Discovery and optimization of RO-85, a novel drug-like, potent, and selective P2X3 receptor antagonist

doi:10.1016/j.bmcl.2009.12.044

Bioorganic & Medicinal Chemistry Letters

Volume 20, Issue 3, 1 February 2010, Pages 1031-1036

https://doi.org/10.1016/j.bmcl.2009.12.044 Get rights and content

Abstract

Despite the extensive literature describing the role of the ATP-gated P2X₃ receptors in a variety of physiological processes the potential of antagonists as therapeutic agents has been limited by the lack of drug-like selective molecules. In this paper we report the discovery and optimization of RO-85, a novel drug-like, potent and selective P2X₃ antagonist. High-throughput screening of the Roche compound collection identified a small hit series of heterocyclic amides from a large parallel synthesis library. Rapid optimization, facilitated by high-throughput synthesis, focusing on increasing potency and improving drug-likeness resulted in the discovery of RO-85.

Graphical abstract

The discovery and optimization of RO-85, a novel drug-like, potent and selective P2X₃ antagonist is described.

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Gefapixant/AF-219, a selective inhibitor of the P2X3 receptor, is the first new drug other than dextromethorphan to be approved for the treatment of refractory chronic cough (RCC) in nearly 60 years. To date, seven P2X subtypes (P2X1-7) activated by extracellular ATP have been cloned, and subtype selectivity of P2X inhibitors is a prerequisite for reducing side effects. We previously identified the site and mechanism of action of Gefapixant/AF-219 on the P2X3 receptor, which occupies a pocket consisting of the left flipper (LF) and lower body (LB) domains. However, the mechanism by which AF-219 selectively acts on the P2X3 receptor is unknown. Here, we combined mutagenesis, chimera construction, molecular simulations, covalent occupation and chemical synthesis, and find that the negative allosteric site of AF-219 at P2X3 is also present in other P2X subtypes, at least for P2X1, P2X2 and P2X4. By constructing each chimera of AF-219 sensitive P2X3 and insensitive P2X2 subtypes, the insensitive P2X2 subtype was made to acquire the inhibitory properties of AF-219 and AF-353, an analog of AF-219 with higher affinity. Our results suggest that the selectivity of AF-219/AF-353 for P2X3 over the other P2X subtypes is determined by a combination of the accessibility of P2X3 binding site and the internal shape of this pocket, a finding that could provide new perspectives for drug design against P2X3-mediated diseases such as RCC, idiopathic pulmonary fibrosis, hypertension and overactive bladder disorder.
Bicyclic 5-5 Systems: Three Heteroatoms 1:2
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This article reviews the advances in the synthesis and reactivity of the bicyclic 5-5 heterocycles with 1:2-heteroatom orientation reported in the period 2006–18, since the publication of CHEC-III. New synthetic methodologies in the development of these heterocycles are discussed and their applications are highlighted. There is an emphasis on the use of these heterocycles in medicinal chemistry where they offer novel startpoints for the development of new drugs.
Identification of aurintricarboxylic acid as a potent allosteric antagonist of P2X1 and P2X3 receptors
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Citation Excerpt :
These results were supported by experiments with A-317491, a potent, selective P2X3R and P2X2/3R antagonist: in a variety of animal models, pharmacological P2X3R inhibition attenuated neuropathic and chronic inflammatory pain but had no effect on nociception in models of acute or postoperative pain (Jarvis et al., 2002). Recent interest in P2XRs has led to the development of several new drug-like antagonists for P2X3Rs in the last few years (Bolcskei and Farkas, 2014; Brotherton-Pleiss et al., 2010; Cantin et al., 2012; Carter et al., 2009; Cho et al., 2013; Ford and Undem, 2013; Gever et al., 2010; Jahangir et al., 2009; Jung et al., 2013, 2017). However, to date AF-219 (formerly RO4926219) is the only P2X3R antagonist under clinical development (Abdulqawi et al., 2015; Muller, 2015).
The homotrimeric P2X3 receptor, one of the seven members of the ATP-gated P2X receptor family, plays a crucial role in sensory neurotransmission. P2X3 receptor antagonists have been identified as promising drugs to treat chronic cough and are suggested to offer pain relief in chronic pain such as neuropathic pain. Here, we analysed whether compounds affect P2X3 receptor activity by high-throughput screening of the Spectrum Collection of 2000 approved drugs, natural products and bioactive substances. We identified aurintricarboxylic acid (ATA) as a nanomolar-potency antagonist of P2X3 receptor-mediated responses. Two-electrode voltage clamp electrophysiology-based concentration–response analysis and selectivity profiling revealed that ATA strongly inhibits the rP2X1 and rP2X3 receptors (with IC₅₀ values of 8.6 nM and 72.9 nM, respectively) and more weakly inhibits P2X2/3, P2X2, P2X4 or P2X7 receptors (IC₅₀ values of 0.76 μM, 22 μM, 763 μM or 118 μM, respectively). Patch-clamp analysis of mouse DRG neurons revealed that ATA inhibited native P2X3 and P2X2/3 receptors to a similar extent than rat P2X3 and P2X2/3 receptors expressed in Xenopus oocytes. In a radioligand binding assay, up to 30 μM ATA did not compete with [³H]-ATP for rP2X3 receptor binding, indicating a non-competitive mechanism of action. Molecular docking studies, site-directed mutagenesis and concentration–response analysis revealed that ATA binds to the negative allosteric site of the hP2X3 receptor. In summary, ATA as a drug-like pharmacological tool compound is a nanomolar-potency, allosteric antagonist with selectivity towards αβ-methylene-ATP-sensitive P2X1 and P2X3 receptors.
Characterization of the antagonist actions of 5-BDBD at the rat P2X4 receptor
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Citation Excerpt :
These advances had also prompted the search for new antagonists with high selectively and potency for P2XRs. For example, NF449 is a selective antagonist for the P2X1R [29], whereas AF-353, RO-4, RO-85 and RO-51 are good and selective P2X3R antagonists [5,6,14,19]. For the P2X7R there are several specific antagonists available, including A-438079 or A-70003, which are good candidates to be used as therapeutically agents [10].
P2X receptors (P2XRs) are a family of ATP-gated ionic channels that are expressed in numerous excitable and non-excitable cells. Despite the great advance on the structure and function of these receptors in the last decades, there is still lack of specific and potent antagonists for P2XRs subtypes, especially for the P2X4R. Here, we studied in detail the effect of the P2X4R antagonist 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) on ATP-induced currents mediated by the rat P2X4R and compared its specificity among another rat P2XRs. We found that 5-BDBD is a potent P2X4R antagonist, with an IC₅₀ of 0.75 μM when applied for 2 min prior and during ATP stimulation. Moreover, at 10 μM concentration, 5-BDBD did not affect the ATP-induced P2X2aR, P2X2bR, and P2X7R current amplitude or the pattern of receptor desensitization. However, at 10 μM concentration but not 0.75 μM 5-BDBD inhibited the P2X1R and P2X3R-gated currents by 13 and 35% respectively. Moreover, we studied the effects of 5-BDBD in long-term potentiation experiments performed in rat hippocampal slices, finding this antagonist can partially decrease LTP, a response that is believed to be mediated in part by endogenous P2X4Rs. These results indicate that 5-BDBD could be used to study the endogenous effects of the P2X4R in the central nervous system and this antagonist can discriminate between P2X4R and other P2XRs, when they are co-expressed in the same tissue.
Molecular mechanism underlying the subtype-selectivity of competitive inhibitor NF110 and its distinct potencies in human and rat P2X3 receptors
2018, Science Bulletin
P2X receptors are a family of extracellular ATP-gated trimeric cation channels that is widely distributed in human tissues. Quite some drug candidates targeting P2X receptors have entered into preclinical or main phases of clinical trials, but many of them failed due to low subtype-selectivity or species differences in pharmacological activities between human and experimental animals. Here, we identified the distinct inhibitory efficacies of NF110, a competitive inhibitor, between the rat (rP2X3) and human (hP2X3) P2X3 receptors. We demonstrated that this difference is determined by two amino acids located in the dorsal fin (DF) domain of P2X3 receptors. As revealed by mutagenesis, metadynamics, and covalent modification, NF110-mediated rP2X3 inhibition may be through a filling in the cavity formed by the DF, left flipper (LF) and lower body (LB) to partially, rather than fully, occupy the ATP-binding pocket. Moreover, substitution of residues located in the DF and/or LF domains of the rP2X2 receptor, a NF110-insensitive subtype, with the equivalent amino acids of rP2X3, bestowed the sensitivity of rP2X2 to NF110. The critical roles of the DF and LF domains in channel gating of P2X and low conservativity in residue sequences of those two domains raise the possibility that small molecules differentially interacting with the residues of the DF and LF domains of different P2X receptors may modulate channel’s activity in a subtype-selective manner. However, the possible species-specificity of P2X inhibitors/modulators makes it more complex when interpreting the preclinical data into clinical researches. Nevertheless, our data provide new insights into the subtype-selectivity of competitive inhibitors and their distinct potencies in the human and experimental animals, both of which are extremely important in the drug discovery of P2X receptors.
Pyrrolinone derivatives as a new class of P2X3 receptor antagonists. Part 1: Initial structure-activity relationship studies of a hit from a high throughput screening
2018, Bioorganic and Medicinal Chemistry Letters
The P2X3 receptor is primarily expressed in the peripheral sensory nerves, and therefore, antagonists of this receptor may be useful for the treatment of chronic pain. Pyrrolinone derivatives have been identified as a novel class of P2X3 receptor antagonists. A lead structure with moderate activity was discovered through a high-throughput screening assay. A structure-activity study led to the discovery of several P2X3 receptor antagonists. Compound 34 showed potent and specific antagonistic activity and analgesic efficacy.

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Discovery and optimization of RO-85, a novel drug-like, potent, and selective P2X3 receptor antagonist

Abstract

Graphical abstract

Bioorg. Med. Chem. Lett.

Bioorg. Med. Chem. Lett.

Jpn. J. Pharmacol.

Pfluegers Arch.

Mol. Pharmacol.

Nature

J. Neurosci.

Nucleic Acids Res.

Discovery and optimization of RO-85, a novel drug-like, potent, and selective P2X₃ receptor antagonist