John D. Lesnick

Pharmacological pleiotropism of the human recombinant α1A-adrenoceptor : implications for α1-adrenoceptor classification

Three fully‐defined α1‐adrenoceptors (α1A, α1B and α1D) have been established in pharmacological and molecular studies. A fourth α1‐adrenoceptor, the putative α1L‐adrenoceptor, has been defined in functional but not molecular studies, and has been proposed to mediate contraction of human lower urinary tract tissues; its relationship to the three fully characterized α1‐adrenoceptors is not known. In the present study, binding affinities were estimated by displacement of [3H]‐prazosin in membrane homogenates of Chinese hamster ovary (CHO‐K1) cells stably expressing the human α1A‐, α1B‐ and α1D‐adrenoceptors and were compared with affinity estimates obtained functionally in identical cells by measuring inhibition of noradrenaline (NA)‐stimulated accumulation of [3H]‐inositol phosphates. For the α1A‐adrenoceptor, binding studies revealed a pharmacological profile typical for the classically defined α1A‐adrenoceptor, such that prazosin, RS‐17053, WB4101, 5‐methylurapidil, Rec15/2739 and S‐niguldipine all displayed subnanomolar affinity. A different profile of affinity estimates was obtained in inositol phosphates accumulation studies: prazosin, WB4101, 5‐methylurapidil, RS‐17053 and S‐niguldipine showed 10 to 40 fold lower affinity than in membrane binding. However, affinity estimates were not ‘frameshifted’, as tamsulosin, indoramin and Rec15/2739 yielded similar, high affinity estimates in binding and functional assays. In contrast, results from human α1B‐ and α1D‐adrenoceptors expressed in CHO‐K1 cells gave antagonist affinity profiles in binding and functional assays that were essentially identical. A concordance of affinity estimates from the functional (inositol phosphates accumulation) studies of the α1A‐adrenoceptor in CHO‐K1 cells was found with estimates published recently from contractile studies in human lower urinary tract tissues (putative α1L‐adrenoceptor). These data show that upon functional pharmacological analysis, the cloned α1A‐adrenoceptor displays pharmacological recognition properties consistent with those of the putative α1L‐adrenoceptor. Why this profile differs from that obtained in membrane binding, and whether it explains the α1L‐adrenoceptor pharmacology observed in many native tissues, requires further investigation.

GDC-0980 Is a Novel Class I PI3K/mTOR Kinase Inhibitor with Robust Activity in Cancer Models Driven by the PI3K Pathway

Alterations of the phosphoinositide-3 kinase (PI3K)/Akt signaling pathway occur broadly in cancer via multiple mechanisms including mutation of the PIK3CA gene, loss or mutation of phosphatase and tensin homolog (PTEN), and deregulation of mammalian target of rapamycin (mTOR) complexes. The dysregulation of this pathway has been implicated in tumor initiation, cell growth and survival, invasion and angiogenesis, thus, PI3K and mTOR are promising therapeutic targets for cancer. We discovered GDC-0980, a selective, potent, orally bioavailable inhibitor of Class I PI3 kinase and mTOR kinase (TORC1/2) with excellent pharmacokinetic and pharmaceutical properties. GDC-0980 potently inhibits signal transduction downstream of both PI3K and mTOR, as measured by pharmacodynamic (PD) biomarkers, thereby acting upon two key pathway nodes to produce the strongest attainable inhibition of signaling in the pathway. Correspondingly, GDC-0980 was potent across a broad panel of cancer cell lines, with the greatest potency in breast, prostate, and lung cancers and less activity in melanoma and pancreatic cancers, consistent with KRAS and BRAF acting as resistance markers. Treatment of cancer cell lines with GDC-0980 resulted in G1 cell-cycle arrest, and in contrast to mTOR inhibitors, GDC-0980 induced apoptosis in certain cancer cell lines, including those with direct pathway activation via PI3K and PTEN. Low doses of GDC-0980 potently inhibited tumor growth in xenograft models including those with activated PI3K, loss of LKB1 or PTEN, and elicited an exposure-related decrease in PD biomarkers. These preclinical data show that GDC-0980 is a potent and effective dual PI3K/mTOR inhibitor with promise for the clinic. Mol Cancer Ther; 10(12); 2426–36. ©2011 AACR.

Ligand Efficacy and Potency at Recombinant α2 Adrenergic Receptors: Agonist-Mediated [35s]gtpγs Binding

Abstract Alpha-2 adrenergic receptors (α2 AR) mediate incorporation of guanosine 5′-O-(γ-thio)triphosphate ([35S]GTPγS) into isolated membranes via receptor-catalyzed exchange of [35S]GTPγS for GDP. In the current study, we used [35S]GTPγS incorporation to characterize the intrinsic activity and potency of agonists and antagonists at the cloned mouse α2a/d and human α2a, α2b, and α2c ARs. Full agonists increased [35S]GTPγS binding to membranes by 2- to 3-fold. Antagonists did not increase [35S]GTPγS binding but competitively inhibited agonist-stimulated [35S]GTPγS binding. Compounds with intrinsic activities less than that of the full agonists norepinephrine (NE) or epinephrine (EPI) were capable of antagonizing agonist-stimulated [35S]GTPγS binding. The agonistic properties of a number of α2 AR ligands were characterized at each α2 AR subtype. The rank order of agonist potency for selected compounds at the human receptors (with intrinsic activity compared with NE, defined as 1.0) was: α2a: Dexmedetomidine (0.73) > guanabenz (0.38) > UK-14304 (1.02) > clonidine (0.32) > ST-91 (0.63) > NE (1.00). α2b: Dexmedetomidine (1.10) > clonidine (0.18) > guanabenz (0.71) > NE (1.00) > ST-91 (0.44) > UK-14304 (0.59). α2c: Dexmedetomidine (1.03) > NE (1.00) > UK-14304 (0.75) > ST-91 (0.32) ≥ clonidine (0.23) ≫ guanabenz (0). This report provides a functional characterization of adrenergic receptor ligands at human and mouse α2a/d AR. It also illustrates the utility of [35S]GTPγS incorporation as a functional marker of receptor activation.

Discovery of (Thienopyrimidin-2-yl)aminopyrimidines as Potent, Selective, and Orally Available Pan-PI3-Kinase and Dual Pan-PI3-Kinase/mTOR Inhibitors for the Treatment of Cancer.

The PI3K/AKT/mTOR pathway has been shown to play an important role in cancer. Starting with compounds 1 and 2 (GDC-0941) as templates, (thienopyrimidin-2-yl)aminopyrimidines were discovered as potent inhibitors of PI3K or both PI3K and mTOR. Structural information derived from PI3K gamma-ligand cocrystal structures of 1 and 2 were used to design inhibitors that maintained potency for PI3K yet improved metabolic stability and oral bioavailability relative to 1. The addition of a single methyl group to the optimized 5 resulted in 21, which had significantly reduced potency for mTOR. The lead compounds 5 (GNE-493) and 21 (GNE-490) have good pharmacokinetic (PK) parameters, are highly selective, demonstrate knock down of pathway markers in vivo, and are efficacious in xenograft models where the PI3K pathway is deregulated. Both compounds were compared in a PI3K alpha mutated MCF7.1 xenograft model and were found to have equivalent efficacy when normalized for exposure.

Molecular cloning, genomic characterization and expression of novel human α1A-adrenoceptor isoforms

We have isolated and characterized from human prostate novel splice variants of the human α1A‐adrenoceptor, several of which generate truncated products and one isoform, α1A‐4, which has the identical splice site as the three previously described isoforms. Long‐PCR on human genomic DNA showed that the α1A‐4 exon is located between those encoding the α1A‐1 and α1A‐3 variants. CHO‐K1 cells stably expressing α1A‐4 showed ligand binding properties similar to those of the other functional isoforms as well as agonist‐stimulated inositol phosphate accumulation. Quantitative PCR analyses revealed that α1A‐4 is the most abundant isoform expressed in the prostate with high levels also detected in liver and heart.

Cardiovascular effects of rilmenidine, moxonidine and clonidine in conscious wild‐type and D79N α2A‐adrenoceptor transgenic mice

We investigated the cardiovascular effects of rilmenidine, moxonidine and clonidine in conscious wild‐type and D79N α2A‐adrenoceptor mice. The in vitro pharmacology of these agonists was determined at recombinant (human) α2‐adrenoceptors and at endogenous (dog) α2A‐adrenoceptors. In wild‐type mice, rilmenidine, moxonidine (100, 300 and 1000 μg kg−1, i.v.) and clonidine (30, 100 and 300 μg kg−1, i.v.) dose‐dependently decreased blood pressure and heart rate. In D79N α2A‐adrenoceptor mice, responses to rilmenidine and moxonidine did not differ from vehicle control. Clonidine‐induced hypotension was absent, but dose‐dependent hypertension and bradycardia were observed. In wild‐type mice, responses to moxonidine (1 mg kg−1, i.v.) were antagonized by the non‐selective, non‐imidazoline α2‐adrenoceptor antagonist, RS‐79948‐197 (1 mg kg−1, i.v.). Affinity estimates (pKi) at human α2A‐, α2B‐ and α2C‐adrenoceptors, respectively, were: rilmenidine (5.80, 5.76 and 5.33), moxonidine (5.37, <5 and <5) and clonidine (7.21, 7.16 and 6.87). In a [35S]‐GTPγS incorporation assay, moxonidine and clonidine were α2A‐adrenoceptor agonists (pEC50/intrinsic activity relative to noradrenaline): moxonidine (5.74/0.85) and clonidine (7.57/0.32). In dog saphenous vein, concentration‐dependent contractions were observed (pEC50/intrinsic activity relative to noradrenaline): rilmenidine (5.83/0.70), moxonidine (6.48/0.98) and clonidine (7.22/0.83). Agonist‐independent affinities were obtained with RS‐79948‐197. Thus, expression of α2A‐adrenoceptors is a prerequisite for the cardiovascular effects of moxonidine and rilmenidine in conscious mice. There was no evidence of I1‐imidazoline receptor‐mediated effects. The ability of these compounds to act as α2A‐adrenoceptor agonists in vitro supports this conclusion.

Rational Design of Phosphoinositide 3-Kinase α Inhibitors That Exhibit Selectivity over the Phosphoinositide 3-Kinase β Isoform

Of the four class I phosphoinositide 3-kinase (PI3K) isoforms, PI3Kα has justly received the most attention for its potential in cancer therapy. Herein we report our successful approaches to achieve PI3Kα vs PI3Kβ selectivity for two chemical series. In the thienopyrimidine series of inhibitors, we propose that select ligands achieve selectivity derived from a hydrogen bonding interaction with Arg770 of PI3Kα that is not attained with the corresponding Lys777 of PI3Kβ. In the benzoxepin series of inhibitors, the selectivity observed can be rationalized by the difference in electrostatic potential between the two isoforms in a given region rather than any specific interaction.

Discovery of Novel PI3-Kinase δ Specific Inhibitors for the Treatment of Rheumatoid Arthritis: Taming CYP3A4 Time-Dependent Inhibition

PI3Kδ is a lipid kinase and a member of a larger family of enzymes, PI3K class IA(α, β, δ) and IB (γ), which catalyze the phosphorylation of PIP2 to PIP3. PI3Kδ is mainly expressed in leukocytes, where it plays a critical, nonredundant role in B cell receptor mediated signaling and provides an attractive opportunity to treat diseases where B cell activity is essential, e.g., rheumatoid arthritis. We report the discovery of novel, potent, and selective PI3Kδ inhibitors and describe a structural hypothesis for isoform (α, β, γ) selectivity gained from interactions in the affinity pocket. The critical component of our initial pharmacophore for isoform selectivity was strongly associated with CYP3A4 time-dependent inhibition (TDI). We describe a variety of strategies and methods for monitoring and attenuating TDI. Ultimately, a structure-based design approach was employed to identify a suitable structural replacement for further optimization.

Potent and highly selective benzimidazole inhibitors of PI3-kinase delta.

Inhibition of PI3Kδ is considered to be an attractive mechanism for the treatment of inflammatory diseases and leukocyte malignancies. Using a structure-based design approach, we have identified a series of potent and selective benzimidazole-based inhibitors of PI3Kδ. These inhibitors do not occupy the selectivity pocket between Trp760 and Met752 that is induced by other families of PI3Kδ inhibitors. Instead, the selectivity of the compounds for inhibition of PI3Kδ relative to other PI3K isoforms appears to be due primarily to the strong interactions these inhibitors are able to make with Trp760 in the PI3Kδ binding pocket. The pharmacokinetic properties and the ability of compound 5 to inhibit the function of B-cells in vivo are described.

The Rational Design of Selective Benzoxazepin Inhibitors of the α-Isoform of Phosphoinositide 3-Kinase Culminating in the Identification of (S)-2-((2-(1-Isopropyl-1H-1,2,4-triazol-5-yl)-5,6-dihydrobenzo[f]imidazo[1,2-d][1,4]oxazepin-9-yl)oxy)propanamide (GDC-0326)

Inhibitors of the class I phosphoinositide 3-kinase (PI3K) isoform PI3Kα have received substantial attention for their potential use in cancer therapy. Despite the particular attraction of targeting PI3Kα, achieving selectivity for the inhibition of this isoform has proved challenging. Herein we report the discovery of inhibitors of PI3Kα that have selectivity over the other class I isoforms and all other kinases tested. In GDC-0032 (3, taselisib), we previously minimized inhibition of PI3Kβ relative to the other class I insoforms. Subsequently, we extended our efforts to identify PI3Kα-specific inhibitors using PI3Kα crystal structures to inform the design of benzoxazepin inhibitors with selectivity for PI3Kα through interactions with a nonconserved residue. Several molecules selective for PI3Kα relative to the other class I isoforms, as well as other kinases, were identified. Optimization of properties related to drug metabolism then culminated in the identification of the clinical candidate GDC-0326 (4).