Selective inhibitors of mTORC1 activate 4EBP1 and suppress tumor growth.

The clinical benefits of pan-mTOR active-site inhibitors are limited by toxicity and relief of feedback inhibition of receptor expression. To address these limitations, we designed a series of compounds that selectively inhibit mTORC1 and not mTORC2. These 'bi-steric inhibitors' comprise a rapamycin-like core moiety covalently linked to an mTOR active-site inhibitor. Structural modification of these components modulated their affinities for their binding sites on mTOR and the selectivity of the bi-steric compound. mTORC1-selective compounds potently inhibited 4EBP1 phosphorylation and caused regressions of breast cancer xenografts. Inhibition of 4EBP1 phosphorylation was sufficient to block cancer cell growth and was necessary for maximal antitumor activity. At mTORC1-selective doses, these compounds do not alter glucose tolerance, nor do they relieve AKT-dependent feedback inhibition of HER3. Thus, in preclinical models, selective inhibitors of mTORC1 potently inhibit tumor growth while causing less toxicity and receptor reactivation as compared to pan-mTOR inhibitors.

Preclinical to clinical translation of CNS transporter occupancy of TD-9855, a novel norepinephrine and serotonin reuptake inhibitor.

BACKGROUND: Monoamine reuptake inhibitors exhibit unique clinical profiles that reflect distinct engagement of the central nervous system (CNS) transporters. METHODS: We used a translational strategy, including rodent pharmacokinetic/pharmacodynamic modeling and positron emission tomography (PET) imaging in humans, to establish the transporter profile of TD-9855, a novel norepinephrine and serotonin reuptake inhibitor. RESULTS: TD-9855 was a potent inhibitor of norepinephrine (NE) and serotonin 5-HT uptake in vitro with an inhibitory selectivity of 4- to 10-fold for NE at human and rat transporters. TD-9855 engaged norepinephrine transporters (NET) and serotonin transporters (SERT) in rat spinal cord, with a plasma EC50 of 11.7 ng/mL and 50.8 ng/mL, respectively, consistent with modest selectivity for NET in vivo. Accounting for species differences in protein binding, the projected human NET and SERT plasma EC50 values were 5.5 ng/mL and 23.9 ng/mL, respectively. A single-dose, open-label PET study (4-20mg TD-9855, oral) was conducted in eight healthy males using the radiotracers [(11)C]-3-amino-4- [2-[(di(methyl)amino)methyl]phenyl]sulfanylbenzonitrile for SERT and [(11)C]-(S,S)-methylreboxetine for NET. The long pharmacokinetic half-life (30-40 h) of TD-9855 allowed for sequential assessment of SERT and NET occupancy in the same subject. The plasma EC50 for NET was estimated to be 1.21 ng/mL, and at doses of greater than 4 mg the projected steady-state NET occupancy is high (>75%). After a single oral dose of 20mg, SERT occupancy was 25 (±8)% at a plasma level of 6.35 ng/mL. CONCLUSIONS: These data establish the CNS penetration and transporter profile of TD-9855 and inform the selection of potential doses for future clinical evaluation.

Combined inhibition of KRASG12C and mTORC1 kinase is synergistic in non-small cell lung cancer.

Current KRASG12C (OFF) inhibitors that target inactive GDP-bound KRASG12C cause responses in less than half of patients and these responses are not durable. A class of RASG12C (ON) inhibitors that targets active GTP-bound KRASG12C blocks ERK signaling more potently than the inactive-state inhibitors. Sensitivity to either class of agents is strongly correlated with inhibition of mTORC1 activity. We have previously shown that PI3K/mTOR and ERK-signaling pathways converge on key cellular processes and that inhibition of both pathways is required for inhibition of these processes and for significant antitumor activity. We find here that the combination of a KRASG12C inhibitor with a selective mTORC1 kinase inhibitor causes synergistic inhibition of Cyclin D1 expression and cap-dependent translation. Moreover, BIM upregulation by KRASG12C inhibition and inhibition of MCL-1 expression by the mTORC1 inhibitor are both required to induce significant cell death. In vivo, this combination causes deep, durable tumor regressions and is well tolerated. This study suggests that the ERK and PI3K/mTOR pathways each mitigate the effects of inhibition of the other and that combinatorial inhibition is a potential strategy for treating KRASG12C-dependent lung cancer.

Mechanisms of response and tolerance to active RAS inhibition in KRAS-mutant NSCLC.

Resistance to inactive state-selective RASG12C inhibitors frequently entails accumulation of RASGTP, rendering effective inhibition of active RAS potentially desirable. Here, we evaluated the anti-tumor activity of the RAS(ON) multi-selective tri-complex inhibitor RMC-7977 and dissected mechanisms of response and tolerance in KRASG12C-mutant NSCLC. Broad-spectrum, reversible RASGTP inhibition with or without concurrent covalent targeting of active RASG12C yielded superior and differentiated antitumor activity across diverse co-mutational KRASG12C-mutant NSCLC mouse models of primary or acquired RASG12C(ON) or (OFF) inhibitor resistance. Interrogation of time-resolved single cell transcriptional responses established an in vivo atlas of multi-modal acute and chronic RAS pathway inhibition in the NSCLC ecosystem and uncovered a regenerative mucinous transcriptional program that supports long-term tumor cell persistence. In patients with advanced KRASG12C-mutant NSCLC, the presence of mucinous histological features portended poor response to sotorasib or adagrasib. Our results have potential implications for personalized medicine and the development of rational RAS inhibitor-anchored therapeutic strategies.

Translational and Therapeutic Evaluation of RAS-GTP Inhibition by RMC-6236 in RAS-Driven Cancers.

RAS-driven cancers comprise up to 30% of human cancers. RMC-6236 is a RAS(ON) multi-selective noncovalent inhibitor of the active, GTP-bound state of both mutant and wild-type variants of canonical RAS isoforms with broad therapeutic potential for the aforementioned unmet medical need. RMC-6236 exhibited potent anticancer activity across RAS-addicted cell lines, particularly those harboring mutations at codon 12 of KRAS. Notably, oral administration of RMC-6236 was tolerated in vivo and drove profound tumor regressions across multiple tumor types in a mouse clinical trial with KRASG12X xenograft models. Translational PK/efficacy and PK/PD modeling predicted that daily doses of 100 mg and 300 mg would achieve tumor control and objective responses, respectively, in patients with RAS-driven tumors. Consistent with this, we describe here objective responses in two patients (at 300 mg daily) with advanced KRASG12X lung and pancreatic adenocarcinoma, respectively, demonstrating the initial activity of RMC-6236 in an ongoing phase I/Ib clinical trial (NCT05379985). SIGNIFICANCE: The discovery of RMC-6236 enables the first-ever therapeutic evaluation of targeted and concurrent inhibition of canonical mutant and wild-type RAS-GTP in RAS-driven cancers. We demonstrate that broad-spectrum RAS-GTP inhibition is tolerable at exposures that induce profound tumor regressions in preclinical models of, and in patients with, such tumors. This article is featured in Selected Articles from This Issue, p. 897.

A novel class of 3-(phenoxy-phenyl-methyl)-pyrrolidines as potent and balanced norepinephrine and serotonin reuptake inhibitors: synthesis and structure-activity relationships.

A series of 3-(phenoxy-phenyl-methyl)-pyrrolidine analogues were discovered to be potent and balanced norepinephrine (NE) and serotonin (5-hydroxytryptamine, 5-HT) reuptake inhibitors. Several of these compounds were identified to have suitable in vitro pharmacokinetic properties for an orally dosed and CNS-targeted drug. Compound 39b, in particular, was identified as a potent NET and SERT reuptake inhibitor (NSRI) with minimal off-target activity and demonstrated robust efficacy in the spinal nerve ligation model of pain behavior.

Discovery of RMC-5552, a Selective Bi-Steric Inhibitor of mTORC1, for the Treatment of mTORC1-Activated Tumors.

Hyperactivation of mTOR kinase by mutations in the PI3K/mTOR pathway or by crosstalk with other mutant cancer drivers, such as RAS, is a feature of many tumors. Multiple allosteric inhibitors of mTORC1 and orthosteric dual inhibitors of mTORC1 and mTORC2 have been developed as anticancer drugs, but their clinical utility has been limited. To address these limitations, we have developed a novel class of "bi-steric inhibitors" that interact with both the orthosteric and the allosteric binding sites in order to deepen the inhibition of mTORC1 while also preserving selectivity for mTORC1 over mTORC2. In this report, we describe the discovery and preclinical profile of the development candidate RMC-5552 and the in vivo preclinical tool compound RMC-6272. We also present evidence that selective inhibition of mTORC1 in combination with covalent inhibition of KRASG12C shows increased antitumor activity in a preclinical model of KRASG12C mutant NSCLC that exhibits resistance to KRASG12C inhibitor monotherapy.

Chemical remodeling of a cellular chaperone to target the active state of mutant KRAS.

The discovery of small-molecule inhibitors requires suitable binding pockets on protein surfaces. Proteins that lack this feature are considered undruggable and require innovative strategies for therapeutic targeting. KRAS is the most frequently activated oncogene in cancer, and the active state of mutant KRAS is such a recalcitrant target. We designed a natural product-inspired small molecule that remodels the surface of cyclophilin A (CYPA) to create a neomorphic interface with high affinity and selectivity for the active state of KRASG12C (in which glycine-12 is mutated to cysteine). The resulting CYPA:drug:KRASG12C tricomplex inactivated oncogenic signaling and led to tumor regressions in multiple human cancer models. This inhibitory strategy can be used to target additional KRAS mutants and other undruggable cancer drivers. Tricomplex inhibitors that selectively target active KRASG12C or multiple RAS mutants are in clinical trials now (NCT05462717 and NCT05379985).

Tumor-selective effects of active RAS inhibition in pancreatic ductal adenocarcinoma.

Broad-spectrum RAS inhibition holds the potential to benefit roughly a quarter of human cancer patients whose tumors are driven by RAS mutations. However, the impact of inhibiting RAS functions in normal tissues is not known. RMC-7977 is a highly selective inhibitor of the active (GTP-bound) forms of KRAS, HRAS, and NRAS, with affinity for both mutant and wild type (WT) variants. As >90% of human pancreatic ductal adenocarcinoma (PDAC) cases are driven by activating mutations in KRAS, we assessed the therapeutic potential of RMC-7977 in a comprehensive range of PDAC models, including human and murine cell lines, human patient-derived organoids, human PDAC explants, subcutaneous and orthotopic cell-line or patient derived xenografts, syngeneic allografts, and genetically engineered mouse models. We observed broad and pronounced anti-tumor activity across these models following direct RAS inhibition at doses and concentrations that were well-tolerated in vivo. Pharmacological analyses revealed divergent responses to RMC-7977 in tumor versus normal tissues. Treated tumors exhibited waves of apoptosis along with sustained proliferative arrest whereas normal tissues underwent only transient decreases in proliferation, with no evidence of apoptosis. Together, these data establish a strong preclinical rationale for the use of broad-spectrum RAS inhibition in the setting of PDAC.

Prediction of human serotonin and norepinephrine transporter occupancy of duloxetine by pharmacokinetic/pharmacodynamic modeling in the rat.

Translation of central nervous system occupancy and clinical effect from animal models to humans has remained elusive for many pharmacological targets. The current studies evaluate the ability of a rodent pharmacokinetic/pharmacodynamic (PK/PD) modeling approach to translate ex vivo occupancy determined in rats to that observed after positron emission tomography (PET) imaging in humans for the dual serotonin transporter (SERT) and norepinephrine transporter (NET) inhibitor duloxetine. Ex vivo transporter occupancy in rat spinal cord was evaluated after single oral doses of 0.3 to 60 mg/kg. A novel methodology, based on the initial rates of association of transporter selective radioligands to tissue homogenates, was developed and validated for the assessment of ex vivo transporter occupancy. Duloxetine exhibited selectivity for occupancy of SERT over NET in rat spinal cord with ED(50) values of 1 and 9 mg/kg, respectively. Corresponding EC(50) values for the inhibition of SERT and NET based on unbound duloxetine spinal cord concentrations were 0.5 and 8 nM. An effect compartment PK/PD modeling approach was used to analyze the relationship between the time course of duloxetine plasma concentration and SERT and NET occupancy. Duloxetine inhibited SERT and NET in rat spinal cord with a plasma EC(50) of 2.95 and 59.0 ng/ml, respectively. Similar plasma EC(50) values for the inhibition of SERT (2.29-3.7 ng/ml) have been reported from human PET studies. This study illustrates the value of translational PK/PD modeling approaches and suggests that the preclinical modeling approach used in the current study is capable of predicting plasma concentrations associated with 50% occupancy of SERT in the human central nervous system.

Discovery, oral pharmacokinetics and in vivo efficacy of velusetrag, a highly selective 5-HT(4) receptor agonist that has achieved proof-of-concept in patients with chronic idiopathic constipation.

Utilization of Theravance's multivalent approach to drug discovery towards 5-HT(4) receptor agonists with a focus on identification of neutral (non-charged at physiological pH) secondary binding groups is described. Optimization of a quinolone-tropane primary binding group with a chiral 2-propanol linker to a range of neutral secondary binding group motifs, for binding affinity and functional potency at the 5-HT(4) receptor, selectivity over the 5-HT(3) receptor, oral pharmacokinetics, and in vivo efficacy in models of GI motility, afforded velusetrag (TD-5108). Velusetrag has achieved proof-of-concept in patients with chronic idiopathic constipation.

Discovery, oral pharmacokinetics and in vivo efficacy of a highly selective 5-HT4 receptor agonist: clinical compound TD-2749.

Further application of our multivalent approach to drug discovery directed to 5-HT(4) receptor agonists is described. Optimization of the linker and secondary binding amine in the indazole-tropane primary binding group series, for binding affinity and functional potency at the 5-HT(4) receptor, selectivity over the 5-HT(3) receptor, oral pharmacokinetics, and in vivo efficacy in models of GI motility, resulted in the identification of clinical compound TD-2749.

THRX-198321 is a bifunctional muscarinic receptor antagonist and beta2-adrenoceptor agonist (MABA) that binds in a bimodal and multivalent manner.

Biphenyl-2-yl-carbamic acid 1-{9-[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydro-quinolin-5-yl)-ethylamino]-nonyl}-piperidin-4-yl ester (THRX-198321) is a single molecule composed of a muscarinic acetylcholine receptor (mAChR) antagonist moiety, represented by the fragment MA, linked by a C9 polymethylene chain to a ß(2)-adrenoceptor (ß(2)AR) agonist moiety, represented by the fragment 8-hydroxy-5-((R)-1-hydroxy-2-methylamino-ethyl)-1H-quinolin-2-one (BA). THRX-198321 exhibited high affinity for mAChR (M(2) pK(I,App) = 10.57 ± 0.09; M(3) pK(I,App) = 10.07 ± 0.11) and ß(2)AR (pK(I,App) = 9.54 ± 0.15), with potent mAChR antagonist (M(2) pK(I,Fn) = 9.69 ± 0.23; M(3) pK(I,Fn) = 10.05 ± 0.17) and ß(2)AR agonist (pEC(50) = 9.25 ± 0.02) activities. Consistent with multivalent interactions, THRX-198321 binding affinity was >300-fold higher at mAChR and 29-fold higher at ß(2)AR relative to its monovalent fragments biphenyl carbamic acid piperidinyl ester (MA) and BA, respectively. THRX-198321 was a competitive antagonist at mAChR (M(2) pK(B) = 9.98 ± 0.13; M(3) pK(B) = 10.31 ± 0.89), whereas THRX-198321 agonist activity at ß(2)AR was competitively inhibited by propranolol. Interactions of THRX-198321 with an allosteric site on mAChR and a novel extracellular allosteric site on ß(2)AR, respectively, were detected by measuring THRX-198321-evoked changes in the dissociation rates for the orthosteric radioligands, [N-methyl-(3)H]scopolamine methyl chloride (M(2) pEC(50,diss) = 6.73 ± 0.10; M(3) pEC(50,diss) = 5.02 ± 0.14) and [4,6-propyl-(3)H]dihydroalprenolol (ß(2)AR pEC(50,diss) = 3.82 ± 0.25). The carbostyril-linker fragment (BA-L) binds to the allosteric site of mAChR (M(2) pEC(50,diss) = 5.06 ± 0.03; M(3) pEC(50,diss) = 4.15 ± 0.25), whereas the MA fragment binds to the allosteric site of ß(2)AR (pEC(50,diss) = 3.60 ± 0.18). Collectively, these observations suggest that THRX-198321 exhibits a multivalent bimodal orientation in the orthosteric and allosteric binding pockets of mAChR and ß(2)AR, a phenomenon that may be unique to this class of molecule.

Efficacy of a Novel Bi-Steric mTORC1 Inhibitor in Models of B-Cell Acute Lymphoblastic Leukemia.

The mechanistic target of rapamycin (mTOR) is a kinase whose activity is elevated in hematological malignancies. mTOR-complex-1 (mTORC1) phosphorylates numerous substrates to promote cell proliferation and survival. Eukaryotic initiation factor 4E (eIF4E)-binding proteins (4E-BPs) are mTORC1 substrates with an integral role in oncogenic protein translation. Current pharmacological approaches to inhibit mTORC1 activity and 4E-BP phosphorylation have drawbacks. Recently we described a series of bi-steric compounds that are potent and selective inhibitors of mTORC1, inhibiting 4E-BP phosphorylation at lower concentrations than mTOR kinase inhibitors (TOR-KIs). Here we report the activity of the mTORC1-selective bi-steric inhibitor, RMC-4627, in BCR-ABL-driven models of B-cell acute lymphoblastic leukemia (B-ALL). RMC-4627 exhibited potent and selective inhibition of 4E-BP1 phosphorylation in B-ALL cell lines without inhibiting mTOR-complex-2 (mTORC2) activity. RMC-4627 suppressed cell cycle progression, reduced survival, and enhanced dasatinib cytotoxicity. Compared to a TOR-KI compound, RMC-4627 was more potent, and its effects on cell viability were sustained after washout in vitro. Notably, a once-weekly, well tolerated dose reduced leukemic burden in a B-ALL xenograft model and enhanced the activity of dasatinib. These preclinical studies suggest that intermittent dosing of a bi-steric mTORC1-selective inhibitor has therapeutic potential as a component of leukemia regimens, and further study is warranted.

Shp1 Loss Enhances Macrophage Effector Function and Promotes Anti-Tumor Immunity.

Shp1, encoded by the gene Ptpn6, is a protein tyrosine phosphatase that transduces inhibitory signals downstream of immunoreceptors in many immune cell types. Blocking Shp1 activity represents an exciting potential immunotherapeutic strategy for the treatment of cancer, as Shp1 inhibition would be predicted to unleash both innate and adaptive immunity against tumor cells. Antibodies blocking the interaction between CD47 on tumor cells and SIRPα on macrophages enhance macrophage phagocytosis, show efficacy in preclinical tumor models, and are being evaluated in the clinic. Here we found that Shp1 bound to phosphorylated peptide sequences derived from SIRPα and transduced the anti-phagocytic signal, as Shp1 loss in mouse bone marrow-derived macrophages increased phagocytosis of tumor cells in vitro. We also generated a novel mouse model to evaluate the impact of global, inducible Ptpn6 deletion on anti-tumor immunity. We found that inducible Shp1 loss drove an inflammatory disease in mice that was phenotypically similar to that seen when Ptpn6 is knocked out from birth. This indicates that acute perturbation of Shp1 in vivo could drive hyperactivation of immune cells, which could be therapeutically beneficial, though at the risk of potential toxicity. In this model, we found that Shp1 loss led to robust anti-tumor immunity against two immune-rich syngeneic tumor models that are moderately inflamed though not responsive to checkpoint inhibitors, MC38 and E0771. Shp1 loss did not promote anti-tumor activity in the non-inflamed B16F10 model. The observed activity in MC38 and E0771 tumors was likely due to effects of both innate and adaptive immune cells. Following Shp1 deletion, we observed increases in intratumoral myeloid cells in both models, which was more striking in E0771 tumors. E0771 tumors also contained an increased ratio of effector to regulatory T cells following Shp1 loss. This was not observed for MC38 tumors, though we did find increased levels of IFNγ, a cytokine produced by effector T cells, in these tumors. Overall, our preclinical data suggested that targeting Shp1 may be an attractive therapeutic strategy for boosting the immune response to cancer via a mechanism involving both innate and adaptive leukocytes.

Allosteric Inhibition of SHP2 Stimulates Antitumor Immunity by Transforming the Immunosuppressive Environment.

The protein tyrosine phosphatase SHP2 binds to phosphorylated signaling motifs on regulatory immunoreceptors including PD-1, but its functional role in tumor immunity is unclear. Using preclinical models, we show that RMC-4550, an allosteric inhibitor of SHP2, induces antitumor immunity, with effects equivalent to or greater than those resulting from checkpoint blockade. In the tumor microenvironment, inhibition of SHP2 modulated T-cell infiltrates similar to checkpoint blockade. In addition, RMC-4550 drove direct, selective depletion of protumorigenic M2 macrophages via attenuation of CSF1 receptor signaling and increased M1 macrophages via a mechanism independent of CD8+ T cells or IFNγ. These dramatic shifts in polarized macrophage populations in favor of antitumor immunity were not seen with checkpoint blockade. Consistent with a pleiotropic mechanism of action, RMC-4550 in combination with either checkpoint or CSF1R blockade caused additive antitumor activity with complete tumor regressions in some mice; tumors intrinsically sensitive to SHP2 inhibition or checkpoint blockade were particularly susceptible. Our preclinical findings demonstrate that SHP2 thus plays a multifaceted role in inducing immune suppression in the tumor microenvironment, through both targeted inhibition of RAS pathway-dependent tumor growth and liberation of antitumor immune responses. Furthermore, these data suggest that inhibition of SHP2 is a promising investigational therapeutic approach. SIGNIFICANCE: Inhibition of SHP2 causes direct and selective depletion of protumorigenic M2 macrophages and promotes antitumor immunity, highlighting an investigational therapeutic approach for some RAS pathway-driven cancers.

RAS nucleotide cycling underlies the SHP2 phosphatase dependence of mutant BRAF-, NF1- and RAS-driven cancers.

Oncogenic alterations in the RAS/RAF/MEK/ERK pathway drive the growth of a wide spectrum of cancers. While BRAF and MEK inhibitors are efficacious against BRAFV600E-driven cancers, effective targeted therapies are lacking for most cancers driven by other pathway alterations, including non-V600E oncogenic BRAF, RAS GTPase-activating protein (GAP) NF1 (neurofibromin 1) loss and oncogenic KRAS. Here, we show that targeting the SHP2 phosphatase (encoded by PTPN11) with RMC-4550, a small-molecule allosteric inhibitor, is effective in human cancer models bearing RAS-GTP-dependent oncogenic BRAF (for example, class 3 BRAF mutants), NF1 loss or nucleotide-cycling oncogenic RAS (for example, KRASG12C). SHP2 inhibitor treatment decreases oncogenic RAS/RAF/MEK/ERK signalling and cancer growth by disrupting SOS1-mediated RAS-GTP loading. Our findings illuminate a critical function for SHP2 in promoting oncogenic RAS/MAPK pathway activation in cancers with RAS-GTP-dependent oncogenic BRAF, NF1 loss and nucleotide-cycling oncogenic KRAS. SHP2 inhibition is a promising molecular therapeutic strategy for patients with cancers bearing these oncogenic drivers.

P2Y receptor regulation of cultured rat cerebral cortical cells: calcium responses and mRNA expression in neurons and glia.

1 We have investigated increases in cytosolic Ca(2+) in response to nucleotides in mixed rat cerebrocortical cultures (neurons and glia in similar numbers) and in essentially neuron-free glial cultures. 2 In both cultures, the agonist-response profile was 2-methylthioADP(2MeSADP)>2-methylthioATP(2MeSATP)>ADP>ATP>adenosine 5'-O-(3-thiotriphosphate), consistent with a P2Y(1) receptor. The maximal responses to 2MeSADP, 2MeSATP and ADP were identical, but that to ATP was higher. 3 Suramin, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid, reactive blue 2 (RB2), and adenosine biphosphate (A3P5P) were antagonists with apparent pA(2) values of 5.5 for suramin, 6.4 for RB2, and 4.7 for A3P5P. 4 Single cell imaging divided the cells from the mixed neuronal-glial cultures into two populations: responsive (neurons) and unresponsive (glial cells) to high [K(+)]. The response of cells to nucleotides was almost exclusively limited to those not responsive to high K(+). 5 In the presence of extracellular Mn(2+), the response of the mixed cultures to 30 mM K(+) and 20 micro M Bay K 8644 was attenuated. However, when 2MeSADP was added there was no reduction in response in cultures previously loaded with Mn(2+). This further indicated that the 2MeSADP response was not in the neurons. 6 Reverse transcriptase-polymerase chain reaction studies detected transcripts for P2Y(1), P2Y(4) and P2Y(6) in RNA preparations from embryonic rat cortex, and from both mixed and glial cultures. P2Y(2) transcripts were not detected in the embryonic cortex. 7 Based on this and previous work, it is proposed that the principal P2Y influences in the brain are on cytosolic Ca(2+) in glial cells and presynaptic sites on neurons.