Semisynthetic preparation of C‑nucleoside 5´-triphosphates as bacterial RNA polymerase substrates

Interest in natural products as pharmaceutical leads has recently resurfaced due to increasing understanding towards the biosynthesis of natural compounds as well as advancements in methodologies that facilitate the semisynthetic preparation of novel compounds. Natural C-nucleosides are one class of bioactive compounds that have seen their biosynthetic background being elucidated in the last few years. Furthermore, the higher stability of the C-nucleosides against enzymatic and chemical hydrolysis compared to the N-nucleosides makes these compounds interesting for their potential medicinal properties. The presented thesis focuses on semisynthesis of C-nucleoside 5´-triphosphates for studying their activity against RNA polymerases (RNAP) that are common drug targets for nucleoside-based pharmaceuticals. The natural C-nucleosides showdomycin, oxazinomycin and pseudouridimycin were produced as starting materials by cultivating strains of Streptomyces soil bacteria. Additionally, a small compound library of showdomycin analogues was prepared, by modifying the maleimide-type base moiety. The chemical 5´-phosphorylation was carried out by using the commonly used Yoshikawa-Ludwig protocol. However, the unmodified showdomycin unexpectedly isomerized during the phosphorylation affording isoshowdomycin-5´-triphosphate. In the RNAP assays, the efficiency of oxazinomycin-5´-triphosphate as a substrate was comparable to that of uridine-5´-triphosphate. In addition, in certain sequence contexts the incorporation of oxazinomycin to the RNA arrested the transcription process. On the other hand, the most promising showdomycin analogue, 4-(ethylthio)showdomycin-5´-triphosphate showed modest inhibition activity and slight selectivity towards the bacterial RNAP.C-nukleosidi-5´-trifosfaattien puolisynteettinen valmistus substraateiksi bakteriaaliselle RNA polymeraasille Mielenkiinto luonnonyhdisteiden käyttöä kohtaan lääkekehityksen johtoyhdisteinä on viime aikoina kohonnut uudelleen. Tähän ovat osaltaan vaikuttaneet lisääntynyt ymmärrys luonnonyhdisteiden biosynteesiä kohtaan sekä kehittyneet menetelmät, jotka helpottavat uusien yhdisteiden puolisynteettistä valmistusta. Luonnolliset Cnukleosidit ovat yksi bioaktiivinen yhdisteryhmä, jonka biosynteettinen alkuperä on selventynyt viimeisimpien vuosien aikana. Lisäksi C-nukleosidien suurempi pysyvyys entsymaattista ja kemiallista hydrolyysiä kohtaan verrattuna Nnukleosideihin tekee näistä yhdisteistä mielenkiintoisia mahdollisten lääkinnällisten ominaisuuksiensa vuoksi. Tämä väistöskirjatyö keskittyy C-nukleosidien 5´-trifosfaattien puolisynteesiin niiden aktiivisuuden tutkimiseksi RNA polymeraasien (RNAP) kanssa, jotka ovat yleinen nukleosidipohjaisten lääkeaineiden vaikutuskohde. Lähtöaineiksi tuotettiin luonnollisiin C-nukleosideihin kuuluvat showdomysiini, oksatsinomysiini ja pseudouridimysiini viljelemällä Streptomyces-suvun maaperäbakteereja. Lisäksi valmistettiin pieni, showdomysiini-johdannaisista koostuva yhdistekirjasto modifioimalla sen maleimidi-tyyppistä emäsosaa. Valmistetut C-nukleosidit fosforyloitiin kemiallisesti käyttäen yleisestä Yoshikawa-Ludwig -menetelmää. Muokkaamaton showdomysiini kuitenkin isomeroitui odottamattomasti fosforylaation aikana antaen tuotteeksi iso-showdomysiini-5´-trifosfaatin. RNAP–kokeissa oksatsinomysiini-5´- trifosfaatin kyky toimia substraattina oli verrattavissa uridiini-5´-trifosfaattiin. Lisäksi oksatsinomysiinin liittyminen RNA:han tietyissä emäsjärjestyskonteksteissa keskeytti transkription. Sen sijaan showdomysiinijohdannaisista lupaavin, 4- (etyylitio)showdomysiini osoitti kohtalaista inhibitioaktiivisuutta ja lievää selektiivisyyttä bakteeriperäistä RNA polymeraasia kohtaan

The DNA polymerase of bacteriophage YerA41 replicates its T-modified DNA in a primer-independent manner

Yersinia phage YerA41 is morphologically similar to jumbo bacteriophages. The isolated genomic material of YerA41 could not be digested by restriction enzymes, and used as a template by conventional DNA polymerases. Nucleoside analysis of the YerA41 genomic material, carried out to find out whether this was due to modified nucleotides, revealed the presence of a ca 1 kDa substitution of thymidine with apparent oligosaccharide character. We identified and purified the phage DNA polymerase (DNAP) that could replicate the YerA41 genomic DNA even without added primers. Cryo-electron microscopy (EM) was used to characterize structural details of the phage particle. The storage capacity of the 131 nm diameter head was calculated to accommodate a significantly longer genome than that of the 145 577 bp genomic DNA of YerA41 determined here. Indeed, cryo-EM revealed, in contrast to the 25 angstrom in other phages, spacings of 33-36 angstrom between shells of the genomic material inside YerA41 heads suggesting that the heavily substituted thymidine increases significantly the spacing of the DNA packaged inside the capsid. In conclusion, YerA41 appears to be an unconventional phage that packages thymidine-modified genomic DNA into its capsids along with its own DNAP that has the ability to replicate the genome.Peer reviewe

The role of the maleimide ring system on the structure-activity relationship of showdomycin

Showdomycin produced by Streptomyces showdoensis ATCC 15227 is a C-nucleoside microbial natural product with antimicrobial and cytotoxic properties. The unique feature of showdomycin in comparison to other nucleosides is its maleimide base moiety, which has the distinct ability to alkylate nucleophilic thiol groups by a Michael addition reaction. In order to understand structure-activity relationships of showdomycin, we synthesized a series of derivatives with modifications in the maleimide ring at the site of alkylation to moderate its reactivity. The showdomycin congeners were designed to retain the planarity of the base ring system to allow Watson-Crick base pairing and preserve the nucleosidic character of the compounds. Consequently, we synthesized triphosphates of showdomycin derivatives and tested their activity against RNA polymerases. Bromo, methylthio, and ethylthio derivatives of showdomycin were incorporated into RNA by bacterial and mitochondrial RNA polymerases and somewhat less efficiently by the eukaryotic RNA polymerase II. Showdomycin derivatives acted as uridine mimics and delayed further extension of the RNA chain by multi-subunit, but not mitochondrial RNA polymerases. Bioactivity profiling indicated that the mechanism of action of ethylthioshowdomycin was altered, with approximately 4-fold reduction in both cytotoxicity against human embryonic kidney cells and antibacterial activity against Escherichia coli. In addition, the ethylthio derivative was not inactivated by medium components or influenced by addition of uridine in contrast to showdomycin. The results explain how both the maleimide ring and the nucleoside nature contribute to the bioactivity of showdomycin and demonstrates for the first time that the two activities can be separated.</p

The DNA polymerase of bacteriophage YerA41 replicates its T-modified DNA in a primer-independent manner

Yersinia phage YerA41 is morphologically similar to jumbo bacteriophages. The isolated genomic material of YerA41 could not be digested by restriction enzymes, and used as a template by conventional DNA polymerases. Nucleoside analysis of the YerA41 genomic material, carried out to find out whether this was due to modified nucleotides, revealed the presence of a ca 1 kDa substitution of thymidine with apparent oligosaccharide character. We identified and purified the phage DNA polymerase (DNAP) that could replicate the YerA41 genomic DNA even without added primers. Cryo-electron microscopy (EM) was used to characterize structural details of the phage particle. The storage capacity of the 131 nm diameter head was calculated to accommodate a significantly longer genome than that of the 145 577 bp genomic DNA of YerA41 determined here. Indeed, cryo-EM revealed, in contrast to the 25 angstrom in other phages, spacings of 33-36 angstrom between shells of the genomic material inside YerA41 heads suggesting that the heavily substituted thymidine increases significantly the spacing of the DNA packaged inside the capsid. In conclusion, YerA41 appears to be an unconventional phage that packages thymidine-modified genomic DNA into its capsids along with its own DNAP that has the ability to replicate the genome

PDE6D Inhibitors with a New Design Principle Selectively Block K-Ras Activity

The trafficking chaperone PDE6D (also referred to as PDE delta) has been nominated as a surrogate target for K-Ras4B (hereafter K-Ras). Arl2-assisted unloading of K-Ras from PDE6D in the perinuclear area is significant for correct K-Ras localization and therefore activity. However, the unloading mechanism also leads to the undesired ejection of PDE6D inhibitors. To counteract ejection, others have recently optimized inhibitors for picomolar affinities; however, cell penetration generally seems to remain an issue. To increase resilience against ejection, we engineered a "chemical spring" into prenyl-binding pocket inhibitors of PDE6D. Furthermore, cell penetration was improved by attaching a cell-penetration group, allowing us to arrive at micromolar in cellulo potencies in the first generation. Our model compounds, Deltaflexin-1 and -2, selectively disrupt K-Ras, but not H-Ras membrane organization. This selectivity profile is reflected in the antiproliferative activity on colorectal and breast cancer cells, as well as the ability to block sternness traits of lung and breast cancer cells. While our current model compounds still have a low in vitro potency, we expect that our modular and simple inhibitor redesign could significantly advance the development of pharmacologically more potent compounds against PDE6D and related targets, such as UNC119 in the future

PDE6D Inhibitors with a New Design Principle Selectively Block K-Ras Activity

The trafficking chaperone PDE6D (also referred to as PDE?) has been nominated as a surrogate target for K-Ras4B (hereafter K-Ras). Arl2-assisted unloading of K-Ras from PDE6D in the perinuclear area is significant for correct K-Ras localization and therefore activity. However, the unloading mechanism also leads to the undesired ejection of PDE6D inhibitors. To counteract ejection, others have recently optimized inhibitors for picomolar affinities; however, cell penetration generally seems to remain an issue. To increase resilience against ejection, we engineered a "chemical spring" into prenyl-binding pocket inhibitors of PDE6D. Furthermore, cell penetration was improved by attaching a cell-penetration group, allowing us to arrive at micromolar in cellulo potencies in the first generation. Our model compounds, Deltaflexin-1 and -2, selectively disrupt K-Ras, but not H-Ras membrane organization. This selectivity profile is reflected in the antiproliferative activity on colorectal and breast cancer cells, as well as the ability to block stemness traits of lung and breast cancer cells. While our current model compounds still have a low in vitro potency, we expect that our modular and simple inhibitor redesign could significantly advance the development of pharmacologically more potent compounds against PDE6D and related targets, such as UNC119 in the future

Nukleosidien ja niiden analogien N-glykosidisen sidoksen synteesi

N-glykosidisen sidoksen muodostamiseksi nukleosidisynteesissä on kehitetty monia erilaisia menetelmiä. Tutkielmassa käsiteltäviä nukleosidien N-glykosylaatiomenetelmiä ovat nukleoemäksen metallisuolan glykosylaatio, fuusiomenetelmä, Hilbert-Johnson-reaktio, silyyli-Hilbert-Johnson-menetelmä eli Vorbrüggen-menetelmä, syklisten enolieetterien aktivointi, Mitsunobu-reaktio, palladium- ja kultakatalyyttiset nukleosidikondensaatiot, intramolekulaarinen N-glykosylaatio ja Click-kemiaan perustuvat nukleosidianalogien synteesimenetelmät. Lisäksi kirjallisessa osassa käsitellään kemiallisesti katalysoitua ja entsymaattista transglykosylaatiota ja nukleosidien syynteesimenetelmiä, joissa emäsosa syklisoidaan sokerin 1-aseman substituentin, kuten amiiniryhmän ympärille. Vorbrüggen-menetelmää käytetään nykyään eniten nukleosidien N-glykosylaatiossa, mutta epätavallisten nukleosidianalogien kuten paikkaisomeerien synteeseissä jokin muu menetelmä voi olla käyttökelpoisempi. Tutkielman kokeellisessa osassa esitetään kahden deoksikoformysiinin analogin valmistamiseksi kehitettyjä synteesimenetelmiä. Deoksikoformysiini luokitellaan adenosiinianalogiksi, joka toimii tehokkaana inhibiittorina adenosiinideaminaasi-entsyymeille ja jota käytetään myös karvasoluleukemian hoidossa. Synteesin kohteina olevien analogien tarkoitus olisi toimia spesifisenä inhibiittorina tyypin 2 adenosiinideaminaasille (ADA2). Tällaista inhibiittoria voitaisiin käyttää ADA2-entsyymin toimintaa sekä deoksikoformysiinin lääkeainevaikutusta koskevissa tutkimuksissa. Synteesin kohteena olevat yhdisteet olivat deoksikormysiinin α-anomeeri sekä 5´-aminosubstituoitu analogi. Kyseisten yhdisteiden N-glykosylaatiota varten valmistettiin sopivasti modifioidut ja suojatut sokeriyksiköt. Koformysiiniyhdisteiden bisyklisen emäsosan synteesi osoittautui kuitenkin ongelmalliseksi eikä N-glykosylaatiovaihetta täten ehditty suorittamaan. Emäsosan synteesivaiheita täytyy optimoida tai vaihtoehtoisesti emäsosa voidaan hankkia kaupallisesti.Siirretty Doriast

Stability of the Phosphotriester PDE6D Inhibitors

peer reviewedAbstract The kinetics for the cleavage of the phosphotriester PDE6D inhibitors 1 (Deltaflexin-2) and 3 (Deltaflexin-1) and their derivatives 2, 4 and 5 is presented under various conditions in aqueous solutions. The conversion of the phosphotriesters (1?5) into the phosphodiesters 1**?5** was detected to take place as a major degradation process. In the absence of enzyme, the 4-acetylthio-2,2-dimethyl-3-oxobutyl protected compounds (1, 4 and 5) are one order of magnitude more stable than the 4-acetylthio-2-ethoxycarbonyl-3-oxo-2-methylbutyl protected ones (2 and 3). In cell culture (DMEM) containing fetal bovine serum and l-glutamine, an intermediary formation of S?S-dimer competed with the removal of the protecting group after deacetylation of the starting material. In addition, the susceptibility of the compounds to amine nucleophiles as well as their stability under acidic and basic condition were determined in non-aqueous solutions

Discovery of the Showdomycin Gene Cluster from <i>Streptomyces showdoensis</i> ATCC 15227 Yields Insight into the Biosynthetic Logic of C‑Nucleoside Antibiotics

Nucleoside antibiotics are a large class of pharmaceutically relevant chemical entities, which exhibit a broad spectrum of biological activities. Most nucleosides belong to the canonical N-nucleoside family, where the heterocyclic unit is connected to the carbohydrate through a carbon–nitrogen bond. However, atypical C-nucleosides were isolated from <i>Streptomyces</i> bacteria over 50 years ago, but the molecular basis for formation of these metabolites has been unknown. Here, we have sequenced the genome of <i>S. showdoensis</i> ATCC 15227 and identified the gene cluster responsible for showdomycin production. Key to the detection was the presence of <i>sdmA</i>, encoding an enzyme of the pseudouridine monophosphate glycosidase family, which could catalyze formation of the C-glycosidic bond. Sequence analysis revealed an unusual combination of biosynthetic genes, while inactivation and subsequent complementation of <i>sdmA</i> confirmed the involvement of the locus in showdomycin formation. The study provides the first steps toward generation of novel C-nucleosides by pathway engineering