The tachykinin receptors neurokinin 1 (NK1R) and neurokinin 2 (NK2R) are G protein-coupled receptors that bind preferentially to the natural peptide ligands substance P and neurokinin A, respectively, and have been targets for drug development. Despite sharing a common C-terminal sequence of Phe-X-Gly-Leu-Met-NH2 that helps direct biological function, the peptide ligands exhibit some degree of cross-reactivity toward each other\'s non-natural receptor. Here, we investigate the detailed structure-activity relationships of the ligand-bound receptor complexes that underlie both potent activation by the natural ligand and cross-reactivity. We find that the specificity and cross-reactivity of the peptide ligands can be explained by the interactions between the amino acids preceding the FxGLM consensus motif of the bound peptide ligand and two regions of the receptor: the β-hairpin of the extracellular loop 2 (ECL2) and a N-terminal segment leading into transmembrane helix 1. Positively charged sidechains of the ECL2 (R177 of NK1R and K180 of NK2R) are seen to play a vital role in the interaction. The N-terminal positions 1 to 3 of the peptide ligand are entirely dispensable. Mutated and chimeric receptor and ligand constructs neatly swap around ligand specificity as expected, validating the structure-activity hypotheses presented. These findings will help in developing improved agonists or antagonists for NK1R and NK2R.

Neurokinin/tachykinin receptors are classified as the G-protein coupled receptor superfamily. The neurokinin 2 receptor (NK2R) is widely expressed in different tissues. NK2R is associated with a range of biological events, such as inflammation, smooth muscle contraction, intestinal motor functions, and asthma. Despite these diverse activities, no approved drugs targeting NK2R have been developed yet. Our study focuses on finding potential inhibitors for NK2R using virtual screening, molecular docking, and ADME (absorption, distribution, metabolism, and excretion) approaches. We used a homology modeling approach and AlphaFold DB to obtain the three-dimensional structure of mouse and human NK2R proteins, respectively. The homology model of NK2R was predicted using MODELLER v10.3 and further refined and validated using the 3Drefine tool and RAMPAGE server, respectively. Molecular docking was performed using a library of 910 structurally similar molecules to four NK1R antagonists: aprepitant, casopitant, fosaprepitant, and rolapitant. Molecular docking revealed six small molecules that displayed high Chemscore fitness scores, and binding energies with desirable ligand-NK2R interactions. The evaluation of the in silico ADME profile, solubility, and permeability of the ligand molecules has revealed that the small molecules are potentially nontoxic and have the chance of exhibiting biological activity after oral administration. Further experimental studies (in vitro and in vivo assays) are required to evaluate the effectiveness of these inhibitors as therapeutic targets.

Radiopharmaceutical development hinges on the affinity and selectivity of the biological component for the intended target. An analogue of the neuropeptide Substance P (SP), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-[Thi8,Met(O2)11]-SP (DOTA-[Thi8,Met(O2)11]SP), in the theranostic pair [68Ga]Ga-/ [213Bi]Bi-DOTA-[Thi8,Met(O2)11]SP has shown promising clinical results in the treatment of inoperable glioblastoma. As the theranostic targeting component, modifications to SP that affect the selectivity of the resulting analogue for the intended target (neurokinin-1 receptor [NK1R]) could be detrimental to its therapeutic potential. In addition to other closely related tachykinin receptors (neurokinin-2 receptor [NK2R] and neurokinin-3 receptor [NK3R]), SP can activate a mast cell expressed receptor Mas-related G protein-coupled receptor subtype 2 (MRGPRX2), which has been implicated in allergic-type reactions. Therefore, activation of these receptors by SP analogues has severe implications for their therapeutic potential. Here, the receptor selectivity of DOTA-[Thi8,Met(O2)11]SP was examined using inositol phosphate accumulation assay in HEK293-T cells expressing NK1R, NK2R, NK3R or MRGPRX2. DOTA-[Thi8,Met(O2)11]SP had similar efficacy and potency as native SP at NK1R, but displayed greater NK1R selectivity. DOTA-[Thi8,Met(O2)11]SP was unable to elicit significant activation of the other tachykinin receptors nor MRGPRX2 at high concentrations nor did it display antagonistic behaviour at these receptors. DOTA-[Thi8,Met(O2)11]SP, therefore has high potency and selectivity for NK1R, supporting its potential for targeted theranostic use in glioblastoma multiforme and other conditions characterised by NK1R overexpression.

The feasibility of eliciting defecation and urination after intranasal (IN) or sublingual (SL) delivery of a small peptide NK2 receptor agonist, [Lys5, MeLeu9, Nle10]-NKA(4-10), was examined using prototype formulations in dogs. In anesthetized animals, administration of 100 or 300 µg/kg IN or 2.0-6.7 mg/kg SL increased colorectal peak pressure and area under the curve. Peak bladder pressure was also increased at the same doses, and this was accompanied by highly efficient voiding at normal physiological bladder pressure. The onset of these effects was rapid (≤2.5 min), and the primary contractions lasted ∼25 min, returning to baseline in <60 min. Slight hypotension lasting a few minutes and causing <10% change from baseline was detected after higher doses and was statistically significant after only 100 µg/kg IN. In conscious dogs, there was a dose-related increase in voiding responses and reduction in the latency to urinate and defecate after 300 and 1000 µg/kg IN; emesis was also observed at these doses. SL administration of 6.7 mg/kg induced urination within 10 min, but not defecation or emesis. These findings support the feasibility of developing a convenient dosage form of small peptide NK2 receptor agonists as on-demand defecation or urination therapies.

OBJECTIVE: Neurokinin 2 receptor (NK2R) agonists may be useful for treating bladder and bowel dysfunction via direct contraction of detrusor and gastrointestinal smooth muscle. The NK2R agonist [Lys5, MeLeu9, Nle10]-NKA(4-10) (LMN-NKA) induces urination and defecation, but also produces the potential side effect of dermal flushing in rats. Although LMN-NKA is a NK2R agonist, it also has affinity for neurokinin 1 receptors (NK1R). Therefore, the goal of this study was to determine the neurokinin receptor (NKR) subtypes responsible for LMN-NKA-induced urination, defecation, and flushing by blocking either NK2Rs or NK1Rs before LMN-NKA administration.
METHODS: To accomplish this goal, we developed a simple high-throughput \'rapid detection voiding assay\' to detect rapid-onset drug-induced urination and defecation in rats. In LMN-NKA dose-response experiments, LMN-NKA (10-100 μg/kg, subcutaneous) was injected and urination, defecation, and flushing were monitored for 30 min. For NKR antagonist experiments, vehicle, the NK2R antagonist GR159897, or the NK1R antagonist CP-99,994 were injected before an acclimation period. Following acclimation, saline or 100 μg/kg LMN-NKA were injected, and behavior was observed for 30 min.
RESULTS: LMN-NKA produced dose-related increases in urination, defecation, and flushing. Blocking NK2Rs reduced urination and blocked defecation, without affecting flushing. Blocking NK1Rs did not change LMN-NKA-induced urination or defecation but reduced LMN-NKA-induced flushing.
CONCLUSIONS: Using the rapid detection voiding assay we show that LMN-NKA-induced urination and defecation are mediated by NK2Rs, while flushing is mediated by NK1Rs. Therefore, drugs that are more selective for NK2 vs. NK1Rs should produce rapid-onset urination and defecation without producing the potential side effect of flushing.

OBJECTIVE: The contractile effects of tachykinins on the gastrointestinal tract are well-known, but how they modulate slow-waves, particularly in species capable of emesis, remains largely unknown. We aimed to elucidate the effects of tachykinins on myoelectric and contractile activity of isolated gastrointestinal tissues of the Suncus murinus.
METHODS: The effects of substance P (SP), neurokinin (NK)A, NKB and selective NK1 (CP122,721, CP99,994), NK2 (SR48,968, GR159,897) and NK3 (SB218,795, SB222,200) receptor antagonists on isolated stomach, duodenum, ileum and colon segments were studied. Mechanical contractile activity was recorded using isometric force displacement transducers. Electrical pacemaker activity was recorded using a microelectrode array.
RESULTS: Compared with NKA, SP induced larger contractions in stomach tissue and smaller contractions in intestinal segments, where oscillation magnitudes increased in intestinal segments, but not the stomach. CP122,721 and GR159,897 inhibited electrical field stimulation-induced contractions of the stomach, ileum and colon. NKB and NK3 had minor effects on contractile activity. The inhibitory potencies of SP and NKA on the peristaltic frequency of the colon and ileum, respectively, were correlated with those on electrical pacemaker frequency. SP, NKA and NKB inhibited pacemaker activity of the duodenum and ileum, but increased that of the stomach and colon. SP elicited a dose-dependent contradictive pacemaker frequency response in the colon.
CONCLUSIONS: This study revealed distinct effects of tachykinins on the mechanical and electrical properties of the stomach and colon vs. the proximal intestine, providing a unique aspect on neuromuscular correlation in terms of the effects of tachykinin on peristaltic and pacemaker activity in gastrointestinal-related symptoms.

Exposure to aerosolized citric acid (CA, 150 mM) and prostaglandin E2 (PGE2, 0.43 mM) for 10 min in guinea pigs reportedly produces the distinct cough patterns (Type I vs. II) and ventilatory responses (long-lasting hyperventilation vs. brief tachypnea) even though triggering the same cough numbers. Type I and II coughs are primarily mediated by activation of TRPV1 and EP3 receptors (a PGE2 receptor) of vagal C-fibers respectively. Substance P (SP) and neurokinin A (NKA) released by vagal pulmonary sensory fibers peripherally are capable of affecting CA-induced cough and ventilation via preferentially activating neurokinin 1 and 2 receptors (NK1R and NK2R) respectively. This study aimed to define the impacts of CA- and PGE2-exposure on pulmonary SP and NKA levels and the roles of NK1R and NK2R in modulating CA- and PGE2-evoked cough and ventilatory responses. In unanesthetized guinea pigs, we determined: (1) pulmonary SP and NKA contents induced by the CA- or PGE2-exposure; (2) effects of CP-99994 and SR-48968 (a NK1R and a NK2R antagonist respectively) given by intraperitoneal injection (IP) or aerosol inhalation (IH) on the CA- and PGE2-evoked cough and ventilatory responses; and (3) immunocytochemical expressions of NK1R/NK2R in vagal C-neurons labeled by TRPV1 or EP3 receptors. We found that CA- and PGE2-exposure evoked Type I and II cough respectively associated with different degrees of increases in pulmonary SP and NKA. Applications of CP-99994 and SR-48968 via IP and IH efficiently suppressed the cough responses to CA with less impact on the cough response to PGE2. These antagonists inhibited or blocked the ventilatory response to CA and caused hypoventilation in response to PGE2. Moreover, NK1R and NK2R were always co-expressed in vagal C-neurons labeled by TRPV1 or EP3 receptors. These results suggest that SP and NKA endogenously released by CA- and PGE2-exposure play important roles in generating the cough and ventilatory responses to CA and PGE2, at least in part, via activation of NK1R and NK2R expressed in vagal C-neurons (pulmonary C-neurons).

Neurokinin 2 receptor (NK2R), a G protein-coupled receptor for neurokinin A (NKA), a tachykinin family member, regulates various physiological functions including pain response, relaxation of smooth muscle, dilation of blood vessels, and vascular permeability. However, the precise role and regulation of NK2R expression in cancer cells have not been fully elucidated. In this study, we found that high NK2R gene expression was correlated with the poor survival of colorectal cancer patients, and Interferon (IFN-α/β) stimulation significantly enhanced NK2R gene expression level of colon cancer cells in a Janus kinas 1/2 (JAK 1/2)-dependent manner. NKA stimulation augmented viability/proliferation and phosphorylation of Extracellular-signal-regulated kinase 1/2 (ERK1/2) levels of IFN-α/β-treated colon cancer cells and NK2R blockade by using a selective antagonist reduced the proliferation in vitro. Administration of an NK2R antagonist alone or combined with polyinosinic-polycytidylic acid, a synthetic analog of double-stranded RNA, to CT26-bearing mice significantly suppressed tumorigenesis. NK2R-overexpressing CT26 cells showed enhanced tumorigenesis and metastatic colonization in both lung and liver after the inoculation into mice. These findings indicate that IFN-α/β-mediated NK2R expression is related to the malignancy of colon cancer cells, suggesting that NK2R blockade may be a promising strategy for colon cancers.

Substance P (SP), a product of the tachykinin 1 (Tac1) gene, is expressed in many hypothalamic neurons. Its wake-promoting potential could be mediated through histaminergic (HA) neurons of the tuberomamillary nucleus (TMN), where functional expression of neurokinin receptors (NKRs) waits to be characterized. As in the process of nociception in the peripheral nervous system (PNS) capsaicin-receptor (transient potential vanilloid 1: TRPV1) signalling is amplified by local release of histamine and SP, we tested the involvement of tachykinins in the capsaicin-induced long-lasting enhancement (LLEcaps) of HA neurons firing by investigating selective neurokinin receptor ligands in the hypothalamic mouse brain slice preparation using patch-clamp recordings in cell-attached mode combined with single-cell RT-PCR. We report that the majority of HA neurons respond to SP (EC50 3 nM), express the SP precursor tachykinin 1 (Tac1) gene and at least one of the neurokinin receptors. Responses to selective agonists of three known neurokinin receptors were sensitive to corresponding antagonists. LLEcaps was significantly impaired by the neurokinin receptor antagonists, indicating that in hypothalamus, as in the PNS, release of tachykinins downstream to TRPV1 activation is able to boost the release of histamine. The excitatory action of SP on histaminergic neurons adds another pathway to the noradrenergic and orexinergic ones to synergistically enhance cortical arousal. We show NK1R to play a prominent role on HA neurons and thus the control of wakefulness.

Animal proof of principle study.
Bladder and bowel dysfunction are common after spinal cord injury (SCI) and in the elderly. Neurokinin 2 receptor agonists are known to produce on-demand urination and defecation in adult SCI rats. This study compared the ability of a neurokinin 2 receptor (NK2R) agonist to produce bladder and colorectal contractions in both young adult and aged SCI rats.
Dignify Therapeutics and Integrated Laboratory Systems, Durham, NC USA.
Bladder and colorectal pressure and voiding efficiency were measured in response to the NK2R agonist, [Lys5,Me,Leu9,Nle10]-NKA(4-10) (LMN-NKA), in anesthetized animals. The potency and efficacy of LMN-NKA was examined in young adult and aged SCI (T3 or T9 transected) rats, with young adult and aged spinal intact rats included as controls.
LMN-NKA (3-300 μg/kg i.v.) produced dose-dependent increases in bladder and colorectal pressure in all anesthetized rats. No differences in the bladder or colorectal pressure responses or voiding efficiency were observed with age or after SCI. The level of SCI did not change the pharmacodynamic responses to the agonist.
An NK2R agonist produced similar responses in young adult and aged SCI rats, suggesting this class of agonists could be used as a potential therapy to induce on-demand urination and defecation in aged populations, with or without SCI.