Establishing the pathogenicity of novel mitochondrial DNA sequence variations: a cell and molecular biology approach

Tese de doutoramento em Ciências da Saúde, no ramo de Ciências Biomédicas, apresentada à Faculdade de Medicina da Universidade de CoimbraAs doenças mitocondriais são das doenças metabólicas mais frequentes e representam um grande encargo económico na sociedade. Atualmente, existem mais de 60 mutações no DNA mitocondrial (mtDNA) associadas a patologias. Uma vez que o mtDNA é altamente polimórfico e apresenta características peculiares, a patogenicidade de uma nova alteração detetada, apoia-se numa série de critérios para estabelecer a correlação genótipo-fenótipo. O presente trabalho inclui o estudo de quatro variações de sequência no mtDNA, não classificadas, com potencial efeito deletério, identificadas em quatro doentes portugueses com suspeita de doença mitocondrial e sem parentesco, estudados no Laboratório de Bioquímica Genética, Centro de Neurociências e Biologia Celular e Molecular – Universidade de Coimbra. O doente 1 (P1) apresentou neuropatia ótica bilateral grave. O doente 2 (P2) manifestou deficiência mental grave, atrofia cerebelar, ataxia grave, fácies grosseira, macrocefalia relativa, hipotonia congénita, afasia, e outras características tal como clinodactilia. O doente 3 (P3) apresentou oftalmoplegia externa progressiva crónica. O doente 4 (P4) sofreu morte súbita infantil, com a suspeita posterior de cardiomiopatia. Foi identificada uma variante no mtDNA em cada doente em genes que codificam subunidades dos complexos enzimáticos da fosforilação oxidativa ou RNAs de transferência. As variantes identificadas foram m.8418T>C, p.Leu18Pro (MT-ATP8), m.14771C>A, p.Pro9Thr (MT-CYB), m.7486G>A, mt-tRNASer(UCN) (MT-TS1), m.14706A>G, mt-tRNAGlu (MT-TE), nos doentes 1, 2, 3 e 4, respetivamente. Foram realizados estudos biomoleculares, usando uma abordagem de genómica funcional para avaliar a patogenicidade das variantes no mtDNA de forma a estabelecer o diagnóstico genético dos quatro doentes e clarificar o mecanismo de patogenicidade. Os métodos usados incluíram sequenciação de Sanger, pirosequenciação, sequenciação de nova geração, PCR longo e PCR em tempo real, histologia, histoquímica, cultura de células, espectrofotometria, fluorimetria, Western-blot, eletroforese nativa em gel de acrilamida, tecnologia Seahorse Bioscience® e microscopia de transmissão eletrónica. As amostras investigadas foram obtidas a partir da cultura primária de fibroblastos (derivada de biópsia de pele) dos quatro doentes descritos acima e de três controlos, além de sangue (estudo familiar dos P2 e P4, e controlos), músculo (P3 e P4) e fígado (P4). A avaliação funcional nas células do P1 mostrou um decréscimo nos níveis da proteína A6L (codificada pelo gene MT-ATP8) do complexo V, uma redução do assembly do complexo V, disfunção mitocondrial (nomeadamente alterações na atividade enzimática da cadeia respiratória mitocondrial, consumo de oxigénio, glicólise, níveis de ATP intracelulares, potencial de membrana mitocondrial e produção de espécies reativas de oxigénio), com evidencias de stresse no reticulo endoplasmático. Nas células do P2 foi detetada disfunção mitocondrial, com diminuição dos níveis da proteína citocromo b e da atividade do complexo III. Observou-se também a presença de corpos multilamelares, sugerindo um comprometimento da autofagia. Os fibroblastos do P3 apresentaram redução no assembly dos quatro complexos com subunidades codificadas pelo mtDNA, disfunção mitocondrial, alterações no potencial de membrana mitocondrial e na produção de espécies reativas de oxigénio, bem como a presença de corpos multilamelares. Foram ainda detetadas evidências histoquímicas e morfológicas, sugestivas de doença mitocondrial, no músculo. No P4, a deficiência na atividade enzimática da fosforilação oxidativa foi detetada apenas na biópsia de fígado e músculo, o que sugere a existência de especificidade tecidular. A redução no assembly dos quatro complexos com subunidades codificadas pelo mtDNA foi o resultado mais significativo, além do aumento anormal do tamanho das mitocôndrias. Para além disso, a investigação genética revelou uma segunda alteração (conhecida como “deleção comum”) no mtDNA (P3), descrita em associação com a patologia. Foi ainda detetada uma mutação no gene SNX14 e outra no gene MYBPC3 nos P3 e P4, respetivamente, em estudos paralelos realizados por colaboradores. O trabalho permitiu: (i) confirmar o potencial patogénico das quatro variantes no mtDNA; (ii) reportar variantes com evidências funcionais de patogenicidade; (iii) incluir estas variantes na investigação genética de doentes que apresentem fenótipo semelhante; (iv) contribuir para desenvolvimentos significativos na patogenicidade em doenças mitocondriais. Em conclusão, as evidências sugerem que: (i) as variantes analisadas estão provavelmente envolvidas na disfunção mitocondrial; (ii) défice mitocondrial pode influenciar mecanismos celulares, nomeadamente stresse no reticulo endoplasmático e a macroautofagia; (iii) as doenças mitocondriais são heterogéneas e complexas, com possível dupla origem genética, além das associadas a síndromes de depleção/deleção, já conhecidas. Abstract Mitochondrial dis orders are among the most frequent metabolic disorders and a major burden for society. There are more than 60 con firmed mitochondrial DNA (mtDNA) point mutations associated with several diseases. Since t he mtDNA is highly polymorphic with pe culiar properties, the pathogenicity of a novel sequence variation needs to be determined using a series of criteria , including functional studies, for establish ing genotype/phenotype correlation s . The present study comprises four unclassified mtDNA variants with potential pathogenic effect , identified in four unrelated P ortuguese patients suspected of mitochondrial disease, at Lab oratory of Biochemical Genetics, C enter for neuroscience and cell biology – University of Coimbra . Patient 1 (P1) presented se vere bilateral optic neuropathy. P atient 2 (P2) manifested severe intellectual disability, cerebella r a trophy, severe ataxia, coa r se face, relative macrocephaly, congenital hypotonia, absent speech, and other features such as clinodactyly. Patient 3 (P3) presented chronic progressive external ophthalmoplegia. Finally, patient 4 (P4) was suspected of cardiomyopathy after sudden death. A mtDNA variant has been identified in each patient, affecting genes encoding subunits of oxidative phosphorylation (OXPHOS) enzymatic complexes or variants in mt - tRNA genes. The alterations identified were m.8418T>C, p.Leu18Pro ( MT - ATP8 ), m.14771C>A, p.P ro9Thr ( MT - CYB ), m.7486G>A, mt - tRNA Ser(UCN) ( MT - TS1 ) m.14706A>G, mt - tRNA Glu ( MT - TE ), in patients 1, 2, 3 and 4, respectively. Accordingly, a series of biomolecular studies, using a functional genomics’ approach was conducted for evalu ating the pathogenic ity of the unclassified mtDNA variants, for establish ing the genetic diagnosis of the patients and clarify the pathogenic mechanism. The methods used included Sanger s equencing, pyrosequencing, next generation sequencing, long - range PCR, real - time PCR, his tochemistry, histology, cell culture, western - blot, blue native polyacrylamide gel electrophoresis , spectrophotometry, fluorimetry, Seahorse Bioscience ® technology and tra nsmission electron microscopy. The samples investigated included cultured fibroblasts (derived from skin biopsy) of the four patients described above plus three controls, and blood (family studies of P2 and P4 , and controls ) , muscle (P3 and P4) and liver (P4) samples when available. The functional evaluation in the skin fibroblasts of P1 showed a decrease in A6L protein level of complex V (encoded by MT -ATP8 gene), a reduction of the fully assembled complex V, mitochondrial dysfunction (namely alterations in OXPHOS enzymatic activity and oxygen consumption, glycolysis, intracellular ATP levels, mitochondrial membrane potential and reactive oxygen species production) and evidences of endoplasmic reticulum stress. In the cells of P2, a decrease in the cytochrome b levels and activity of complex III was observed. Moreover, in addition to mitochondrial dysfunction detected, the presence of multilamellar bodies were identified in skin fibroblasts, suggesting autophagy impairment. Skin fibroblasts of P3 presented a reduction in the assembly of the four complexes with subunits encoded by mtDNA, mitochondrial dysfunction, changes in mitochondrial membrane potential and in the production of reactive oxygen species. Also, multilamellar bodies were observed in fibroblasts. Additionally, morphological and histochemical evidences for mitochondrial disease were detected in patient’s muscle. In P4, deficiency of OXPHOS enzymatic activity was only observed in muscle and liver biopsy, suggesting tissue specificity. The reduction in assembly of the four complexes with subunits encoded by mtDNA was the more significant finding detected in patient’s fibroblasts, in addition to abnormal increase of mitochondria’s size. Furthermore, deep er molecular genetic investigation revealed a second mtDNA alteration in P 3, described in association with the pathology, known as “ common deletion”. Also, a muta tion in SNX14 gene (P2) and a mutation in MYBPC3 gene (P4) were detected, in parallel studies performed by other collaborators. The present work allowed: (i) to confirm the high pathogenic potential of the four unclassified mtDNA variants; (ii) to report the variants showing functional evidences for its pathogenicity; (iii) to include these sequence variations in the genetic investigation of the patients presenting similar phenotypes; (iv) to contribute for significant developments in the field of mitochondrial diseases pathogenicity. In conclusion, the evidences taken together suggest that: (i) the mtDNA variants analysed are probably involved in the observed mitochondrial dysfunction; (ii) mitochondrial impairment may influence cellular mechanisms, namely endoplasmic xli reticulum stress and macroautophagy; (iii) mitochondrial diseases are heterogeneous and complex diseases that may have a double genetic origin, besides the known depletion or deletion - associated syndromes.Mitochondrial disorders are among the most frequent metabolic disorders and a major burden for society. There are more than 60 confirmed mitochondrial DNA (mtDNA) point mutations associated with several diseases. Since the mtDNA is highly polymorphic with peculiar properties, the pathogenicity of a novel sequence variation needs to be determined using a series of criteria, including functional studies, for establishing genotype/phenotype correlations. The present study comprises four unclassified mtDNA variants with potential pathogenic effect, identified in four unrelated Portuguese patients suspected of mitochondrial disease, at Laboratory of Biochemical Genetics, Center for neuroscience and cell biology – University of Coimbra. Patient 1 (P1) presented severe bilateral optic neuropathy. Patient 2 (P2) manifested severe intellectual disability, cerebellar atrophy, severe ataxia, coarse face, relative macrocephaly, congenital hypotonia, absent speech, and other features such as clinodactyly. Patient 3 (P3) presented chronic progressive external ophthalmoplegia. Finally, patient 4 (P4) was suspected of cardiomyopathy after sudden death. A mtDNA variant has been identified in each patient, affecting genes encoding subunits of oxidative phosphorylation (OXPHOS) enzymatic complexes or variants in mt-tRNA genes. The alterations identified were m.8418T>C, p.Leu18Pro (MT-ATP8), m.14771C>A, p.Pro9Thr (MT-CYB), m.7486G>A, mt-tRNASer(UCN) (MT-TS1) m.14706A>G, mt - tRNAGlu (MT-TE), in patients 1, 2, 3 and 4, respectively. Accordingly, a series of biomolecular studies, using a functional genomics’ approach was conducted for evaluating the pathogenicity of the unclassified mtDNA variants, for establishing the genetic diagnosis of the patients and clarify the pathogenic mechanism. The methods used included Sanger sequencing, pyrosequencing, next generation sequencing, long-range PCR, real-time PCR, histochemistry, histology, cell culture, western-blot, blue native polyacrylamide gel electrophoresis, spectrophotometry, fluorimetry, Seahorse Bioscience® technology and transmission electron microscopy. The samples investigated included cultured fibroblasts (derived from skin biopsy) of the four patients described above plus three controls, and blood (family studies of P2 and P4, and controls), muscle (P3 and P4) and liver (P4) samples when available. The functional evaluation in the skin fibroblasts of P1 showed a decrease in A6L protein level of complex V (encoded by MT-ATP8 gene), a reduction of the fully assembled complex V, mitochondrial dysfunction (namely alterations in OXPHOS enzymatic activity and oxygen consumption, glycolysis, intracellular ATP levels, mitochondrial membrane potential and reactive oxygen species production) and evidences of endoplasmic reticulum stress. In the cells of P2, a decrease in the cytochrome b levels and activity of complex III was observed. Moreover, in addition to mitochondrial dysfunction detected, the presence of multilamellar bodies were identified in skin fibroblasts, suggesting autophagy impairment. Skin fibroblasts of P3 presented a reduction in the assembly of the four complexes with subunits encoded by mtDNA, mitochondrial dysfunction, changes in mitochondrial membrane potential and in the production of reactive oxygen species. Also, multilamellar bodies were observed in fibroblasts. Additionally, morphological and histochemical evidences for mitochondrial disease were detected in patient’s muscle. In P4, deficiency of OXPHOS enzymatic activity was only observed in muscle and liver biopsy, suggesting tissue specificity. The reduction in assembly of the four complexes with subunits encoded by mtDNA was the more significant finding detected in patient’s fibroblasts, in addition to abnormal increase of mitochondria’s size. Furthermore, deeper molecular genetic investigation revealed a second mtDNA alteration in P3, described in association with the pathology, known as “common deletion”. Also, a mutation in SNX14 gene (P2) and a mutation in MYBPC3 gene (P4) were detected, in parallel studies performed by other collaborators. The present work allowed: (i) to confirm the high pathogenic potential of the four unclassified mtDNA variants; (ii) to report the variants showing functional evidences for its pathogenicity; (iii) to include these sequence variations in the genetic investigation of the patients presenting similar phenotypes; (iv) to contribute for significant developments in the field of mitochondrial diseases pathogenicity. In conclusion, the evidences taken together suggest that: (i) the mtDNA variants analysed are probably involved in the observed mitochondrial dysfunction; (ii) mitochondrial impairment may influence cellular mechanisms, namely endoplasmic reticulum stress and macroautophagy; (iii) mitochondrial diseases are heterogeneous and complex diseases that may have a double genetic origin, besides the known depletion or deletion-associated syndromes.FEDER - COMPETE2020 (Strategic projects: POCI-01-0145-FEDER-007440, HealthyAging2020 CENTRO-01-0145-FEDER000012-N2323 and New Strategies to Manage Brain Diseases 2013 to 2015| CENTRO07-ST24-FEDER-002002/6/8 Programa Operacional Regional do Centro - Projecto Mais Centro

Análise experimental da patogenicidade de variações de sequência novas no genoma mitocondrial

Dissertação de mestrado em Investigação Biomédica (Neurobiologia), apresentada à Faculdade de Medicina da Universidade de CoimbraAs citopatias mitocondriais são um grupo de doenças heterogéneas, causadas por defeitos no sistema mitocondrial de produção de ATP. Este organelo tem o seu próprio genoma (mtDNA) que, quando sofre lesões/mutações, origina subunidades da cadeia respiratória mitocondrial (CRM) anormais, comprometendo o bom funcionamento da fosforilação oxidativa (OXPHOS) e, consequentemente, a síntese de ATP. Assim, é admissível que mutações, em qualquer gene essencial à integridade e funcionamento da CRM, possam causar alterações a nível da estrutura e função mitocondriais. Mutações pontuais no mtDNA incluem as que causam decréscimo na síntese de proteínas mitocondriais e as que afetam alguma das 13 subunidades da CRM, codificadas por aquele genoma. Uma vez que o mtDNA é altamente polimórfico, o significado patogénico de uma nova alteração de sequência detetada, necessita de ser determinado, usando uma série de critérios antes de se poder estabelecer o diagnóstico genético. O objetivo do presente trabalho é avaliar experimentalmente a patogenicidade de 12 novas variações de sequência no mtDNA, detetadas previamente no Laboratório de Bioquímica Genética do Centro de Neurociências e Biologia Celular, da Universidade de Coimbra (Responsável: Professora Doutora Manuela Grazina), em indivíduos portugueses com suspeita de citopatia mitocondrial, no período compreendido entre 1997 e 2010. No sentido de avaliar o impacto das variações de sequência, foram realizados diversos estudos, nomeadamente a verificação da sua ausência em controlos, por sequenciação automática. Foi ainda efetuada a determinação do seu efeito na expressão dos níveis dos transcritos respetivos por PCR em tempo real, na quantidade de proteína, por Western-blot, e na atividade dos complexos da CRM por espectrofotometria, em fibroblastos de cultura. Uma análise prévia in silico demonstrou que 4 das 12 alterações alteram o aminoácido e têm probabilidade de alterar a função da proteína, 2 alteram a estrutura de dois tRNAs e as restantes alteram o codão para outro menos frequentemente usado, no genoma mitocondrial. Os resultados experimentais obtidos no presente trabalho para a alteração m.14771C>A, no gene MT-CYB, que codifica uma subunidade do complexo III da CRM, mostram que, quando comparados com os valores controlo, os níveis do transcrito respetivo estão significativamente diminuídos, por outro lado os níveis da proteína que se encontram ligeiramente diminuídos. No que diz respeito à atividade dos complexos da CRM, verificou-se um aumento significativo na atividade do complexo III, nas células estudadas. É de notar que o presente trabalho representa uma contribuição científica original, dado que permite analisar o impacto das alterações em estudo de uma forma mais detalhada e precisa. Assim, de acordo com os critérios de patogenicidade, podemos concluir que a alteração m.12013G>A é “definitivamente” patogénica, a m.144771C>A obteve uma pontuação que permite atribuir a classificação de “provavelmente” patogénica, e as restantes alterações mantêm a previsão de “potencialmente” patogénicas, até poderem ser efetuados mais estudos.Mitochondrial disorders are a heterogeneous group of disorders that are caused by defects in the mitochondrial ATP production system. This organelle has its own genome (mtDNA), that when injured leads to the formation of abnormal mitochondrial respiratory (CRM) subunits, jeopardizing the proper functioning of oxidative phosphorylation (OXPHOS) and therefore ATP syntheses. Then, mutations in any gene essential for integrity and function of CRM could cause structural and functional mitochondrial alterations. The mtDNA point mutations include those that impair mitochondrial protein synthesis and those that affect any of the 13 CRM subunits encoded by mtDNA. Given the fact that mtDNA is highly polymorphic, the pathogenic significance of a detected novel sequence alteration needs to be determined using a series of criteria before a genetic diagnosis can be established. With this study, we aim to evaluate the pathogenicity of 12 novel mtDNA mutations, previously detected in Biochemical Genetics Laboratory of Center of Neuroscience and Cell Biology, University of Coimbra (Director: Professor Manuela Grazina), found in Portuguese patients suspected of mitochondrial cytophaties, in the period from 1997 to 2010. In order to assess the impact of these sequence variations, the study was conducted to verify their absence in controls, by automated sequencing. Furthermore, the determination of its effect in transcripts expression levels by Real-Time PCR, in protein levels by Western blot and in CRM complexes activity by spectrophotometry, using primary culture fibroblasts, was also performed. A previous in silico analysis showed that 4 of the 12 sequence variations change the amino acid and have probability to alter the protein function, 2 induce alterations of tRNAs structure and the remaining alter the codon to another less frequently used codon. The experimental results obtained for the sequence variation m.14771C>A, located in MT-CYB gene that encodes a complex III subunit of CRM, show that, when compared with control value, the transcript expression levels are significantly decreased and protein levels that are slightly diminished. Regarding CRM to complexes activity, it is significantly increased for complex III, in these cells. It is noteworthy that this study represents an original scientific contribution, allowing to analyze in more detail and precision the impact of these sequence variations. Therefore, according to published pathogenic criteria, we conclude that m.12013G>A mutation is considered “definitely” pathogenic, m.144771C>A mutation is “probably” pathogenic, while the remaining mutations are “potentially” pathogenic, until further studies can be performed

The Importance of Internal Marketing in Companies

Relatório de Estágio do Mestrado em Marketing apresentado à Faculdade de EconomiaEntidade de acolhimento: No âmbito do segundo ano do Mestrado de Marketing da Faculdade de Economia da Universidade de Coimbra, o relatório de estágio tem como objetivo primordial o estudo de um tema e a apresentação das atividades desenvolvidas na instituição selecionada pelo aluno, que neste caso foi a Monstros & Cia (Grupo CH). Esta empresa desempenha funções ao nível da Comunicação, Marketing, Design e Desenvolvimento Web.Objetivos do estágio: Este estágio curricular decorreu de 5 de novembro de 2020 a 17 de março de 2021. Os objetivos definidos com a supervisora da Monstros & Cia foram os seguintes: Desenvolvimento de competências na área da Comunicação; Aplicação dos conhecimentos teóricas na realidade empresarial; Contacto com uma vasta rede de profissionais do setor; Desenvolvimento das soft skills; Criação e Gestão de conteúdos para diferentes meios de comunicação; Rapidez de resposta e apresentação de solução para as solicitações de clientes e Gestão de meios online e offline.Contribuição teórica: O enquadramento teórico realizado neste relatório tem foco no tema do Marketing Interno. Nesta secção do trabalho é abordada a evolução histórica do Marketing Interno, é apresentado o plano de Marketing Interno, as atividades que podem ser desenvolvidas na área e vantagens e desvantagens dessas atividades para as empresas.Contribuição prática: Tendo em conta a temática do Marketing Interno, a principal contribuição prática passou pela criação de um projeto que consistiu no desenho de duas estratégias que poderão vir a ser apresentadas na empresa Monstros & Cia (Grupo CH). Destaca-se também o conjunto de atividades desenvolvidas ao longo de todo o estágio.Análise crítica/conclusões: Todas as tarefas e objetivos delineados foram cumpridos durante o estágio, sendo que todo o trabalho desenvolvido está hoje publicado, seja no jornal interno da empresa como nas redes sociais e websites de clientes da Monstros & Cia. A aprendizagem durante os 5 meses será essencial para introdução no mercado de trabalho, tendo em conta que a inserção num ambiente e contexto empresarial.Host Entity: As part of the second year of the Master in Marketing at the Faculty of Economics, University of Coimbra, the internship report has as its primary objective the study of a topic and the presentation of the activities developed in the institution selected by the student, which in this case was Monstros & Cia (Grupo CH). This company has functions in Communication, Marketing, Design and Web Development.Internship objectives: This internship ran from November 5 of 2020 to March 17 of 2021. The objectives defined with the supervisor of Monstros & Cia were the following: Development of skills in the area of Communication; Application of theoretical knowledge in business reality; Contact with a wide network of professionals in the sector; Development of soft skills; Creation and Management of content for different media; Speed of response and presentation of solution to customer requests and Management of online and offline media.Theoretical contribution: The theoretical framework carried out in this report focuses on the topic of Internal Marketing. In this section of the report is addressed the historical evolution of Internal Marketing, the internal marketing plan is presented, the activities that can be developed in the area and advantages and disadvantages of these activities for companies.Practical contribution: Considering the topic of internal marketing, the main practical contribution was the creation of a project that consisted in the design of two strategies that may be presented in the company Monstros & Cia (Grupo CH). It also stands out the set of activities developed throughout the internship.Critical analysis/conclusions: All tasks and objectives outlined were met during the internship, and all the work developed is now published, both in the company's internal newspaper and in the social media and websites of Monstros & Cia's customers. The learning during the 5 months will be essential for introduction in the labor market, considering the insertion in an environment and business context

Additive Potentiation of R334W-CFTR Function by Novel Small Molecules

The R334W (c.1000C>T, p.Arg334Trp) is a rare cystic fibrosis (CF)-causing mutation for which no causal therapy is currently approved. This mutation leads to a significant reduction of CF transmembrane conductance regulator (CFTR) channel conductance that still allows for residual function. Potentiators are small molecules that interact with CFTR protein at the plasma membrane to enhance CFTR-dependent chloride secretion, representing thus pharmacotherapies targeting the root cause of the disease. Here, we generated a new CF bronchial epithelial (CFBE) cell line to screen a collection of compounds and identify novel potentiators for R334W-CFTR. The active compounds were then validated by electrophysiological assays and their additive effects in combination with VX-770, genistein, or VX-445 were exploited in this cell line and further confirmed in intestinal organoids. Four compounds (LSO-24, LSO-25, LSO-38, and LSO-77) were active in the functional primary screen and their ability to enhance R334W-CFTR-dependent chloride secretion was confirmed using electrophysiological measurements. In silico ADME analyses demonstrated that these compounds follow Lipinski’s rule of five and are thus suggested to be orally bioavailable. Dose–response relationships revealed nevertheless suboptimal efficacy and weak potency exerted by these compounds. VX-770 and genistein also displayed a small potentiation of R334W-CFTR function, while VX-445 demonstrated no potentiator activity for this mutation. In the R334W-expressing cell line, CFTR function was further enhanced by the combination of LSO-24, LSO-25, LSO-38, or LSO-77 with VX-770, but not with genistein. The efficacy of potentiator VX-770 combined with active LSO compounds was further confirmed in intestinal organoids (R334W/R334W genotype). Taken together, these molecules were demonstrated to potentiate R334W-CFTR function by a different mechanism than that of VX-770. They may provide a feasible starting point for the design of analogs with improved CFTR-potentiator activity

Rescue of Mutant CFTR Trafficking Defect by the Investigational Compound MCG1516A

Although some therapeutic progress has been achieved in developing small molecules that correct F508del-CFTR defects, the mechanism of action (MoA) of these compounds remain poorly elucidated. Here, we investigated the effects and MoA of MCG1516A, a newly developed F508del-CFTR corrector. MCG1516A effects on wild-type (WT) and F508del-CFTR were assessed by immunofluorescence microscopy, and biochemical and functional assays both in cell lines and in intestinal organoids. To shed light on the MoA of MCG1516A, we evaluated its additivity to the FDA-approved corrector VX-661, low temperature, genetic revertants of F508del-CFTR (G550E, R1070W, and 4RK), and the traffic-null variant DD/AA. Finally, we explored the ability of MCG1516A to rescue trafficking and function of other CF-causing mutations. We found that MCG1516A rescues F508del-CFTR with additive effects to VX-661. A similar behavior was observed for WT-CFTR. Under low temperature incubation, F508del-CFTR demonstrated an additivity in processing and function with VX-661, but not with MCG1516A. In contrast, both compounds promoted additional effects to low temperature to WT-CFTR. MCG1516A demonstrated additivity to genetic revertant R1070W, while VX-661 was additive to G550E and 4RK. Nevertheless, none of these compounds rescued DD/AA trafficking. Both MCG1516A and VX-661 rescued CFTR processing of L206W- and R334W-CFTR with greater effects when these compounds were combined. In summary, the absence of additivity of MCG1516A to genetic revertant G550E suggests a putative binding site for this compound on NBD1:NBD2 interface. Therefore, a combination of MCG1516A with compounds able to rescue DD/AA traffic, or mimicking the actions of revertant R1070W (e.g., VX-661), could enhance correction of F508del-CFTR defects