Introns, stretches of RNA that are initially transcribed from DNA but spliced out prior to translation, are a basic feature of RNA.
Although less common, some proteins have their own equivalent of introns. Such so-called inteins are translated from mRNA, but splice themselves out of the initial amino acid string before proteins fold up into their final shape.
Now, researchers from the State University of New York (SUNY) at Albany have identified small molecules that could inhibit intein self-splicing from the protein PrP8 in the fungal pathogens Cryptococcus neoformans and Cryptococcus gattii, emerging pathogens that can cause fungal meningitis in immunocompromised patients.
The team published its findings in the January 4, 2021, online issue of the Proceedings of the National Academy of Sciences.
The function of inteins is still largely a mystery. But where they occur, they are "most commonly found in proteins that are critical to cell survival, so splicing inhibitors kill the cell," senior author Marlene Belfort told BioWorld Science.
"Protein splicing is a form of post-translational modification, but rather than a subtle change to a single amino-acid residue, an entire protein (the intein) excises itself and the flanking protein sequences are joined or spliced," she added. "So, if one inhibits protein splicing, the protein that needs to be expressed (joined) has the intein stuck in it and cannot function."
And while inteins are widespread among bacteria and archaea, they do not appear to occur in human proteins -- and within bacteria, they have not been found in the human microbiome, suggesting the possibility that intein targeting could disrupt pathogens without undue effects on their human hosts, or the host microbiome.
Potentially, targeting inteins could play a role in fighting one of the biggest infection disease scourges of humanity -- Mycobacterium tuberculosis, which infects a quarter of the world's population and kills 1.5 million people every year, more people than any other infectious agent.
Several M. tuberculosis proteins contain inteins, and Belfort, a distinguished professor of biological sciences and biomedical sciences at SUNY at Albany and senior advisor to its RNA Institute, and her colleagues had previously shown that the chemotherapy cisplatin could prevent inteins from splicing themselves out of M. tuberculosis proteins, killing the bacteria.
Cisplatin, though, "is very toxic by virtue of its DNA-binding activity," Belfort said, making it unsuitable for the immunocompromised patients. "But in the crystal structure the ligands that bind DNA are missing.... providing a path for development of less toxic anti-intein drugs."
To identify such less-toxic intein drugs, the team turned its attention to fungi. Belfort's laboratory is interested in RNA splicing, and several pathogenic fungi, including C. neoformans and C. gattii as well as Aspergillus fumigatus, have inteins in the protein Prp8, which is a key protein in the RNA spliceosome, placing it "at the crossroads of protein and RNA splicing," Belfort said. Consequently, her lab "had a particular interest in the Prp8 intein in cryptococci because of our interest in Prp8 as an RNA splicing factor."
Clinically, fungal pathogens are becoming more of a public health problem. Candida auris is now considered a top tier urgent threat by the Centers for Disease Control and Prevention (CDC), and other fungal infections, too, are becoming more dire threats.
Intein and outtakes
In the work now published in PNAS, Belfort, co-corresponding author Hongmin Li of the University of Arizona, and their colleagues first developed screening assays to identify proteins that could inhibit intein splicing in Prp8, and used them to screen a library of 300 small-molecule compounds. They identified the organic amide 6G-318S as the strongest intein splicing inhibitor within that library. Chemical optimization resulted in a compound that was less toxic to human cells.
The team confirmed the intein target by demonstrating that 6G-318S repressed the growth of fungi that had inteins in Prp8, but were not as effective against Candida albicans, a fungus that lacks inteins. Furthermore, overexpression of Prp8 led to resistance against G6-318S.
Belfort said that the team is currently optimizing the compound's drug-like properties. Already, she said, the compounds she and her colleagues have identified are "as effective as current antimycotics, with some having additive and even synergistic activities with anti-fungal drugs, which is exciting. At the same time, these compounds are less toxic to cells than cisplatin."