Cyclophilins (Cyp) are proteins that catalyze the interconversion of trans/cis isomers of proline belonging to the peptidyl-prolyl isomerases family (PPIase). In addition to their PPIase activity, Cyps have diverse biological roles and have been implicated in a number of different diseases such as HIV-1 and HCV. Although several Cyp inhibitors have been reported in the literature, none are able to inhibit with high specificity various Cyp isoforms. To facilitate the development of isoform-specific Cyp ligands, we have pursued detailed studies of Cyp dynamics and ligand binding thermodynamics using molecular simulations, biophysical assays and protein X-ray crystallography. Research efforts were focussed on the identification of novel Cyp inhibitors using X-ray crystallographic studies and Surface Plasmon Resonance (SPR) experiments on fragments from an in-house bespoke library of small compounds. These biophysical studies revealed a number of fragments that are able to bind to diverse Cyp isoforms with high micromolar – low millimolar activity. To further examine the binding of these fragments to cyclophilins, identify interactions with the proteins and explain specificity trends from SPR and X-ray results, molecular dynamics (MD) simulations and free energy calculations were pursued. Models of apo and holo Cyps in complex with fragments that we had experimentally tested were set up using the Amber, AmberTools and FESetup software. Free energy calculations were performed using the thermodynamic integration (TI) technique with the Sire/OpenMM software. The results were analysed with custom scripts. Correlations between computed and measured binding energies, and calculated and observed binding modes were analysed to help develop guidelines for the development of isoform specific cyclophilin ligands. A detailed comparison of the merits and drawbacks of the experimental and computational techniques used in this work has also been made, and strategies for effective combination of the methodologies in structure-based projects are outlined.

La découverte et le développement d'un médicament est un processus long et très coûteux. Pour améliorer ce processus, une nouvelle approche de criblage et de conception rationnelle de nouveaux ligands est apparue durant la dernière décennie. Elle se base sur le criblage par des techniques de biophysique structurale, de petites molécules organiques appelées "fragments". Ces fragments sont caractérisés par une faible affinité pour la cible mais par une grande efficacité de liaison. Cette approche a été mise en place au CBS et appliquée à la découverte de nouveaux inhibiteurs des cyclophilines humaines. Les cyclophilines (Cyps) sont des protéines ubiquitaires chez l'Homme. Elles possèdent une activité peptidyl-prolyl cis-trans isomèrase et aident au repliement des protéines. Il a été montré que le développement d'inhibiteurs de ces protéines pourrait déboucher sur de nouveaux traitements pour des pathologies telles que le VIH, le VHC, certains cancers ou Alzheimer. Nous avons découvert, par un criblage RMN puis une validation par cristallographie aux rayons X, 14 fragments-touches millimolaires sur les Cyps. Ces fragments-touches ont permis de déterminer une nouvelle famille chimique d'inhibiteurs des Cyps. Le meilleur inhibiteur possède une activité 1-10 µM sur CypA, B et D, et de la dizaine de micromolaires sur la réplication du VHC dans les cellules. Un brevet a été déposé sur cette famille chimique. / Drug discovery and development is a long and expensive process. To improve this process, a new approach of creening and rational drug design appeared during the last decade. This approach is based on screening of small organic compounds called "fragments" by structural biophysic techniques. These fragments are characterized by low affinity for the target but high ligand efficiency. This approach was implemented at the CBS and applied to find new human cyclophilin inhibitors. Cyclophilins (Cyps) are ubiquitous proteins in Human. They have a peptidyl-prolyl cis-transisomerase activity and help protein folding. It was shown that development of inhibitors for these proteins could lead to new treatments against HIV, HCV, cancers or Alzheimer diseases for example. We discovered 14 fragment hits in the millimolar range on the Cyps by NMR screening and further validation by X-ray crystallography. With these fragment hits, we identified a new family of chemical inhibitors of the Cyps. The best inhibitor has 1-10 µM activity on CypA, B and D, and around 10 micromolar activity on HCV replication in cells. A patent has been deposited for this family of chemical inhibitors.

FKBP-65 is a member of the immunophilin class of proteins consisting primarily of the cyclophilins and the FKBP's which bind the immunosuppressant drugs cyclosporin A and FK506, respectively. Immunophilins possess peptidylprolyl cis-trans isomerase (PPiase) activity which is inhibited upon binding of their respective macrolides. Specific cellular targets of most immunophilins and the role of PPiase activity in vivo remain largely unknown. FKBP-65 has been proposed as a molecular chaperone of tropoelastin (TE), the soluble precursor of elastin (Davis et al. 1998). TE contains 12% proline residues, many of which are found in VPGVG repeats. When P2 is in the trans conformation, these motifs form repeated type-II ~-turns and ~-spirals resulting in selfassociation of TE via an inverse temperature-dependent transition known as coacervation. Coacervation can be monitored by turbidity increases at 300 nm. We have used purified recombinant FKBP-65 in coacervation assays with chick aorta TE to show that FKBP65 specifically affects the coacervation characteristics of TE in a concentration-dependant manner. The overall extent of coacervation of TE could be increased by more than 2-fold over controls by inclusion of nM amounts of FKBP-65 in the assay. Also, FKBP-65 decreases the coacervation onset temperature of TE by 5-l 0°C. Structural evidence suggests that the influence of FKBP65 on tropoelastin coacervation may be due to its ability to increase the ~ structural content of tropoelastin. These results suggest that FKBP-65 may be a physiologically relevant, TE-specific molecular chaperone. / Thesis / Master of Science (MSc)

Diss. (sammanfattning) Linköping : Linköpings universitet, 2007. / Härtill 4 uppsatser.

Les dernières décennies ont été marquées par l’émergence ou la réémergence d’un nombre croissant de virus pathogènes. Malheureusement, les antiviraux actuellement sur le marché ciblent un nombre restreint de virus ; il y a donc un besoin urgent de développer des antiviraux à large spectre. Les cyclophilines sont des protéines cellulaires impliquées dans un grand nombre de processus biologiques, qui possèdent une activité enzymatique peptidyl-prolyl cis-trans isomérase (PPIase). Elles sont également impliquées dans la réplication de virus appartenant à des familles éloignées et constituent donc une cible de choix pour le développement d'antiviraux à large spectre. Toutefois, les inhibiteurs de cyclophilines disponibles possèdent de nombreux inconvénients qui rendent leur utilisation clinique difficile.Par une stratégie de "fragment-based drug design", nous avons généré une nouvelle famille d'inhibiteurs de cyclophilines, les SMCypI ("Small-Molecule Cyclophilin Inhibitors"), complètement différents de tous les inhibiteurs de cyclophilines existants. La cristallographie de ces composés a montré qu'ils se fixaient dans les deux poches voisines du site actif des cyclophilines et qu'ils inhibaient leur activité PPIase. Ces composés n’étaient pas immunosuppressifs et bloquaient in vitro l'infection par le VIH, le VHC et les Coronavirus.L'activité anti-VHC du C31, composé le plus actif sur l'activité PPIase des cyclophilines, a été caractérisée. Le C31 était un inhibiteur pan-génotypique du VHC, doté d’une haute barrière contre la résistance et présentant une activité additive avec les inhibiteurs du VHC approuvés. Nous avons montré que le C31 bloquait l'infection par le VHC en rompant l'interaction entre la protéine virale NS5A et la cyclophiline A de façon PPIase-dépendante. Enfin, le C31 était actif sur la réplication des virus zika, de la dengue, de la fièvre jaune et du Nil Occidental.L'activité des SMCypI a été caractérisée sur l'infection par le Coronavirus 229E. De manière intéressante, l’inhibition de l’activité PPIase était nécessaire, mais pas suffisante pour l’activité antivirale. Une étude de la relation structure-activité des composés a révélé qu'un groupement chimique situé à l'interface entre les deux poches du site actif des cyclophilines jouait un rôle clé dans l'effet anti-coronavirus. Le F836 a été identifié comme le composé le plus actif, qui bloquait l'effet cytopathique et la quantité d'ARN du HCoV-229E avec la même efficacité que l'alisporivir, sans toxicité associée. Ce composé bloquait l'entrée du HCoV-229E après l'attachement du virus à la surface cellulaire, et était également actif sur l'entrée des HCoV-OC43 et du MERS-CoV. Nous avons par la suite démontré l’association de la cyclophiline A avec les particules virales. Par l'utilisation de la technologie CRISPR-Cas9, des cellules invalidées pour la cyclophiline A ont été générées. La cyclophiline A apparaissait nécessaire pour l'infection par HCoV-229E et la cible de l'effet antiviral du F836.Les SMCypI constituent un outil pour la compréhension des mécanismes par lesquels les cyclophilines modulent les infections virales et représentent des candidats crédibles pour le développement futur d'antiviraux à large spectre. / Over the past decades, an increasing number of viruses has emerged or re-emerged in humans. Unfortunately, currently approved antiviral drugs target a small set of viruses. Thus, there is an urgent need for the development of broad-spectrum antiviral drugs.Cyclophilins are cellular proteins involved in a large number of biological processes, and in different viral lifecycles from unrelated families. They appear as a potential target for the development of broad-spectrum antiviral approaches. However, currently available cyclophilin inhibitors have drawbacks which limit their clinical use.By means of "fragment-based drug design", we generated a new class of small-molecule cyclophilin inhibitors (SMCypI), unrelated with those already available. Cristallographic studies revealed that the SMCypIs bind to two close pockets of the active site and inhibit cyclophilin PPIase activity. These compounds do not bear immunosuppressive properties and inhibit the replication of HIV, HCV and coronaviruses in vitro.We characterized the anti-HCV activity of C31, the most potent inhibitor of cyclophilin PPIase activity. C31 had pan-genotypic HCV inhibitor properties, with a high barrier to resistance and additive effects with currently approved anti-HCV agents. C31 blocked HCV replication by disrupting the interaction between the nonstructural viral protein NS5A and cyclophilin A in a PPIase-dependent manner. Finally, C31 was active on zika, yellow fever, dengue and West-Nile virus infections.The antiviral activity of the SMCypIs has then been characterized on HCoV-229E infection. Interestingly, PPIase inhibition was necessary, but not sufficient for antiviral effect. A structure-activity relationship study identified a key moiety in the SMCypIs at the interface between the two cyclophilin pockets. F836 has been identified as the most potent compound which inhibited both the cytopathic effect and the intracellular RNA of HCoV-229E without associated cytotoxicity and as potently as alisporivir. This compound targeted HCoV-229E entry at a post-attachment step and was also active on HCoV-OC43 and MERS-CoV strains. We then demonstrated that cyclophilin A was associated with viral particles. By means of CRISPR-Cas9, cell lines depleted for cyclophilin A were generated. Cyclophilin A was identified as a proviral factor for HCoV-229E and was partially involved in F836 antiviral effect. Cyclophilin A expression level was drastically decreased by infection.SMCypIs represent a unique tool to decipher the cellular and molecular mechanisms by which cyclophilins interfere with viral lifecycles, as well as drugable compounds that could find an indication as broad-spectrum antiviral drugs.

Orientador: Celso Eduardo Benedetti / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T05:38:04Z (GMT). No. of bitstreams: 1 Campos_BrunaMedeia_D.pdf: 41386343 bytes, checksum: 8048b677c8a43e7438a1c349c584b9ec (MD5) Previous issue date: 2013 / Resumo: O cancro cítrico, causado pelo fitopatógeno Xanthomonas axonopodis pv. citri (Xac) constitui uma doença que afeta todos os cultivares comerciais de citros e é uma ameaça para a citricultura brasileira. A doença é caracterizada pela formação de pústulas, devido à hiperplasia e hipertrofia induzida pela bactéria. A patogenicidade de Xac é dependente do sistema secretório tipo III, que transloca proteínas efetoras pertencentes à família AvrBs3/PthA para dentro da célula hospedeira. Estudos recentes mostram que PthAs funcionam como fatores de transcrição no hospedeiro, transativando genes específicos da planta que irão beneficiar a bactéria ou desencadear uma resposta de defesa. Com o objetivo de entender melhor o mecanismo de ação de PthAs como ativadores da transcrição, nosso laboratório identificou, através da técnica de duplo-híbrido, várias proteínas de laranja (Citrus sinensis) que interagiram com diferentes PthAs. Entre elas, destacamos uma ciclofilina (CsCyp), que realiza isomerização de resíduos de prolina, uma proteína com domínio tioredoxina (CsTdx), relacionada com interação proteína-proteína e redução de pontes dissulfeto e uma cisteína protease (CsCP), envolvida na resposta de defesa da planta. Este trabalho teve como objetivo a caracterização estrutural e funcional de CsCyp. A resolução da estrutura de CsCyp mostrou que CsCyp pertence ao grupo de ciclofilinas divergentes que possuem um loop adicional (KSGKPLH), duas cisteínas invariáveis (C40 e C168) e um glutamato (E83) conservado. Este último interage com resíduos do loop, estabilizando-o. A função destes elementos era desconhecida até o momento e o trabalho visou elucidar sua atuação na regulação da atividade PPIase da proteína. Neste trabalho, verificamos que C40 e C168 formam uma ponte dissulfeto. Dados de modelagem e simulação suportaram a hipótese de que a formação da ponte dissulfeto induz mudanças conformacionais que quebram a interação do E83 com o loop divergente, levando ao fechamento do sítio ativo de CsCyp. O estudo descreveu, portanto um mecanismo de regulação redox, que controla a atividade PPIase da proteína. Adicionalmente, mostrou-se que CsTdx interage com CsCyp através dos resíduos conservados C40 e C168, sendo críticos para tal interação. Foi mostrado também que CsCyp interage com o domínio C-terminal (CTD) da RNA Polimerase II (RNA Pol II), especificamente com a repetição YSPSAP. Surpreendentemente, através da transformação de plantas com construções para silenciamento e superexpressão de CsCyp, verificamos que plantas de citros com níveis reduzidos de CsCyp apresentaram um aumento dos sintomas do cancro cítrico quando infiltradas com Xac, enquanto que plantas com níveis aumentados de CsCyp apresentaram sintomas reduzidos quando infiltradas com Xac, indicando que CsCyp tem papel importante no desenvolvimento dos sintomas do cancro cítrico. Este trabalho também mostra a expressão e purificação das proteínas CsTdx e CsCP e a comprovação de que CsCP interage com PthAs 2 e 3 através de ensaios de duplo-híbrido / Abstract: Citrus canker, caused by Xanthomonas axonopodis pv. citri (Xac) is a disease that affects most species of the genus Citrus and represents a threat to the Brazilian citrus industry. The disease is characterized by the formation of pustules due to hyperplasia and hypertrophy induced by the bacteria. Xac pathogenicity is dependent on a type III secretory system that translocates effector proteins which belongs to AvrBs3/PthA family inside the host cell. Recent studies showed that these proteins work as transcriptional factors that transactivate specific plant genes which will either benefit the bacteria or trigger defense response. To gain insights into PthA mechanism of action as transcription activators, our laboratory identified that PthA targeted the citrus protein complex comprising the thioredoxin CsTdx, ubiquitin-conjugating enzymes CsUev/Ubc13 and cyclophilin CsCyp. Also, we showed previously that the CsCyp binds the citrus thioredoxin CsTdx and the C-terminal domain of RNA Polymerase II (CTD), and that CsCyp complements the function of Cpr1 and Ess1, two yeast prolyl-isomerases that regulate transcription by the isomerization of proline residues of the regulatory C-terminal domain (CTD) of RNA polymerase II. In this work we solved the 3D structure of CsCyp in complex with its inhibitor cyclosporine A (CsA), showing that CsCyp is a divergent cyclophilin that carries the additional loop KSGKPLH, invariable cysteines C40 and C168, and conserved glutamate E83. Despite the suggested roles in ATP and metal binding, the function of these unique structural elements remains unknown. Here we show that the conserved cysteines form a disulfide bond that inactivates the enzyme, whereas E83, which belongs to the catalytic loop and is also critical for enzyme activity, is anchored to the divergent loop to maintain the active site open. In addition, we demonstrate that C40 and C168 are required for the interaction with CsTdx and that CsCyp binds the citrus CTD YSPSAP repeat. Our data support the model where formation of the C40- C168 disulfide bond induces a conformational change that disrupts the interaction of the divergent and catalytic loops, via E83, causing the active site to close. This suggests a new type of allosteric regulation in divergent cyclophilins, involving disulfide bond formation and a loop displacement mechanism. Moreover, we present evidence that PthA2 inhibits the peptidyl-prolyl cis-trans isomerase (PPIase) activity of CsCyp in a similar fashion as CsA, and that silencing of CsCyp, as well as treatments with CsA, enhance canker lesions in Xac-infected leaves. Given that CsCyp appears to function as a negative regulator of cell growth and that Ess1 negatively regulates transcription elongation in yeast, we propose that PthAs activate host transcription by inhibiting the PPIase activity of CsCyp on the CTD / Doutorado / Genetica de Microorganismos / Doutora em Genética e Biologia Molecular