“Recognizing a surprising fact is the first step towards discovery.” This famous quote from Louis Pasteur is particularly appropriate to describe what led us to study mycolactone, a lipid toxin produced by the human pathogen Mycobacterium ulcerans. M. ulcerans is the causative agent of Buruli ulcer, a neglected tropical disease manifesting as chronic, necrotic skin lesions with a “surprising” lack of inflammation and pain. Decades after its first description, mycolactone has become much more than a mycobacterial toxin. This uniquely potent inhibitor of the mammalian translocon (Sec61) helped reveal the central importance of Sec61 activity for immune cell functions, the spread of viral particles and, unexpectedly, the viability of certain cancer cells.
1. Mycolactone Targets the Sec61 Translocon
After the discovery of mycolactone, identifying its molecular target took a new dimension. Indeed, in addition to providing an effective treatment of BU, it could lead to the generation of novel immunomodulatory molecules. In 2014, Hall et al. made a breakthrough by demonstrating that mycolactone prevents the translocation of model secretory proteins into the endoplasmic reticulum (ER), leading to their degradation in the cytosol by the ubiquitin–proteasome system 
. Using cell-free assays, McKenna et al. then showed that mycolactone selectively affects the step of cotranslational translocation of secreted and integral transmembrane proteins (TMPs) into the ER 
. In eukaryotes, the cotranslational protein translocation pathway is initiated by recognition of signal peptides or transmembrane domains by the signal recognition particle (SRP). The SRP then targets the ribosome-nascent polypeptide complex to the Sec61 translocon. Sec61 is a heterotrimeric complex embedded in the ER membrane that ensures the transport of most secreted proteins (with a signal peptide but no transmembrane domain) and single-spanning TMPs into the ER. TMPs exclusively relying on Sec61 for insertion into the ER membrane include the Type I (with a signal peptide) and the Type II (without a signal peptide and a cytosolic N terminus) TMPs. Instead, the rare subsets of Type III TMPs (without a signal peptide and the opposite N terminal topology) and the C-terminal tail-anchored proteins can use alternative pathways for membrane integration at the ER 
, while mitochondrial membrane proteins depend on independent TIM/TOM complexes for mitochondrial membrane insertion.
In collaboration with Ville Paavilainen (University of Helsinki, Helsinki, Finland), researchers demonstrated that mycolactone prevents the cotranslational translocation of proteins into the ER by directly targeting the Sec61 translocon. Indeed, a single amino acid mutation (R66G) in the pore-forming (alpha) subunit of Sec61 conferred full resistance to mycolactone activity in bioassays 
. Mycolactone was not the first reported inhibitor of Sec61. In 2005, Garrison et al. had discovered cotransin, a fungal product derivative with the capacity to inhibit Sec61 in a selective, substrate-specific manner 
. Competition assays between cotransin and mycolactone, and mutant Sec61 studies, indicated that the binding sites of the two compounds overlap 
. However, the in vitro translocation assays and global profiling of mycolactone-susceptible proteins in T cells, DCs and sensory neurons showed that, contrary to cotransin, mycolactone is not substrate-selective 
. In addition, these proteomic analyses made it possible to characterize for the first time the signature of the Sec61 blockade at the cellular level 
Consistent with its mechanism of action, most of the detected Sec61 clients (secreted proteins, Type I and Type II TMPs) were massively downregulated by mycolactone in the three tested cell types. In contrast, mycolactone did not affect the cellular levels of Type III TMPs, C-tail anchored and mitochondrial membrane proteins 
. Recent cryo-EM studies suggest that mycolactone and cotransin both operate by maintaining Sec61 in a close, inactive conformation through interactions with its sealing plug and lateral gate. However, the structural determinants of their differential selectivity for Sec61 substrates remain to be elucidated 
2. Sec61 Activity, Immunity and Apoptosis
The identification of mycolactone-resistant Sec61 mutants enabled researchers to examine the role of the translocon in its immunomodulatory and cytotoxic effects. Production of IFN-γ by T cells and activation of the IFN-γ receptor signaling pathway in infected macrophages are key components of anti-mycobacterial immunity 
; both of which were markedly impaired by mycolactone. Expression of the Sec61α-R66G mutant in mycolactone-treated T cells rescued their effector functions in vitro and in vivo. When expressed in macrophages, the mycolactone-resistant mutant restored IFN-γ receptor-mediated anti-microbial responses 
. These experiments demonstrated that Sec61 is the host receptor mediating the diverse immunomodulatory effects of mycolactone. Further, they revealed a novel mechanism of immune evasion evolved by M. ulcerans
. Beyond the control of IFN-γ and IFN-γ receptor production, the proteomic studies showed that inhibiting protein translocation has the potential to suppress inflammatory responses. In mouse models, the systemic administration of mycolactone could be used therapeutically to limit chronic skin inflammation, rheumatoid arthritis and inflammatory pain 
). While less potent than natural mycolactone in the conditions used, mini-mycolactone also conferred significant protection in these disease models, confirming the potential of mycolactone-derived structures as prospective immunosuppressants.
Notably, the Sec61α-R66G mutant protected mycolactone-treated cells from undergoing apoptosis 
, showing that mycolactone cytotoxicity is a late consequence of Sec61 blockade. Looking at the proteins that were up-regulated by mycolactone in treated cells, researchers identified the hallmarks of cytosolic and ER stress responses. Thapsigargin, tunicamycin, and MG132 are canonical ER stressors targeting Ca2+
ATPases, protein glycosylation or the proteasome, respectively, which trigger an unfolded protein response (UPR) to restore protein homeostasis. Like them, mycolactone upregulated the UPR in treated cells, leading to the expression of the pro-apoptotic factor C/EBP homologous protein (Chop). This provided an explanation for how a prolonged exposure to saturating amounts of mycolactone can lead to cell apoptosis (Figure 1
). However, unlike canonical ER stressors, mycolactone did not augment in parallel the expression of the ER chaperone GRP78/BiP 
. Because BiP increases the cell’s ability to resolve ER stress and prevents the transition from protective to terminal UPR, this suggested that ER stress caused by mycolactone is more prone to evolve towards apoptosis.
3. Sec61 Blockers for Oncology
The observation that mycolactone causes terminal UPR led us to investigate whether the proteotoxic impact of Sec61 blockade could also be exploited therapeutically. Despite the fact that considerable advances have been made over the years, the plasma cell malignancy multiple myeloma (MM) still remains an incurable disease. The current first line of treatment consists of proteasome inhibitors (bortezomib and derivatives) and immunomodulators (lenalidomide and derivatives), but eventually the majority of MM patients will relapse over time because of the generation of drug-resistant cancerous cells 
. Therefore, the development of novel drugs with different mechanisms of action is vital to turn the tide of the battle against MM. Researches reasoned that Sec61 blockade may represent a novel therapeutic approach of interest in MM, by inducing proteotoxic stress responses while preventing the expression of membrane receptors that are key to MM cell division and dissemination.
With regard to this, recent work in the laboratory established protein translocation inhibition as a novel, useful tool against MM. By broadly inhibiting Sec61 with mycolactone, researchers showed that the translocon’s activity has a central role in determining MM cells’ fate. Indeed, mycolactone efficiently reduced MM cell line production of immunoglobulins and multiple type I/II TMP receptors such as CD138, a hallmark of MM that allows the survival of cancerous cells in the bone marrow by promoting growth factor signaling. Mycolactone treatment also decreased MM cell expression of the pro-survival IL-6 receptor and CD40, whose activation stimulates IL-6 production. As a later effect, mycolactone induced a pro-apoptotic ER stress-response in MM cell lines and tumors isolated from MM patients. This was used as proof of concept to show that Sec61 inhibitors have the potential to be used as an anti-cancer treatment against MM, alone or in combination with currently used chemotherapies 
. Strikingly, mycolactone combined with bortezomib significantly delayed MM tumor growth in mice without significant toxicity. Equally importantly, mycolactone showed a synergistic action with lenalidomide, and was even effective at inducing cell death in bortezomib- and or lenalinomide-resistant cells 
, giving hope for the treatment of relapsed/refractory MM.
As described in the previous sections, type III TMPs translocate into the ER in a Sec61-independent manner, and their levels are therefore not blunted by Sec61 inhibitors. The plasma cell-specific B cell maturation antigen (BCMA) belongs to the class of type III TMPs and ever since its overexpression and activation have been associated with MM, it has attracted great attention from the medical research community. As a result, BCMA is currently used as a biomarker for MM diagnosis and tracking. Furthermore, BCMA is used as a target to treat MM through specific antibodies and chimeric antigen receptor (CAR)-T cell therapy, both of which are giving promising results in clinics for an efficient and durable MM cancer treatment 
. As expected, researchers found that mycolactone does not decrease BCMA expression via MM cell lines, but surprisingly its expression was greatly increased after treatment in a dose-dependent manner 
. Even though researchers do not have mechanistic insights as to why mycolactone increases BCMA levels, researchers hypothesize that this is a secondary effect of the stress response induced by the Sec61 blocker. Nonetheless, it stands to reason that the effect of mycolactone on BCMA may potentiate the anti-BCMA therapies that are currently emerging against MM, giving mycolactone an additional key role in MM treatment.
Mycolactone is not the only Sec61 inhibitor that showed promising results by targeting clinically relevant proteins in the Oncology field. In fact, the cyclodepsipeptide apratoxins, a class of molecules isolated from marine cyanobacteria, showed a significantly strong antiproliferative activity in different cancer cells and only later was its mechanism of action revealed. Like cotransin and mycolactone, apratoxin A inhibits the secretion pathway through the direct inhibition of Sec61 
. The ability of apratoxin A to inhibit vascular endothelial growth factor (VEGF)-A expression inspired Cai et al. to test its anti-angiogenic effect, a hallmark of solid tumors. The group showed that a synthetic analog of apratoxin A, which was improved through structure–activity relationship studies and named apratoxin S10, inhibits angiogenesis and cancer cell growth in vitro. The antiangiogenic effect was mainly due to the ability of apratoxin S10 to downregulate VEGF receptor on endothelial cells while the anti-tumor effect was shown to result from a reduction in secretion of VEGF-A and IL-6, which have a role in both promoting tumor cell growth and the formation of new blood vessels 
. The dual anti-tumor and anti-angiogenic properties of apratoxin A are extremely promising for solid tumor treatments, but the extensive pancreatic toxicity of the molecule in vivo 
has blocked it from further clinical studies. How it compares to mycolactone and cotransin, in terms of Sec61 substrate selectivity and mechanism of inhibition, remains largely unknown.
As mentioned above, a different molecule belonging to the same family, the cyclodepsipeptide natural product cotransin, which was first discovered through a Sec61-unrelated screen of molecules that aimed at discovering inhibitors of the expression of cell adhesion molecules, proved to be a selective Sec61 inhibitor. Unlike mycolactone, cotransin only inhibits a subset of Sec61 clients, including the vascular cell adhesion molecule 1 (VCAM-1) and human epidermal growth factor receptor 3 (HER3), by preventing the recognition of specific signal sequences at the N-terminus end of target proteins by the translocon 
. HER3 is a therapeutically interesting Sec61 client because it has a key role in tumorigenesis in several types of tumors, induces cell proliferation and has been linked to chemotherapy resistance. Much effort has been devoted to targeting HER3 through diverse methods against different types of cancers 
. Ruiz-Saenz et al. showed that CT8, which belongs to the class of cotransins, selectively inhibits the expression of HER3 by blocking its translocation in the ER and subsequently inducing its proteasomal degradation in the cytosol, with no effect on the expression of other protein members of the HER family. This selective effect was in fact due to the specific sequence of the N-terminal signal peptide of HER3, as part of the mechanism of action of cotransins, and proved to enhance the pro-apoptotic effects of chemotherapeutic drugs currently used against breast cancer and other solid tumors 
What researchers learned from the action of these natural products shed light on the great therapeutic potential of Sec61 blockers: their anti-tumor, pro-apoptotic properties, together with the possibility of developing specific inhibitors that target selective clinically relevant Sec61 proteins to avoid/minimize normal cell toxicity, show that Sec61 inhibitors may make great contributions to the field of Oncology in the near future.