Small-molecule inhibitors for the Prp8 intein as antifungal agents.

Self-splicing proteins, called inteins, are present in many human pathogens, including the emerging fungal threats Cryptococcus neoformans (Cne) and Cryptococcus gattii (Cga), the causative agents of cryptococcosis. Inhibition of protein splicing in Cryptococcus sp. interferes with activity of the only intein-containing protein, Prp8, an essential intron splicing factor. Here, we screened a small-molecule library to find addititonal, potent inhibitors of the Cne Prp8 intein using a split-GFP splicing assay. This revealed the compound 6G-318S, with IC50 values in the low micromolar range in the split-GFP assay and in a complementary split-luciferase system. A fluoride derivative of the compound 6G-318S displayed improved cytotoxicity in human lung carcinoma cells, although there was a slight reduction in the inhibition of splicing. 6G-318S and its derivative inhibited splicing of the Cne Prp8 intein in vivo in Escherichia coli and in C. neoformans Moreover, the compounds repressed growth of WT C. neoformans and C. gattii In contrast, the inhibitors were less potent at inhibiting growth of the inteinless Candida albicans Drug resistance was observed when the Prp8 intein was overexpressed in C. neoformans, indicating specificity of this molecule toward the target. No off-target activity was observed, such as inhibition of serine/cysteine proteases. The inhibitors bound covalently to the Prp8 intein and binding was reduced when the active-site residue Cys1 was mutated. 6G-318S showed a synergistic effect with amphotericin B and additive to indifferent effects with a few other clinically used antimycotics. Overall, the identification of these small-molecule intein-splicing inhibitors opens up prospects for a new class of antifungals.

Spliceosomal Prp8 intein at the crossroads of protein and RNA splicing.

The spliceosome is a large ribonucleoprotein complex that removes introns from pre-mRNAs. At its functional core lies the essential pre-mRNA processing factor 8 (Prp8) protein. Across diverse eukaryotes, this protein cofactor of RNA catalysis harbors a self-splicing element called an intein. Inteins in Prp8 are extremely pervasive and are found at 7 different sites in various species. Here, we focus on the Prp8 intein from Cryptococcus neoformans (Cne), a human fungal pathogen. We solved the crystal structure of this intein, revealing structural homology among protein splicing sequences in eukaryotes, including the Hedgehog C terminus. Working with the Cne Prp8 intein in a reporter assay, we find that the biologically relevant divalent metals copper and zinc inhibit intein splicing, albeit by 2 different mechanisms. Copper likely stimulates reversible modifications on a catalytically important cysteine, whereas zinc binds at the terminal asparagine and the same critical cysteine. Importantly, we also show that copper treatment inhibits Prp8 protein splicing in Cne. Lastly, an intein-containing Prp8 precursor model is presented, suggesting that metal-induced protein splicing inhibition would disturb function of both Prp8 and the spliceosome. These results indicate that Prp8 protein splicing can be modulated, with potential functional implications for the spliceosome.

SufB intein of Mycobacterium tuberculosis as a sensor for oxidative and nitrosative stresses.

Inteins are mobile genetic elements that self-splice at the protein level. Mycobacteria have inteins inserted into several important genes, including those corresponding to the iron-sulfur cluster assembly protein SufB. Curiously, the SufB inteins are found primarily in mycobacterial species that are potential human pathogens. Here we discovered an exceptional sensitivity of Mycobacterium tuberculosis SufB intein splicing to oxidative and nitrosative stresses when expressed in Escherichia coli. This effect results from predisposition of the intein's catalytic cysteine residues to oxidative and nitrosative modifications. Experiments with a fluorescent reporter system revealed that reactive oxygen species and reactive nitrogen species inhibit SufB extein ligation by forcing either precursor accumulation or N-terminal cleavage. We propose that splicing inhibition is an immediate, posttranslational regulatory response that can be either reversible, by inducing precursor accumulation, or irreversible, by inducing N-terminal cleavage, which may potentially channel mycobacteria into dormancy under extreme oxidative and nitrosative stresses.

Exploring Intein Inhibition by Platinum Compounds as an Antimicrobial Strategy.

Inteins, self-splicing protein elements, interrupt genes and proteins in many microbes, including the human pathogen Mycobacterium tuberculosis Using conserved catalytic nucleophiles at their N- and C-terminal splice junctions, inteins are able to excise out of precursor polypeptides. The splicing of the intein in the mycobacterial recombinase RecA is specifically inhibited by the widely used cancer therapeutic cisplatin, cis-[Pt(NH3)2Cl2], and this compound inhibits mycobacterial growth. Mass spectrometric and crystallographic studies of Pt(II) binding to the RecA intein revealed a complex in which two platinum atoms bind at N- and C-terminal catalytic cysteine residues. Kinetic analyses of NMR spectroscopic data support a two-step binding mechanism in which a Pt(II) first rapidly interacts reversibly at the N terminus followed by a slower, first order irreversible binding event involving both the N and C termini. Notably, the ligands of Pt(II) compounds that are required for chemotherapeutic efficacy and toxicity are no longer bound to the metal atom in the intein adduct. The lack of ammine ligands and need for phosphine represent a springboard for future design of platinum-based compounds targeting inteins. Because the intein splicing mechanism is conserved across a range of pathogenic microbes, developing these drugs could lead to novel, broad range antimicrobial agents.

Cisplatin protects mice from challenge of Cryptococcus neoformans by targeting the Prp8 intein.

The Prp8 intein is one of the most widespread eukaryotic inteins, present in important pathogenic fungi, including Cryptococcus and Aspergillus species. Because the processed Prp8 carries out essential and non-redundant cellular functions, a Prp8 intein inhibitor is a mechanistically novel antifungal agent. In this report, we demonstrated that cisplatin, an FDA-approved cancer drug, significantly arrested growth of Prp8 intein-containing fungi C. neoformans and C. gattii, but only poorly inhibited growth of intein-free Candida species. These results suggest that cisplatin arrests fungal growth through specific inhibition of the Prp8 intein. Cisplatin was also found to significantly inhibit growth of C. neoformans in a mouse model. Our results further showed that cisplatin inhibited Prp8 intein splicing in vitro in a dose-dependent manner by direct binding to the Prp8 intein. Crystal structures of the apo- and cisplatin-bound Prp8 inteins revealed that two degenerate cisplatin molecules bind at the intein active site. Mutation of the splicing-site residues led to loss of cisplatin binding, as well as impairment of intein splicing. Finally, we found that overexpression of the Prp8 intein in cryptococcal species conferred cisplatin resistance. Overall, these results indicate that the Prp8 intein is a novel antifungal target worth further investigation.

The dynamic intein landscape of eukaryotes.

BACKGROUND: Inteins are mobile, self-splicing sequences that interrupt proteins and occur across all three domains of life. Scrutiny of the intein landscape in prokaryotes led to the hypothesis that some inteins are functionally important. Our focus shifts to eukaryotic inteins to assess their diversity, distribution, and dissemination, with the aim to comprehensively evaluate the eukaryotic intein landscape, understand intein maintenance, and dissect evolutionary relationships. RESULTS: This bioinformatics study reveals that eukaryotic inteins are scarce, but present in nuclear genomes of fungi, chloroplast genomes of algae, and within some eukaryotic viruses. There is a preponderance of inteins in several fungal pathogens of humans and plants. Inteins are pervasive in certain proteins, including the nuclear RNA splicing factor, Prp8, and the chloroplast DNA helicase, DnaB. We find that eukaryotic inteins frequently localize to unstructured loops of the host protein, often at highly conserved sites. More broadly, a sequence similarity network analysis of all eukaryotic inteins uncovered several routes of intein mobility. Some eukaryotic inteins appear to have been acquired through horizontal transfer with dsDNA viruses, yet other inteins are spread through intragenomic transfer. Remarkably, endosymbiosis can explain patterns of DnaB intein inheritance across several algal phyla, a novel mechanism for intein acquisition and distribution. CONCLUSIONS: Overall, an intriguing picture emerges for how the eukaryotic intein landscape arose, with many evolutionary forces having contributed to its current state. Our collective results provide a framework for exploring inteins as novel regulatory elements and innovative drug targets.