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 Streptomyces bacteria over 50 years ago, but the molecular basis for formation of these metabolites has been unknown. Here, we have sequenced the genome of S. showdoensis ATCC 15227 and identified the gene cluster responsible for showdomycin production. Key to the detection was the presence of sdmA, 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 sdmA confirmed the involvement of the locus in showdomycin formation. The study provides the first steps toward generation of novel C-nucleosides by pathway engineering.

The trafficking chaperone PDE6D (also referred to as PDEδ) 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.

Pseudouridimycin (PUM), a selective inhibitor of bacterial RNA polymerase has been previously detected in microbial-extracts of two strains of Streptomyces species (strain ID38640 and ID38673). Here, we isolated PUM and its deoxygenated analogue desoxy-pseudouridimycin (dPUM) from Streptomyces albus DSM 40763, previously reported to produce the metabolite strepturidin (STU). The isolated compounds were characterized by HRMS and spectroscopic techniques and they selectively inhibited transcription by bacterial RNA polymerase as previously reported for PUM. In contrast, STU could not be detected in the cultures of S. albus DSM 40763. As the reported characteristics reported for STU are almost identical with that of PUM, the existence of STU was questioned. We further sequenced the genome of S. albus DSM 40763 and identified a gene cluster that contains orthologs of all PUM biosynthesis enzymes but lacks the enzymes that would conceivably allow biosynthesis of STU as an additional product.

Oxazinomycin is a C-nucleoside antibiotic that is produced by Streptomyces hygroscopicus and closely resembles uridine. Here, we show that the oxazinomycin triphosphate is a good substrate for bacterial and eukaryotic RNA polymerases (RNAPs) and that a single incorporated oxazinomycin is rapidly extended by the next nucleotide. However, the incorporation of several successive oxazinomycins or a single oxazinomycin in a certain sequence context arrested a fraction of the transcribing RNAP. The addition of Gre RNA cleavage factors eliminated the transcriptional arrest at a single oxazinomycin and shortened the nascent RNAs arrested at the polythymidine sequences suggesting that the transcriptional arrest was caused by backtracking of RNAP along the DNA template. We further demonstrate that the ubiquitous C-nucleoside pseudouridine is also a good substrate for RNA polymerases in a triphosphorylated form but does not inhibit transcription of the polythymidine sequences. Our results collectively suggest that oxazinomycin functions as a Trojan horse substrate and its inhibitory effect is attributable to the oxygen atom in the position corresponding to carbon five of the uracil ring.

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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.

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 Å in other phages, spacings of 33–36 Å 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.

Pseudouridimycin is a microbial C-nucleoside natural product that specifically inhibits bacterial RNA polymerases by binding to the active site and competing with uridine triphosphate for the nucleoside triphosphate (NTP) addition site. Pseudouridimycin consists of 5′-aminopseudouridine and formamidinylated, N-hydroxylated Gly−Gln dipeptide moieties to allow Watson−Crick base pairing and to mimic protein−ligand interactions of the triphosphates of NTP, respectively. The metabolic pathway of pseudouridimycin has been studied in Streptomyces species, but no biosynthetic steps have been characterized biochemically. Here, we show that the flavin-dependent oxidase SapB functions as a gate-keeper enzyme selecting pseudouridine (K M = 34 μM) over uridine (K M = 901 μM) in the formation of pseudouridine aldehyde. The pyridoxal phosphate (PLP)-dependent SapH catalyzes transamination, resulting in 5′-aminopseudouridine with a preference for arginine, methionine, or phenylalanine as cosubstrates as amino group donors. The binary structure of SapH in complex with pyridoxamine-5′-phosphate and site-directed mutagenesis identified Lys289 and Trp32 as key residues for catalysis and substrate binding, respectively. The related C-nucleoside oxazinomycin was accepted as a substrate by SapB with moderate affinity (K M = 181 μM) and was further converted by SapH, which opens possibilities for metabolic engineering to generate hybrid C-nucleoside pseudouridimycin analogues in Streptomyces.