Coibamide A Targets Sec61 to Prevent Biogenesis of Secretory and Membrane Proteins

Coibamide A (CbA) is a marine natural product with potent antiproliferative activity against human cancer cells and a unique selectivity profile. Despite promising antitumor activity, the mechanism of cytotoxicity and specific cellular target of CbA remain unknown. Here, we develop an optimized synthetic CbA photoaffinity probe (photo-CbA) and use it to demonstrate that CbA directly targets the Sec61 alpha subunit of the Sec61 protein translocon. CbA binding to Sec61 results in broad substratenonselective inhibition of ER protein import and potent cytotoxicity against specific cancer cell lines. CbA targets a lumenal cavity of Sec61 that is partially shared with known Sec61 inhibitors, yet profiling against resistance conferring Sec61 alpha mutations identified from human HCT116 cells su ests a distinct binding mode for CbA. Specifically, despite conferring strong resistance to all previously known Sec61 inhibitors, the Sec61 alpha mutant R66I remains sensitive to CbA. A further unbiased screen for Sec61 alpha resistance mutations identified the CbA-resistant mutation S71P, which confirms nonidentical binding sites for CbA and apratoxin A and supports the susceptibility of the Sec61 plug region for channel inhibition. Remarkably, CbA, apratoxin A, and ipomoeassin F do not display comparable patterns of potency and selectivity in the NCI60 panel of human cancer cell lines. Our work connecting CbA activity with selective prevention of secretory and membrane protein biogenesis by inhibition of Sec61 opens up possibilities for developing new Sec61 inhibitors with improved druglike properties that are based on the coibamide pharmacophore.Peer reviewe

Marine Natural Products as Molecular Probes to Study Cancer Cell Stress and Proteostasis

Natural products are compounds isolated from living organisms and represent a large source of novel drug leads. Nearly 60% of current FDA-approved anticancer drugs are direct derivatives of, or are inspired by, natural products. This work represents the biological characterization of three cytotoxic marine natural products and their use as pharmacological tools to study patterns of cell stress and proteotoxicity in human cancer cells. The macrolactone natural product mandelalide A was previously reported to be a direct inhibitor of mitochondrial ATP synthase. We show that mandelalide A is also an indirect activator of AMP-activated protein kinase (AMPK), the major sensor of energy stress in mammalian cells. Using representative mouse embryonic fibroblasts (MEFs) and human cancer cells we examined the phosphorylation status of key residues in the AMPK signal transduction pathway in response to the mandelalides. We conclude that ATP-depletion by mandelalide A induces early activation of AMPK that likely serves as a protective response to restore energy balance and promote cell survival. The majority of this work represents the study of a distinct type of cell stress that occurs when cellular proteostasis is disrupted. For these studies we utilized coibamide A and apratoxin A, two complex secondary metabolites that act as direct inhibitors of Sec61-mediated cotranslational translocation and are therefore able to disrupt protein biosynthesis at the start of the cellular secretory pathway. Under normal conditions proteostasis is maintained in the ER by the unfolded protein response (UPR); when there is an abundance of nascent protein synthesis into the ER the UPR is activated. This is a highly conserved process in normal mammalian cells and can be used in cancer cells for a survival advantage. We show that canine osteosarcoma cells have atypical expression of glucose-regulated protein (GRP)78, a central mediator of the UPR. Apratoxin A was used to compare the action of direct and putative inhibitors of GRP78 in normal canine osteoblast progenitors and canine osteosarcoma cells. We show that carboplatin-resistant canine osteosarcoma cells retain sensitivity to several small molecule inhibitors of chaperone protein function indicating that several nodes in the canine osteosarcoma chaperome could be a relevant chemotherapeutic target for the future treatment of aggressive bone cancers in dogs. Finally, although coibamide A and apratoxin A are considered broad-spectrum inhibitors of Sec61 function, with limited substrate selectivity, we provide further evidence that the two compounds function differently and are not identical. We used a proteomic approach to compare the secretomes of human glioblastoma cells treated with coibamide A and apratoxin A and showed that while many Sec61 substrates overlap there are notable differences between the two natural products. We also take advantage of advances in the total synthesis of coibamide A to probe the substrate selectivity of several diastereomers of the coibamide A structure. We investigated the ability of four coibamide variants to inhibit protein secretion and induce cytotoxicity. From this pharmacological characterization we find that modifications to the parent coibamide structure can produce distinct differences in the bioactivity profile. In summary these data add to the biological characterization of several lead marine natural products as molecular probes to target cell survival signaling in preclinical cancer models

Targeting of HER/ErbB family proteins using broad spectrum Sec61 inhibitors coibamide A and apratoxin A

Coibamide A is a potent cancer cell toxin and one of a select group of natural products that inhibit protein entry into the secretory pathway via a direct inhibition of the Sec61 protein translocon. Many Sec61 client proteins are clinically relevant drug targets once trafficked to their final destination in or outside the cell, however the use of Sec61 inhibitors to block early biosynthesis of specific proteins is at a pre-clinical stage. In the present study we evaluated the action of coibamide A against human epidermal growth factor receptor (HER, ErbB) proteins in representative breast and lung cancer cell types. HERs were selected for this study as they represent a family of Sec61 clients that is frequently dysregulated in human cancers, including coibamide-sensitive cell types. Although coibamide A inhibits biogenesis of a broad range of Sec61 substrate proteins in a presumed substrate nonselective manner, endogenous HER3 (ErbB-3) and EGFR (ErbB-1) proteins were more sensitive to coibamide A, and the related Sec61 inhibitor apratoxin A, than HER2 (ErbB-2). Despite this rank order of sensitivity (HER3 > EGFR > HER2), Sec61-dependent inhibition by coibamide A was sufficient to decrease cell surface expression of HER2. We report that coibamide Aor apratoxin A-mediated block of HER3 entry into the secretory pathway is unlikely to be mediated by the HER3 signal peptide alone. HER3 (G11L/S15L), that is fully resistant to the highly substrate-selective cotransin analogue CT8, was more resistant than wild-type HER3 but only at low coibamide A (3 nM) concentrations; HER3 (G11L/S15L) expression was inhibited by higher concentrations of either natural product. Timeand concentration-dependent decreases in HER protein expression induced a commensurate reduction in AKT/MAPK signaling in breast and lung cancer cell types and loss in cell viability. Coibamide A potentiated the cytotoxic efficacy of small molecule kinase inhibitors lapatinib and erlotinib in breast and lung cancer cell types, respectively. These data indicate that natural product modulators of Sec61 function have value as chemical probes to interrogate HER/ErbB signaling in treatment-resistant human cancers.Peer reviewe

Sexualidade na deficiência intelectual: uma análise das percepções de mães de adolescentes especiais Sexuality in intellectual disabilities: an analysis of the perceptions of mothers of special adolescents

Adolescência é a fase transitória entre infância e idade adulta, momento importante do desenvolvimento humano, marcado por mudanças físicas, psicológicas e sociais relativas ao início da sexualidade. Este momento geralmente é conturbado e o poderá ser ainda mais para adolescentes com deficiência intelectual (DI) por confrontar com preconceitos e mistificações estabelecidas há tempos. A maneira infantilizante e discriminatória de serem tratados pela família e sociedade influenciam as percepções das mães de filhos com DI. Assim, objetivando investigar as concepções que mães de jovens com DI têm sobre a sexualidade deles e como elas irão refletir na adoção de práticas de educação sexual, foram entrevistadas 20 mães de adolescentes entre 12 a 18 anos, de ambos os sexos, com diagnóstico de DI, atendidos numa clínica escola localizada no estado do Espírito Santo. Analisando as entrevistas, percebeu-se em 12 respostas, a ideia de ausência de sexualidade na pessoa com DI, trazendo uma postura infantilizadora e superprotetora dessas mães em relação aos filhos, considerando-os com pouca possibilidade de desenvolver interesses e comportamentos sexuais. Quanto às concepções das mães nas manifestações sexuais de seus filhos, 15 delas revelaram entender que a sexualidade deles é diferente da de pessoas sem deficiência intelectual. Percebeu-se que 12 das 20 mães nunca orientaram seus filhos sexualmente, alegando que não compreenderiam. Em geral, as mães não reconhecem uma identidade sexual em seus filhos e, por conseguinte, não fornecem uma educação sexual, reproduzindo a concepção social e cultural que nega a existência da sexualidade quando associada à DI.<br>Adolescence is the transitional phase between childhood and adulthood, an important moment of human development, marked by physical, psychological and social changes at the beginning of sexuality. This moment is often troubled and may be even more so for teenagers with intellectual disability (ID) by confronting preconceptions and mystification established long ago. The infantilizing and discriminatory way they may be treated by family and society influence the perceptions of mothers of children with ID. Thus, to investigate the conceptions that mothers of young people with ID have about their sexuality and how they will reflect the adoption of sexual education practices, 20 mothers of adolescents aged 12 to 18 years, of both sexes with a diagnosis of ID were interviewed at a clinical school located in the state of Espírito Santo. Analyzing the interviews, 12 answers indicated the idea of absence of sexuality in the person with ID, fostering an infantilized and overprotective attitude of these mothers to their children; it was considered unlikely that they would develop interest in sex or present sexual behaviors. As for the conceptions of mothers on the sexual manifestations of their children, 15 of them revealed understanding that their sexuality is different from that of people without intellectual disability. It was noted that 12 of the 20 mothers had never oriented their children about sex, saying they would not be capable of understanding. In general, the mothers do not recognize a sexual identity in their children and, therefore, do not provide them with sexual education, reproducing the social and cultural concept that denies the existence of sexuality when associated with ID