If a fully formed protein is known to mediate disease, it might seem like a good idea to keep the protein from developing in the first place, to nip it in the bud, so to speak. And, in fact, agents have been found that can prevent protein translation by stalling the ribosome, the general-purpose molecular machines that synthesize proteins from messenger RNA transcripts. Unfortunately, these agents are like the proverbial monkey wrench in the works. They disrupt the ribosome no matter what protein it is making.
Finer translation-stalling tools may be possible, however. That’s the upshot of a new paper prepared by researchers at the University of California, Berkeley, and Pfizer Worldwide Research and Development. These researchers report that they have found a chemical compound that halts the production of a small set of proteins but does not disturb general protein production.
This compound, the researchers assert, could point to a new drug search strategy: Find compounds that target undesired proteins before they are even made. The researchers add that this strategy could be especially valuable in the development of small molecules targeting disease-mediating proteins that were previously thought to be undruggable.
Details about the new compound and its mechanism of action appeared March 21 in the journal PLOS Biology in an article entitled “Selective Stalling of Human Translation through Small-Molecule Engagement of the Ribosome Nascent Chain.”
“Here, we demonstrate that the compound PF-06446846 inhibits translation of PCSK9 [proprotein convertase subtilisin/kexin type 9] by inducing the ribosome to stall around codon 34, mediated by the sequence of the nascent chain within the exit tunnel,” wrote the article’s authors. “We further show that PF-06446846 reduces plasma PCSK9 and total cholesterol levels in rats following oral dosing.”
When delivered orally to rats, the small molecule lowered low-density lipoprotein cholesterol (LDL-C) levels, much the way statins do, though by a different mechanism: by lowering the production of the protein PCSK9.
While antibiotics like erythromycin are known to stall the ribosome, they halt production of most proteins, said Jamie Cate, Ph.D., one of two senior authors, a UC Berkeley professor of molecular and cell biology and of chemistry and a faculty scientist at Lawrence Berkeley National Laboratory.
The chemical in this instance stalls the ribosome only when it’s producing the protein PCSK9 and a couple of dozen others out of the tens of thousands of proteins the body produces, as shown by a relatively new technique called ribosomal profiling.
“PCSK9 was just where we started. Now we can think about how to come up with other small molecules that hit proteins that nobody has been able to target before because, maybe, they have a floppy part, or they don’t have a nook or cranny where you can bind a small molecule to inhibit them,” Cate said. “This research is saying, we may be able to just prevent the synthesis of the protein in the first place.”
Cate suspects that the small molecule in the current study, a multiringed chlorinated compound, could serve as a template, like a key blank that can be machined to open a specific lock.
“We now have this key blank that we can cut in a number of different ways to try to go after undruggable proteins in a number of different disease states,” Cate said. “No one really thought that would have been possible before.”
The small molecule was discovered by Pfizer labs through live-cell screening for compounds that lower production of the protein PCSK9, which regulates the recycling of the LDL receptor. Knocking out the protein is known to lower blood levels of LDL-C, the so-called “bad” cholesterol, presumably lowering risk of cardiovascular disease. PCSK9 inhibitors, mostly monoclonal antibodies, actually lower LDL better than the well-known statins, though they have to be injected into the bloodstream.
When it became clear that the chemical was acting on the ribosome, Spiros Liras, Ph.D., vp of medicinal chemistry at Pfizer, approached Cate and Jennifer Doudna, Ph.D., both leaders in the field of ribosome function and translation, to establish a collaboration through UC Berkeley’s California Institute for Quantitative Bioscience (QB3) to further investigate the questions of selectivity and mechanism of action. Cate is also director of UC Berkeley’s Center for RNA Systems Biology, while Doudna is a professor of molecular and cell biology and of chemistry, a Howard Hughes Medical Institute investigator, and executive director of the Innovative Genomics Institute.
“Pfizer brought a significant depth of knowledge and resources to the collaboration, including fundamental cell biology, disease-relevant expertise, chemical biology, and medicinal chemistry,” said Liras. “We aimed at building a strong cross-institutional collaboration that would complement our strengths in drug discovery with UC Berkeley’s strengths in ribosome biochemistry and structural biology.”
In the PLOS Biology paper, Cate, Robert Dullea, Ph.D., at Pfizer, and their teams at UC Berkeley and Pfizer describe how the drug interacts with the ribosome to halt protein production.
According to Cate, the ribosome assembles amino acids into a chain inside a tunnel that holds about 30 to 40 amino acids before the end begins to poke out of the tunnel. The chemical studied appears to bind to specific amino acid sequences of the growing protein within that tunnel in the ribosome and makes them kink enough to stop progress down the tunnel, halting protein synthesis.
“We found that the proteins that are stalled are too short to stick outside the ribosome,” Cate said. “So we think the compound is actually trapping this snake-like chain, the starting part of the protein, in the tunnel—not completely blocking the tunnel, but just partially blocking it, in a way that prevents this particular protein from making its way out.”
While it’s still unclear what the two dozen proteins affected have in common that makes them susceptible to stalling by the small molecule, Cate sees these findings as clear evidence that ribosomal stalling can occur very specifically, something most researchers thought unlikely.
“We think that we now have enough understanding of the mechanism that we have our foot in the door to explore the relevance of this biology more broadly,” said Cate.
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