regulated by the Bcl-2 family of proteins. Inhibition of the anti-apoptotic members represents a strategy to induce apoptotic cell death in cancer cells. We have measured the membrane binding properties of a series of peptides, including modified α/ß-peptides, designed to exhibit enhanced membrane permeability to allow cell entry and improved access for engagement of Bcl-2 family members. The peptide cargo is based on the pro-apoptotic protein Bim, which interacts with all anti-apoptotic proteins to initiate apoptosis. The α/ß-peptides contained cyclic ß-amino acid residues designed to increase their stability and membrane-permeability. Dual polarisation interferometry was used to study the binding of each peptide to two different model membrane systems designed to mimic either the plasma membrane or the outer mitochondrial membrane. The impact of each peptide on the model membrane structure was also investigated, and the results demonstrated that the modified peptides had increased affinity for the mitochondrial membrane and significantly altered the structure of the bilayer. The results also showed that the presence of an RRR motif significantly enhanced the ability of the peptides to bind to and insert into the mitochondrial membrane mimic, and provide insights into the role of selective membrane targeting of peptides.

The emerging roles of autophagy in intestinal epithelial cells and its links to inflammatory bowel disease.

Landmark genome-wide association studies (GWAS) identified that mutations in autophagy genes correlated with inflammatory bowel disease (IBD), a heterogenous disease characterised by prolonged inflammation of the gastrointestinal tract, that can reduce a person's quality of life. Autophagy, the delivery of intracellular components to the lysosome for degradation, is a critical cellular housekeeping process that removes damaged proteins and turns over organelles, recycling their amino acids and other constituents to supply cells with energy and necessary building blocks. This occurs under both basal and challenging conditions such as nutrient deprivation. An understanding of the relationship between autophagy, intestinal health and IBD aetiology has improved over time, with autophagy having a verified role in the intestinal epithelium and immune cells. Here, we discuss research that has led to an understanding that autophagy genes, including ATG16L, ATG5, ATG7, IRGM, and Class III PI3K complex members, contribute to innate immune defence in intestinal epithelial cells (IECs) via selective autophagy of bacteria (xenophagy), how autophagy contributes to the regulation of the intestinal barrier via cell junctional proteins, and the critical role of autophagy genes in intestinal epithelial secretory subpopulations, namely Paneth and goblet cells. We also discuss how intestinal stem cells can utilise autophagy. Importantly, mouse studies have provided evidence that autophagy deregulation has serious physiological consequences including IEC death and intestinal inflammation. Thus, autophagy is now established as a key regulator of intestinal homeostasis. Further research into how its cytoprotective mechanisms can prevent intestinal inflammation may provide insights into the effective management of IBD.

Genotype-Tailored ERK/MAPK Pathway and HDAC Inhibition Rewires the Apoptotic Rheostat to Trigger Colorectal Cancer Cell Death.

The EGFR/RAS/MEK/ERK signaling pathway (ERK/MAPK) is hyperactivated in most colorectal cancers. A current limitation of inhibitors of this pathway is that they primarily induce cytostatic effects in colorectal cancer cells. Nevertheless, these drugs do induce expression of proapoptotic factors, suggesting they may prime colorectal cancer cells to undergo apoptosis. As histone deacetylase inhibitors (HDACis) induce expression of multiple proapoptotic proteins, we examined whether they could synergize with ERK/MAPK inhibitors to trigger colorectal cancer cell apoptosis. Combined MEK/ERK and HDAC inhibition synergistically induced apoptosis in colorectal cancer cell lines and patient-derived tumor organoids in vitro, and attenuated Apc-initiated adenoma formation in vivo. Mechanistically, combined MAPK/HDAC inhibition enhanced expression of the BH3-only proapoptotic proteins BIM and BMF, and their knockdown significantly attenuated MAPK/HDAC inhibitor-induced apoptosis. Importantly, we demonstrate that the paradigm of combined MAPK/HDAC inhibitor treatment to induce apoptosis can be tailored to specific MAPK genotypes in colorectal cancers, by combining an HDAC inhibitor with either an EGFR, KRASG12C or BRAFV600 inhibitor in KRAS/BRAFWT; KRASG12C, BRAFV600E colorectal cancer cell lines, respectively. These findings identify a series of ERK/MAPK genotype-tailored treatment strategies that can readily undergo clinical testing for the treatment of colorectal cancer.

Targeting the BCL-2-regulated apoptotic pathway for the treatment of solid cancers.

The deregulation of apoptosis is a key contributor to tumourigenesis as it can lead to the unwanted survival of rogue cells. Drugs known as the BH3-mimetics targeting the pro-survival members of the BCL-2 protein family to induce apoptosis in cancer cells have achieved clinical success for the treatment of haematological malignancies. However, despite our increasing knowledge of the pro-survival factors mediating the unwanted survival of solid tumour cells, and our growing BH3-mimetics armamentarium, the application of BH3-mimetic therapy in solid cancers has not reached its full potential. This is mainly attributed to the need to identify clinically safe, yet effective, combination strategies to target the multiple pro-survival proteins that typically mediate the survival of solid tumours. In this review, we discuss current and exciting new developments in the field that has the potential to unleash the full power of BH3-mimetic therapy to treat currently recalcitrant solid malignancies.

Discovery, development and application of drugs targeting BCL-2 pro-survival proteins in cancer.

The discovery of a new class of small molecule compounds that target the BCL-2 family of anti-apoptotic proteins is one of the great success stories of basic science leading to translational outcomes in the last 30 years. The eponymous BCL-2 protein was identified over 30 years ago due to its association with cancer. However, it was the unveiling of the biochemistry and structural biology behind it and its close relatives' mechanism(s)-of-action that provided the inspiration for what are now known as 'BH3-mimetics', the first clinically approved drugs designed to specifically inhibit protein-protein interactions. Herein, we chart the history of how these drugs were discovered, their evolution and application in cancer treatment.

BECLIN1: Protein Structure, Function and Regulation.

BECLIN1 is a well-established regulator of autophagy, a process essential for mammalian survival. It functions in conjunction with other proteins to form Class III Phosphoinositide 3-Kinase (PI3K) complexes to generate phosphorylated phosphatidylinositol (PtdIns), lipids essential for not only autophagy but other membrane trafficking processes. Over the years, studies have elucidated the structural, biophysical, and biochemical properties of BECLIN1, which have shed light on how this protein functions to allosterically regulate these critical processes of autophagy and membrane trafficking. Here, we review these findings and how BECLIN1's diverse protein interactome regulates it, as well as its impact on organismal physiology.

Influenza A virus infection-induced macroautophagy facilitates MHC class II-restricted endogenous presentation of an immunodominant viral epitope.

CD4+ T cells recognize peptides presented by major histocompatibility complex class II molecules (MHC-II). These peptides are generally derived from exogenous antigens. Macroautophagy has been reported to promote endogenous antigen presentation in viral infections. However, whether influenza A virus (IAV) infection-induced macroautophagy also leads to endogenous antigen presentation through MHC-II is still debated. In this study, we show that IAV infection leads to endogenous presentation of an immunodominant viral epitope NP311-325 by MHC-II to CD4+ T cells. Mechanistically, such MHC-II-restricted endogenous IAV antigen presentation requires de novo protein synthesis as it is inhibited by the protein synthesis inhibitor cycloheximide, and a functional ER-Golgi network as it is totally blocked by Brefeldin A. These results indicate that MHC-II-restricted endogenous IAV antigen presentation is dependent on de novo antigen and/or MHC-II synthesis, and transportation through the ER-Golgi network. Furthermore, such endogenous IAV antigen presentation by MHC-II is enhanced by TAP deficiency, indicating some antigenic peptides are of cytosolic origin. Most importantly, the bulk of such MHC-II-restricted endogenous IAV antigen presentation is blocked by autophagy inhibitors (3-MA and E64d) and deletion of autophagy-related genes, such as Beclin1 and Atg7. We have further demonstrated that in dendritic cells, IAV infection prevents autophagosome-lysosome fusion and promotes autophagosome fusion with MHC class II compartment (MIIC), which likely promotes endogenous IAV antigen presentation by MHC-II. Our results provide strong evidence that IAV infection-induced autophagosome formation facilitates endogenous IAV antigen presentation by MHC-II to CD4+ T cells. The implication for influenza vaccine design is discussed.

Optimization of Benzothiazole and Thiazole Hydrazones as Inhibitors of Schistosome BCL-2.

Limited therapeutic options are available for the treatment of human schistosomiasis caused by the parasitic Schistosoma flatworm. The B cell lymphoma-2 (BCL-2)-regulated apoptotic cell death pathway in schistosomes was recently characterized and shown to share similarities with the intrinsic apoptosis pathway in humans. Here, we exploit structural differences in the human and schistosome BCL-2 (sBCL-2) pro-survival proteins toward a novel treatment strategy for schistosomiasis. The benzothiazole hydrazone scaffold previously employed to target human BCL-XL was repurposed as a starting point to target sBCL-2. We utilized X-ray structural data to inform optimization and then applied a scaffold-hop strategy to identify the 5-carboxamide thiazole hydrazone scaffold (43) with potent sBCL-2 activity (IC50 30 nM). Human BCL-XL potency (IC50 13 nM) was inadvertently preserved during the optimization process. The lead analogues from this study exhibit on-target activity in model fibroblast cell lines dependent on either sBCL-2 or human BCL-XL for survival. Further optimization of the thiazole hydrazone class is required to exhibit activity in schistosomes and enhance the potential of this strategy for treating schistosomiasis.

The role of BCL-2 family proteins and therapeutic potential of BH3-mimetics in malignant pleural mesothelioma.

Introduction: With limited recent therapeutic changes, malignant pleural mesothelioma (MPM) is associated with poor survival and death within 12 months, making it one of the most lethal malignancies. Due to unregulated asbestos use in developing countries and home renovation exposures, cases of MPM are likely to present for decades. As MPM is largely driven by dysregulation of tumor suppressor genes, researchers have examined other mechanisms of subverting tumor proliferation and spread. Over-expression of pro-survival BCL-2 family proteins impairs cells from undergoing apoptosis, and BH3-mimetics  targeting them are a novel treatment option across various cancers, though have not been widely investigated in MPM.Areas covered: This review provides an overview of MPM and its current treatment landscape. It summarizes the role of BCL-2 family proteins in tumorigenesis and the therapeutic potential of BH3-mimetics . Finally, it discusses the role of BCL-2 proteins in MPM and the pre-clinical rationale for investigating BH3-mimetics as a therapeutic strategy.Expert opinion: As a disease without readily actionable oncogene driver mutations and with modest benefit from immune checkpoint inhibition, novel therapeutic options are urgently needed for MPM. Hence, BH3-mimetics provide a promising treatment option, with evidence supporting dependence on pro-survival BCL-2 proteins for MPM cell survival.

A preliminary assessment of oral monepantel's tolerability and pharmacokinetics in individuals with treatment-refractory solid tumors.

PURPOSE: Monepantel is an approved veterinary anthelmintic with a strong safety profile. Preclinical evidence suggests novel mTOR pathway-associated anticancer activity. An open-label Phase I trial assessed tolerability, pharmacokinetics, pharmacodynamics and PET-CT imaging following oral Zolvix® monepantel administration to adults with treatment refractory, progressing and unresectable solid tumors. METHODS: Subjects were scheduled to daily home-based monepantel administration for 28 days in a 3 + 3 dose escalation study (5.0, 25.0 and 62.5 mg/kg bw). RESULTS: Of 41 reported drug-related AEs, 68% were Grade 1 and 24% were Grade 2; 35 AEs related to gastrointestinal effects including very poor palatability. DLT and MTD could not be determined due to early termination. Myelosuppression was not observed at the lowest level tested. Three of four Cohort 1 subjects had reduced mTOR pathway marker p-RPS6KB1 levels in PBMCs and achieved RECISTv1.1 SD by CT; one had progressive bony metastases by FDG-PET. One subject recorded PD on day 28, correlating with no detectable plasma monepantel from day 7. Monepantel sulfone dominated monepantel in pharmacokinetics. Both Cohort 2 subjects withdrew early due to AEs and the trial was terminated. CONCLUSIONS: Short-term 5 mg/kg bw monepantel administration provides a combined steady-state trough plasma monepantel and monepantel sulfone concentration of 0.5 µM. Gastrointestinal AEs including very poor palatability are concerning and suggested to be resolved by future drug product reformulation. RECISTv1.1, p-RPS6KB1 and plasma tumor marker outcomes provide preliminary evidence of anticancer activity.

Diversity in the intrinsic apoptosis pathway of nematodes.

Early studies of the free-living nematode C. elegans informed us how BCL-2-regulated apoptosis in humans is regulated. However, subsequent studies showed C. elegans apoptosis has several unique features compared with human apoptosis. To date, there has been no detailed analysis of apoptosis regulators in nematodes other than C. elegans. Here, we discovered BCL-2 orthologues in 89 free-living and parasitic nematode taxa representing four evolutionary clades (I, III, IV and V). Unlike in C. elegans, 15 species possess multiple (two to five) BCL-2-like proteins, and some do not have any recognisable BCL-2 sequences. Functional studies provided no evidence that BAX/BAK proteins have evolved in nematodes, and structural studies of a BCL-2 protein from the basal clade I revealed it lacks a functionally important feature of the C. elegans orthologue. Clade I CED-4/APAF-1 proteins also possess WD40-repeat sequences associated with apoptosome assembly, not present in C. elegans, or other nematode taxa studied.

Crosstalk between apoptosis and autophagy signaling pathways.

The fate of a cell is determined by multiple signaling pathways in response to a range of stimuli. Probably the most prominent cell death mechanism is apoptosis which can be triggered by both internal stresses, as well as extracellular stimuli, and is executed by two well-characterized pathways, the intrinsic and the extrinsic apoptosis pathways. Although autophagy can also lead to cell death under certain circumstances, its major function is as a cell survival process. Given that the life of a cell is at stake, it is not surprising that there is significant molecular crosstalk between these pathways. The nature of these interconnections is diverse and ranges from protein-protein interactions and post-translational modifications through to the degradation of molecular components by different proteins and organelles. In this chapter we review these mechanisms in detail.

A small molecule interacts with VDAC2 to block mouse BAK-driven apoptosis.

Activating the intrinsic apoptosis pathway with small molecules is now a clinically validated approach to cancer therapy. In contrast, blocking apoptosis to prevent the death of healthy cells in disease settings has not been achieved. Caspases have been favored, but they act too late in apoptosis to provide long-term protection. The critical step in committing a cell to death is activation of BAK or BAX, pro-death BCL-2 proteins mediating mitochondrial damage. Apoptosis cannot proceed in their absence. Here we show that WEHI-9625, a novel tricyclic sulfone small molecule, binds to VDAC2 and promotes its ability to inhibit apoptosis driven by mouse BAK. In contrast to caspase inhibitors, WEHI-9625 blocks apoptosis before mitochondrial damage, preserving cellular function and long-term clonogenic potential. Our findings expand on the key role of VDAC2 in regulating apoptosis and demonstrate that blocking apoptosis at an early stage is both advantageous and pharmacologically tractable.

The Structural Biology of Bcl-xL.

Interactions between the pro-survival and pro-apoptotic members of the Bcl-2 family of proteins dictate whether a cell lives or dies. Much of our knowledge of the molecular details of these interactions has come from biochemical and structural studies on the pro-survival protein Bcl-xL. The first high-resolution structure of any Bcl-2 family member was of Bcl-xL, which revealed the conserved topology amongst all family members. Subsequent structures of Bcl-xL complexes with pro-apoptotic ligands demonstrated the general features of all pro-survival:pro-apoptotic complexes. Structural studies involving Bcl-xL were also the basis for the discovery of the first small-molecule pro-survival protein inhibitors, leading ultimately to the development of a new class of drugs now successfully used for cancer treatment in the clinic. This article will review our current knowledge of the structural biology of Bcl-xL and how this has impacted our understanding of the molecular details of the intrinsic apoptotic pathway.

BCL-XL and MCL-1 are the key BCL-2 family proteins in melanoma cell survival.

Malignant melanoma is one of the most difficult cancers to treat due to its resistance to chemotherapy. Despite recent successes with BRAF inhibitors and immune checkpoint inhibitors, many patients do not respond or become resistant to these drugs. Hence, alternative treatments are still required. Due to the importance of the BCL-2-regulated apoptosis pathway in cancer development and drug resistance, it is of interest to establish which proteins are most important for melanoma cell survival, though the outcomes of previous studies have been conflicting. To conclusively address this question, we tested a panel of established and early passage patient-derived cell lines against several BH3-mimetic drugs designed to target individual or subsets of pro-survival BCL-2 proteins, alone and in combination, in both 2D and 3D cell cultures. None of the drugs demonstrated significant activity as single agents, though combinations targeting MCL-1 plus BCL-XL, and to a lesser extent BCL-2, showed considerable synergistic killing activity that was elicited via both BAX and BAK. Genetic deletion of BFL-1 in cell lines that express it at relatively high levels only had minor impact on BH3-mimetic drug sensitivity, suggesting it is not a critical pro-survival protein in melanoma. Combinations of MCL-1 inhibitors with BRAF inhibitors also caused only minimal additional melanoma cell killing over each drug alone, whilst combinations with the proteasome inhibitor bortezomib was more effective in multiple cell lines. Our data show for the first time that therapies targeting specific combinations of BCL-2 pro-survival proteins, namely MCL-1 plus BCL-XL and MCL-1 plus BCL-2, could have significant benefit for the treatment of melanoma.

Structural insights into BCL2 pro-survival protein interactions with the key autophagy regulator BECN1 following phosphorylation by STK4/MST1.

BECN1/Beclin 1 is a critical protein in the initiation of autophagosome formation. Recent studies have shown that phosphorylation of BECN1 by STK4/MST1 at threonine 108 (T108) within its BH3 domain blocks macroautophagy/autophagy by increasing BECN1 affinity for its negative regulators, the anti-apoptotic proteins BCL2/Bcl-2 and BCL2L1/Bcl-xL. It was proposed that this increased binding is due to formation of an electrostatic interaction with a conserved histidine residue on the anti-apoptotic molecules. Here, we performed biophysical studies which demonstrated that a peptide corresponding to the BECN1 BH3 domain in which T108 is phosphorylated (p-T108) does show increased affinity for anti-apoptotic proteins that is significant, though only minor (<2-fold). We also determined X-ray crystal structures of BCL2 and BCL2L1 with T108-modified BECN1 BH3 peptides, but only showed evidence of an interaction between the BH3 peptide and the conserved histidine residue when the histidine flexibility was restrained due to crystal contacts. These data, together with molecular dynamics studies, indicate that the histidine is highly flexible, even when complexed with BECN1 BH3. Binding studies also showed that detergent can increase the affinity of the interaction. Although this increase was similar for both the phosphorylated and non-phosphorylated peptides, it suggests factors such as membranes could impact on the interaction between BECN1 and BCL2 proteins, and therefore, on the regulation of autophagy. Hence, we propose that phosphorylation of BECN1 by STK4/MST1 can increase the affinity of the interaction between BECN1 and anti-apoptotic proteins and this interaction can be stabilized by local environmental factors. Abbreviations: asu: asymmetric unit; BH3: BCL2/Bcl-2 homology 3; DAPK: death associated protein kinase; MD: molecular dynamics; MST: microscale thermophoresis; NMR: nuclear magnetic resonance; PDB: protein data bank; p-T: phosphothreonine; SPR: surface plasmon resonance; STK4/MST1: serine/threonine kinase 4.