Bacterial ‘Docking Domains’ May Open New Paths to Next-Generation HDAC Inhibitors

Researchers have uncovered the molecular mechanism bacteria use to build a family of natural anticancer compounds, a discovery that could help scientists engineer improved versions of histone deacetylase (HDAC) inhibitors for cancer treatment.

The study, published in Nature Communications, identifies the long-elusive biosynthetic pathway for FR-901375, a naturally occurring HDAC inhibitor closely related to the approved lymphoma drug Romidepsin. The findings also explain how bacteria “mix and match” components of these molecules to generate structurally diverse compounds, providing a blueprint for designing new drug candidates.

HDAC inhibitors block histone deacetylases—enzymes that help regulate which genes are switched on or off inside cells. By inhibiting these enzymes, the drugs can reactivate genes that suppress tumor growth or trigger cancer cell death. Romidepsin (Istodax) is already approved to treat certain T-cell lymphomas, but researchers have long been interested in developing additional members of this drug family that are more selective and effective.

Although FR-901375 has been known for decades, scientists had never identified the bacterial genes or molecular machinery responsible for producing it.

The new study fills that gap.

The researchers identified the previously unknown biosynthetic gene cluster for FR-901375 in Pseudomonas chlororaphis subsp. piscium and used genetic, biochemical, and structural approaches—including AlphaFold modeling, mutagenesis, mass spectrometry, and gene deletion experiments—to determine how the compound is assembled.

Like Romidepsin and related compounds, FR-901375 belongs to a family of cyclic molecules known as depsipeptides. These drugs are built inside bacteria by enormous enzyme complexes called PKS-NRPS hybrids, which combine two natural-product assembly systems to construct the finished molecule.

A key finding was the discovery of how small protein regions known as docking domains allow different sections of this assembly line to communicate. These molecular connectors enable the portion of the machinery that builds a conserved zinc-binding pharmacophore—the business end of the drug that inhibits HDAC enzymes—to link with a second set of enzymes that constructs a variable peptide “cap.” Differences in this cap influence how individual drugs interact with different HDAC enzymes.

“The βHD domain employs a mechanism to facilitate productive engagement of PKS and NRPS subunits, involving direct binding to a conserved epitope on the SLiM-bearing ACP domain,” the authors write.

The researchers found that one docking element, known as the β-hairpin docking (βHD) domain, plays the central role in joining the two biosynthetic systems. Surprisingly, another docking element previously thought to be equally important contributed relatively little to the interaction and was not essential for FR-901375 production inside bacterial cells.

Structural modeling and laboratory experiments showed that the βHD domain directly binds a conserved region of the acyl carrier protein, allowing the growing molecule to be transferred efficiently from one enzyme complex to the next. The same interaction was conserved across several related HDAC inhibitor pathways, suggesting bacteria use a common strategy to generate multiple drug variants.

“The observation that ACP-SLiM and βHD-C di-domains from noncognate depsipeptide HDAC inhibitor assembly lines engage productively supports the view that the interaction epitope between βHD and ACP domains… plays a key role in the biosynthesis of all members of this clinically important family of anticancer agents,” the authors write.

The team also reconstructed how the FR-901375 pathway likely evolved through gene transfer, duplication, and recombination events that modified the peptide-building portion of the biosynthetic machinery while preserving its ability to connect with the conserved pharmacophore assembly system.

According to the researchers, understanding this evolutionary process could help scientists design entirely new HDAC inhibitors using the same modular strategy that bacteria have refined over millions of years.

“Our work delivers deep insight into evolutionary mechanisms underpinning the combinatorial biosynthesis of depsipeptide HDAC inhibitors,” the authors conclude. “Moreover, it provides a rational basis for developing approaches to the creation of analogues of depsipeptide HDAC inhibitors and other hybrid polyketide-nonribosomal peptides via evolution-guided biosynthetic engineering.”

The findings could ultimately accelerate efforts to develop next-generation HDAC inhibitors with improved potency, greater selectivity, and fewer side effects for treating cancer.