Is EC class predictable from reaction mechanism?

We thank the Scottish Universities Life Sciences Alliance (SULSA) and the Scottish Overseas Research Student Awards Scheme of the Scottish Funding Council (SFC) for financial support.Background: We investigate the relationships between the EC (Enzyme Commission) class, the associated chemical reaction, and the reaction mechanism by building predictive models using Support Vector Machine (SVM), Random Forest (RF) and k-Nearest Neighbours (kNN). We consider two ways of encoding the reaction mechanism in descriptors, and also three approaches that encode only the overall chemical reaction. Both cross-validation and also an external test set are used. Results: The three descriptor sets encoding overall chemical transformation perform better than the two descriptions of mechanism. SVM and RF models perform comparably well; kNN is less successful. Oxidoreductases and hydrolases are relatively well predicted by all types of descriptor; isomerases are well predicted by overall reaction descriptors but not by mechanistic ones. Conclusions: Our results suggest that pairs of similar enzyme reactions tend to proceed by different mechanisms. Oxidoreductases, hydrolases, and to some extent isomerases and ligases, have clear chemical signatures, making them easier to predict than transferases and lyases. We find evidence that isomerases as a class are notably mechanistically diverse and that their one shared property, of substrate and product being isomers, can arise in various unrelated ways. The performance of the different machine learning algorithms is in line with many cheminformatics applications, with SVM and RF being roughly equally effective. kNN is less successful, given the role that non-local information plays in successful classification. We note also that, despite a lack of clarity in the literature, EC number prediction is not a single problem; the challenge of predicting protein function from available sequence data is quite different from assigning an EC classification from a cheminformatics representation of a reaction.Publisher PDFPeer reviewe

Phytochemical study and biological activities of diterpenes and derivatives from Plectranthus species

Tese de doutoramento, Farmácia (Química Farmacêutica e Terapêutica), Universidade de Lisboa, Faculdade de Farmácia, 2011This study focused on the research of new bioactive constituents from four species of the Plectranthus plants. Previous works on plants of the genus Plectranthus (Lamiaceæ) evidenced that some of their constituents possess interesting biological activities. The antimicrobial activity of the plant extracts and of the isolated metabolites was thoroughly searched. Antioxidant, anticholinesterase and anti-inflammatory properties of some compounds were also screened. The phytochemical study of the acetone extracts of Plectranthus ornatus Codd., P. ecklonii Benth., P. porcatus Winter & Van Jaarsv and P. saccatus Benth. rendered several terpenoid constituents mostly diterpenes. From P. ornatus three new forskolin-like labdane diterpenes (6-O-acetylforskolin, 1,6-di-O-acetylforskolin and 1,6-di-O-acetyl-9-deoxyforskolin), a new diterpene with the rare halimane skeleton (11R*-acetoxyhalima-5,13E-dien-15-oic acid), and two known labdane diterpenes were isolated; the rhinocerotinoic acid which was found in Plectranthus species for the first time, and plectrornatin C. Six known triterpenoids were also identified as mixtures. The study of P. ecklonii led to the isolation of two known abietanes, sugiol and parvifloron D. Sugiol was obtained from Plectranthus species for the first time. Four known triterpenoids were also identified as mixtures. P. porcatus, a plant not hitherto studied, yield a new spiro-abietane diterpene [(13S,15S)-6β,7α,12α,19-tetrahydroxy-13β,16-cyclo-8-abietene-11,14-dione]. A new beyerane diterpene (ent-7α-acetoxy-15-beyeren-18-oic acid) was isolated from P. saccatus. Attempting to find novel bioactive prototypes from the more potent antibacterial diterpenes, isolated in higher yields, some diterpene derivatives were prepared. Nine new derivatives were obtained from (11R*,13E)-11-acetoxyhalima-5,13-dien-15-oic acid (P. ornatus). A new 2β-(4-hydroxy)benzoyloxy derivative of microstegiol was prepared from parvifloron D (P. ecklonii). From the 7α-acetoxy-6β-hydroxyroyleanone (isolated in the past from P. grandidentatus) thirteen ester derivatives were synthesized, whereof ten were new compounds. The unequivocal chemical structures of pure compounds (natural and derivatives) were deduced from their spectroscopic (IR, MS, 1D and 2D NMR experiments) and physico-chemical data, as well as from literature information. The preliminary antimicrobial activity screenings of all the isolated metabolites showed that several diterpenes inhibited the growth of the Gram positive bacteria tested. In addition, the minimum inhibitory concentration against standard and clinical isolates of sensitive and resistant Staphylococcus and Enterococcus strains was determined for the antibacterial metabolites and their synthesized derivatives. The (11R*,13E)-11-acetoxyhalima-5,13-dien-15-oic acid and its (11R*,13E)-halima-5,13-diene-11,15-diol derivative were the more active halimanes. Parvifloron D was less active than its microstegiol 2β-(4-hydroxy)benzoate derivative, but both showed more potent antibacterial activities than the halimane diterpenoids. The three 12-O-benzoyl esters derivatives of the 7α-acetoxy-6β-hydroxyroyleanone prototype revealed to be more potent growth inhibitors against Staphylococcus and Enterococcus strains than the prototype. The 6β-propionyloxy-12-O-propionyl derivative also showed to be more active against Enterococcus than the viii prototype. Generally, the 12-esters and the 6,12-diesters were more active against Enterococcus than Staphylococcus strains. The hydrophobic extra-interactions with the bacterial targets seem to play an important role on the activity of royleanones derivatives prepared. Taking into account the IC50 values which expressed the scavenging DPPH radical ability, the isolated metabolite parvifloron D as well as 7α-acetoxy-6β-hydroxyroyleanone showed in vitro antioxidant activity. The in vitro acetylcholinesterase assay did not detect any activity for all the newly isolated diterpenes and 7α-acetoxy-6β-hydroxyroyleanone. The COX inhibitor screening assay was tested on 6-O-acetylforskolin, rhinocerotinoic acid, plectrornatin C, (11R*,13E)-halima-5,13-diene-11,15- diol, 11R*-acetoxyhalima-5,13E-dien-15-oic acid and on its methyl ester, for their ability to inhibit COX-2. The preliminary results encourage further studies aiming to confirm and to examine its potential anti-inflammatory activity in a more robust approach.estudo teve como objectivo a pesquisa de novos constituintes bioactivos de quatro espécies de plantas do género Plectranthus. A actividade antimicrobiana dos extractos obtidos e dos metabolitos isolados foi realizada e foram testadas as propriedades anti-oxidante, anti-colinesterase e anti-inflamatória de alguns compostos. O estudo fitoquímico dos extractos de acetona de Plectranthus ornatus Codd., P. ecklonii Benth., P. porcatus Winter & Van Jaarsv. e P. saccatus Benth. originou diversos constituintes terpénicos, principalmente diterpenos. Três novos diterpenos do tipo forskolina (6-O-acetilforskolina; 1,6-di-O-acetilforskolina e 1,6-di-O-acetil-9-deoxiforskolina) foram isolados de P. ornatus. Foram também identificados um novo diterpeno com o raro esqueleto de halimano (ácido 11R*-acetoxihalima-5,13E-dien-15-óico), dois diterpenos labdânicos conhecidos; o ácido rinocerotinóico encontrado pela primeira vez em espécies do género Plectranthus, e a plectrornatina C. Seis triterpenos já conhecidos foram igualmente identificados na forma de misturas. O estudo de P. ecklonii originou o isolamento de dois abietanos conhecidos: o sugiol e a parviflorona D. O sugiol foi isolado pela primeira vez de espécies Plectranthus. Outros quatro triterpenos conhecidos foram identificados também como misturas. A planta P. porcatus, até à data não estudada, originou um novo diterpeno spiro-abietânico [(13S,15S)-6β,7α,12α,19-tetrahidroxi-13β,16-ciclo-8-abietene-11,14-diona]. Um novo diterpeno com esqueleto de beierano (ácido ent-7α-acetoxi-15-beieren-18-óico) foi isolado de P. saccatus. Na tentativa de obter novos protótipos bioactivos, vários derivados foram preparados, a partir dos diterpenos antibacterianos mais potentes e isolados em maior quantidade. Nove novos derivados foram obtidos do ácido (11R*,13E)-11-acetoxihalima-5,13-dien-15-óico (P. ornatus). Um novo derivado 2β-(4-hidroxi)benzoilado do microstegiol, foi preparado a partir da parviflorona D (P. ecklonii). Treze ésteres derivados da 7α-acetoxi-6β-hidroxiroyleanona (isolada anteriormente de P. grandidentatus) foram sintetizados, sendo de assinalar que dez dos derivados são compostos novos. A determinação estrutural dos compostos puros (naturais e derivados) foi deduzida por espectroscopia (IV, EM, RMN 1D e 2D), propriedades físico-químicas e com base na informação obtida da literatura. O estudo preliminar da actividade antimicrobiana de todos os metabolitos isolados, mostrou que diversos diterpenos inibem o crescimento de bactérias de Gram positivo. A concentração mínima inibitória (CMI) dos metabolitos e seus derivados foi determinada em estirpes de Staphylococcus e Enterococcus, tanto em bactérias padrão como em isolados clínicos resistentes e sensíveis a antibióticos. O ácido (11R*,13E)-11-acetoxihalima-5,13-dien-15-óico e o seu derivado (11R*,13E)-halima-5,13-diene-11,15-diol foram os halimanos mais activos. A parviflorona D foi menos activa do que o seu correspondente derivado 2β-(4-hidroxi)benzoilado, mas ambos apresentaram uma actividade antibacteriana mais potente do que os diterpenos com esqueleto de halimano. Os três 12-O-benzoil-ésteres derivados do protótipo 7α-acetoxi-6β-hidroxiroyleanona revelaram ser inibidores mais potentes do que a royleanona-protótipo, contra as estirpes testadas de Staphylococcus e Enterococcus. O derivado 6β-propioniloxi-12-O-propionilo mostrou ser o mais activo contra as estirpes testadas de Enterococcus do que o protótipo. De um modo geral, os derivados 12-ésteres e os 6,12-diésteres foram mais activos contra as estirpes de Enterococcus do que as estirpes de Staphylococcus testadas. As interacções hidrofóbicas com os alvos bacterianos parecem ter um papel importante na actividade antibacteriana dos derivados de royleanona preparados. Os metabolitos parviflorona D e a 7α-acetoxi-6β-hidroxiroyleanona demostraram possuir actividade antioxidante in vitro, tendo em conta os valores de IC50 que expressam a actividade anti-oxidante com base na captura do radical DPPH. Todos os novos diterpenos isolados e derivados obtidos neste trabalho foram testados e não revelaram possuir actividade inibitória da acetilcolinesterase in vitro. A actividade anti-inflamatória foi testada nos compostos 6-O-acetilforskolina, ácido rinocerotinóico, plectrornatina C, (11R*,13E)-halima-5,13-diene-11,15-diol, ácido 11R*-acetoxihalima-5,13E-dien-15-óico e no seu éster metílico, através da sua capacidade de inibir a COX-2. Os resultados preliminares obtidos apoiam a necessidade de estudos futuros de forma a confirmar, explorar e discutir uma potencial actividade anti-inflamatória.The research work was performed, mostly, in the Faculdade de Farmácia da Universidade de Lisboa at the Medicinal Chemistry Group (former Centro de Estudos de Ciências Farmacêuticas – CECF) of the Institute for Medicines and Pharmaceutical Sciences (iMed.UL). Funding to these research centres and the attribution of a Doctoral degree grant (SFRH/BD/19250/2004) were provided by the Fundação para a Ciência e a Tecnologia - Ministério da Ciência, Tecnologia e Ensino Superior (FCT-MCTES)