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. 2021 Aug 24;12(9):1493–1497. doi: 10.1021/acsmedchemlett.1c00378

An Enantiodefined Conformationally Constrained Fatty Acid Mimetic and Potent Inhibitor of ToxT

Lauren E Markham 1, Jessica D Tolbert 1, F Jon Kull 1, Charles R Midgett 1, Glenn C Micalizio 1,*
PMCID: PMC8436414  PMID: 34531958

Abstract

graphic file with name ml1c00378_0005.jpg

The chiral conformation that palmitoleic acid takes when it is bound to ToxT, the master regulator of virulence genes in the bacterial pathogen Vibrio cholerae, was used as inspiration to design a novel class of fatty acid mimetics. The best mimetic, based on a chiral hydrindane, was found to be a potent inhibitor of this target. The synthetic chemistry that enabled these studies was based on the sequential use of a stereoselective annulative cross-coupling reaction and dissolving metal reduction to establish the C13 and C9 stereocenters, respectively.

Keywords: Palmitoleic acid, transcription factor, hydrindane, metallacycle-mediated annulative cross-coupling

Free fatty acids play important roles in biology, serving as signaling agents that regulate diverse processes including inflammation and metabolism.1 Thus, proteins regulated by fatty acids are compelling targets for medicinal chemistry.2 While the natural achiral ligands of these targets are functionally competent, they usually have low binding affinities, due in part to the substantial loss in entropy incurred as they adopt a specific three-dimensional conformation when bound. Here we report an approach for the design and synthesis of conformationally constrained chiral mimetics of palmitoleic acid and show one to be a potent inhibitor of ToxT, a transcription factor that is essential for the bacterial enteropathogen Vibrio cholerae to cause the debilitating diarrheal disease cholera (Figure 1A). As illustrated in Figure 1B, the fatty acid mimetics of interest were based on a chiral hydrindane core skeleton and thought to be accessible by metallacycle-mediated annulative cross-coupling technology3 and subsequent functionalization. Overall, these studies demonstrate that modern metallacycle-mediated coupling technology can be leveraged to deliver structurally unique and conformationally constrained fatty acid mimetics and that the approach taken can drive the discovery of potent inhibitors after the synthesis of a relatively small number of novel compounds.

Figure 1.

Figure 1

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Structure of the ToxT–palmitoleic acid complex and synthesis of a fatty acid mimetic and potent inhibitor of this transcriptional activator.

Fatty acids are known to inhibit bacterial virulence in part by acting on AraC transcription factors.4,5 In V. cholerae, ToxT activates the expression of cholera toxin and the toxin-coregulated pilus, which is inhibited by the unsaturated fatty acid components of bile.6−8 In 2010, the X-ray crystal structure of ToxT was solved,4 revealing a bound molecule of palmitoleic acid, an unsaturated fatty acid shown to be a weak inhibitor of virulence (IC50 estimated to be ∼100 μM). When bound to ToxT, the normally unconstrained fatty acid adopts a “U-shaped” conformation (Figure 2) that is distinct from other conformations it takes when bound to other biological macromolecules.9,10 To develop potent inhibitors of ToxT, it was hypothesized that the synthesis of conformationally constrained chiral ligands that support positioning of the carboxylic acid and saturated hydrocarbon tail similar to that observed in the palmitoleic acid–ToxT structure should offer increased potency and selectivity. Such constrained mimetics would be capable of accessing only a small subset of conformations, an entropically favorable characteristic over the highly flexible natural fatty acid ligands.

Figure 2.

Figure 2

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Structure of palmitoleic acid and its conformations when bound to ToxT, human afamin, and pFABP5. The palmitoleic acid conformations were overlaid by aligning C9 and C10.

As depicted in Figure 3A, the bound conformation of palmitoleic acid shown might be effectively mimicked by a conformationally constrained chiral hydrindane. The bicyclic unsaturated hydrocarbon core of such a species should occupy a similar position in ToxT as palmitoleic acid, and the chirality and substitution should properly orient the carboxylic acid headgroup (projecting from C9) and the hydrophobic tail (projecting from C8). Here, allylic-1,2 strain was reasoned to be a feature that would further support the desired positioning of the side chain at C9, with a gearing effect impacting the preferred orientation of the hydrophobic tail bound to C8. At the start of this inquiry, it was not immediately clear what the optimal length of each side chain should be at C8 and C9 to result in the most potent ToxT inhibitor, and therefore, the synthetic pathway conceived to generate candidates was designed to be flexible with respect to the nature of these side chains (e.g., m and n).

Figure 3.

Figure 3

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Design and asymmetric synthesis of hydrindane-based mimetics of palmitoleic acid for inhibition of ToxT.

As depicted in Figure 3B, Ti-mediated alkoxide-directed annulative cross-coupling of TMS-alkyne 1 with chiral enyne 2 delivered stereodefined hydrindane 3.3 Partial purification of this intermediate was accomplished with silica gel chromatography, and the slightly impure material obtained from this was moved forward in the synthetic sequence. Oxidation at C16 by the action of Dess–Martin periodinane11 was followed by protodesilylation through simple treatment with HCl (1 M) to deliver stereodefined dienone 4 in 18% yield over the three-step sequence. Dissolving metal reduction and kinetic quenching of the resulting conjugated enolate delivered the expected β,γ-unsaturated ketone in 64% yield with the C9 stereocenter being established with very high levels of stereoselectivity (no spectroscopic evidence was found for the other isomer).12 This intermediate was subsequently reduced (NaBH4, MeOH/DCM) at C16 to generate the corresponding homoallylic alcohol and then deoxygenated by conversion to the xanthate (NaH, CS2, MeI) and treatment with the combination of Bu3SnH and AIBN.13 Overall, this sequence of transformations delivered stereodefined hydrindane 5 in 84% yield. Finally, acetal deprotection by exposure to HCl in aqueous THF delivered aldehyde 6 in 69% yield, and oxidation to the carboxylic acid ultimately generated chiral conformationally constrained acid 7 in 83% yield. Notably, from intermediate 5, the side chain projecting from C9 could easily be elongated by Horner–Wadsworth–Emmons olefination14 followed by saponification (6 → 8).

By means of this chemical sequence, a small panel of potential mimetics of palmitoleic acid was prepared featuring members that had minor perturbations in structure, specifically in the lengths and natures of the side chains projecting from C8 and C9 (Figure 4B; also see the Supporting Information). This small collection was then evaluated alongside the known ToxT inhibitors virstatin15 and (±)-916 (Figure 4A) in a cellular assay (V. cholerae El Tor strain C6706) based on a β-galactosidase reporter. This assay has been used to assess the activity of ToxT at the tcpA promoter, one of the central virulence factors of V. cholerae,16 and it has historically been conducted at a few selected concentrations to provide an assessment of potential small-molecule inhibitors of virulence.16

Figure 4.

Figure 4

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Chiral hydrindanes inhibit ToxT activity (El Tor strain). (A, B) Structures of (A) known inhibitors and (B) inhibitors reported in this paper. (C) Scatter plot showing representative dose responses of the ToxT-induced relative β-galactosidase activity in the presence of virstatin (Vir), (±)-9, and the five new compounds (at 5, 50, 500, and 5000 nM). The lines are shown to emphasize the dose-dependent differences between Vir, (±)-9, 11, and ent-11. The bar graphs show controls used for each experiment. All of the trials were normalized to the WT with DMSO. Each plotted data point is the mean ± SD of n = 3 independent trials. None of the compounds were toxic (Table S1).

As depicted in Figure 4C, the established benchmark ToxT inhibitor virstatin showed low to modest inhibition at concentrations up to 5 μM, with minor changes in activity at the four concentrations tested (all ∼20% inhibition).16 The racemic compound (±)-9,17 which has previously been shown to be highly active against the noncirculating classical strain (∼80% inhibition at 500 nM),16 achieved only ∼45% inhibition of this El Tor strain at 500 nM. The varying efficacies of this compound in classical versus El Tor strains is not surprising given the differences in conditions required to induce virulence factor expression under laboratory conditions.

Moving to the evaluation of the novel hydrindanes prepared here, compound 8 showed an inhibition profile similar to (±)-9. Increasing the length of the hydrophobic tail, as in 10, resulted in decreased activity (25% inhibition at 500 nM), while shortening the side chain leading to the carboxylic acid, as in 7, led to increased activity (∼60% inhibition at 500 nM).18 Moving forward from compound 7, increasing the length of the hydrophobic tail by one carbon, as shown in hydrindane 11, led to a substantial increase in activity (∼80% inhibition at 500 nM).

A central hypothesis of these studies is that the absolute stereochemistry of the hydrindane is an important feature of ligand design. To test this, the enantiomer of 11 was prepared and evaluated for its capacity to inhibit ToxT in this β-galactosidase assay. As depicted in Figure 4C, ent-11 is ∼10-fold less potent (∼50% inhibition at 500 nM), revealing that the absolute stereochemistry plays a significant role in determining the inhibitory profile of these fatty acid mimetics.19

Overall, this proof of concept study demonstrates that chiral hydrindane motifs can serve as the core of conformationally constrained chiral mimetics of a natural fatty acid and reveals that modern alkoxide-directed metallacycle-mediated annulative cross-coupling technology is an effective tool for preparing such compounds. Additionally, these studies have produced the most potent known inhibitor of the ToxT transcription factor, which could be a lead to a viable antivirulence therapeutic to combat cholera. Given the broad medicinal relevance of biological macromolecules regulated by fatty acids, we anticipate that future studies will build on and challenge this approach.

Acknowledgments

We gratefully acknowledge financial support of this work by the National Institute of General Medical Sciences and the National Institute of Allergy and Infectious Diseases, National Institutes of Health (GM080266, GM134725 and AI072661).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.1c00378.

  • Procedures and spectroscopic data (PDF)

Author Contributions

Conceptualization: G.C.M., F.J.K., C.R.M. Chemical synthesis: L.E.M. Evaluation of compounds as inhibitors of ToxT: J.D.T. All of the authors participated in writing, editing, and approving the final version of the manuscript.

The authors declare no competing financial interest.

Supplementary Material

ml1c00378_si_001.pdf (2.8MB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ml1c00378_si_001.pdf (2.8MB, pdf)

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