G proteins are key mediators of G protein-coupled receptor signalling, which facilitates a plethora of important physiological processes. The cyclic depsipeptides YM-254890 and FR900359 are the only known specific inhibitors of the Gq subfamily of G proteins; however, no synthetic route has been reported previously for these complex natural products and they are not easily isolated from natural sources. Here we report the first total synthesis of YM-254890 and FR900359, as well as of two known analogues, YM-385780 and YM-385781. The versatility of the synthetic approach also enabled the design and synthesis of ten analogues, which provided the first structure–activity relationship study for this class of compounds. Pharmacological characterization of all the compounds at Gq-, Gi- and Gs-mediated signalling provided succinct information on the structural requirements for inhibition, and demonstrated that both YM-254890 and FR900359 are highly potent inhibitors of Gq signalling, with FR900359 being the most potent. These natural products and their analogues represent unique tools for explorative studies of G protein inhibition.

Multidrug resistance (MDR) remains one of the major impediments for efficacious cancer chemotherapy. Increased efflux of multiple chemotherapeutic drugs by transmembrane ATP‐binding cassette (ABC) transporter superfamily is considered one of the primary causes for cancer MDR, in which the role of P‐glycoprotein (P‐gp/ABCB1) has been most well‐established. The clinical co‐administration of P‐gp drug efflux inhibitors, in combination with anticancer drugs which are P‐gp transport substrates, was considered to be a treatment modality to surmount MDR in anticancer therapy by blocking P‐gp‐mediated multidrug efflux. Extensive attempts have been carried out to screen for sets of nontoxic, selective, and efficacious P‐gp efflux inhibitors. In this review, we highlight the recent achievements in drug design, characterization, structure–activity relationship (SAR) studies, and mechanisms of action of the newly synthetic, potent small molecules P‐gp inhibitors in the past 5 years. The development of P‐gp inhibitors will increase our knowledge of the mechanisms and functions of P‐gp‐mediated drug efflux which will benefit drug discovery and clinical cancer therapeutics where P‐gp transporter overexpression has been implicated in MDR.

Transmembrane signals initiated by a range of extracellular stimuli converge on members of the Gq family of heterotrimeric G proteins, which relay these signals in target cells. Gq family G proteins comprise Gq, G11, G14, and G16, which upon activation mediate their cellular effects via inositol lipid–dependent and –independent signaling to control fundamental processes in mammalian physiology. To date, highly specific inhibition of Gq/11/14 signaling can be achieved only with FR900359 (FR) and YM-254890 (YM), two naturally occurring cyclic depsipeptides. To further development of FR or YM mimics for other Gα subunits, we here set out to rationally design Gα16 proteins with artificial FR/YM sensitivity by introducing an engineered depsipeptide-binding site. Thereby we permit control of G16 function through ligands that are inactive on the WT protein. Using CRISPR/Cas9-generated Gαq/Gα11-null cells and loss- and gain-of-function mutagenesis along with label-free whole-cell biosensing, we determined the molecular coordinates for FR/YM inhibition of Gq and transplanted these to FR/YM-insensitive G16. Intriguingly, despite having close structural similarity, FR and YM yielded biologically distinct activities: it was more difficult to perturb Gq inhibition by FR and easier to install FR inhibition onto G16 than perturb or install inhibition with YM. A unique hydrophobic network utilized by FR accounted for these unexpected discrepancies. Our results suggest that non-Gq/11/14 proteins should be amenable to inhibition by FR scaffold–based inhibitors, provided that these inhibitors mimic the interaction of FR with Gα proteins harboring engineered FR-binding sites.

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Extracellular signals perceived by G protein-coupled receptors are transmitted via G proteins, and subsequent intracellular signaling cascades result in a plethora of physiological responses. The natural product cyclic depsipeptides YM-254890 and FR900359 are the only known compounds that specifically inhibit signaling mediated by the G subfamily. In this study we exploit a newly developed synthetic strategy for this compound class in the design, synthesis, and pharmacological evaluation of eight new analogues of YM-254890. These structure-activity relationship studies led to the discovery of three new analogues, YM-13, YM-14, and YM-18, which displayed potent and selective G inhibitory activity. This provides pertinent information for the understanding of the G inhibitory mechanism by this class of compounds and importantly provides a pathway for the development of labeled YM-254890 analogues.