Design, Synthesis, and Characterization of I-BET567, a Pan-Bromodomain and Extra Terminal (BET) Bromodomain Oral Candidate.

Through regulation of the epigenome, the bromodomain and extra terminal (BET) family of proteins represent important therapeutic targets for the treatment of human disease. Through mimicking the endogenous N-acetyl-lysine group and disrupting the protein-protein interaction between histone tails and the bromodomain, several small molecule pan-BET inhibitors have progressed to oncology clinical trials. This work describes the medicinal chemistry strategy and execution to deliver an orally bioavailable tetrahydroquinoline (THQ) pan-BET candidate. Critical to the success of this endeavor was a potency agnostic analysis of a data set of 1999 THQ BET inhibitors within the GSK collection which enabled identification of appropriate lipophilicity space to deliver compounds with a higher probability of desired oral candidate quality properties. SAR knowledge was leveraged via Free-Wilson analysis within this design space to identify a small group of targets which ultimately delivered I-BET567 (27), a pan-BET candidate inhibitor that demonstrated efficacy in mouse models of oncology and inflammation.

Discovery of a Novel Bromodomain and Extra Terminal Domain (BET) Protein Inhibitor, I-BET282E, Suitable for Clinical Progression.

The functions of the bromodomain and extra terminal (BET) family of proteins have been implicated in a wide range of diseases, particularly in the oncology and immuno-inflammatory areas, and several inhibitors are under investigation in the clinic. To mitigate the risk of attrition of these compounds due to structurally related toxicity findings, additional molecules from distinct chemical series were required. Here we describe the structure- and property-based optimization of the in vivo tool molecule I-BET151 toward I-BET282E, a molecule with properties suitable for progression into clinical studies.

Automated reconstruction of cast-off blood spatter patterns based on Euclidean geometry and statistical likelihood.

Cast-off spatter patterns exhibit linear trails of elliptical stains. These characteristic patterns occur by centrifugal forces that detach drops from a swinging object covered with blood or other liquid. This manuscript describes a method to reconstruct the motion, or swing, of the object. The method is based on stain inspection and Euclidean geometry. The reconstructed swing is represented as a three-dimensional region of statistical likelihood. The reconstruction uncertainty corresponds to the volume of the reconstructed region, which is specific to the uncertainties of the case at hand. Simple numerical examples show that the reconstruction method is able to reconstruct multiple swings that are either intersecting or adjacent to each other. The robustness, spatial convergence, computing time of the reconstruction method is characterized. For the purpose of this study, about 20 cast-off experiments are produced, with motion of the swinging object documented using video and/or accelerometers. The swings follow circular or arbitrary paths, and are either human- or machine-made. The reconstruction results are compared with the experimentally documented swings. Agreement between measured and reconstructed swings is very good, typically within less than 10 cm. The method used in this study is implemented as a numerical code written in an open source language, provided in an open access repository, for purposes of transparency and access.

Discovery of a Bromodomain and Extraterminal Inhibitor with a Low Predicted Human Dose through Synergistic Use of Encoded Library Technology and Fragment Screening.

The bromodomain and extraterminal (BET) family of bromodomain-containing proteins are important regulators of the epigenome through their ability to recognize N-acetyl lysine (KAc) post-translational modifications on histone tails. These interactions have been implicated in various disease states and, consequently, disruption of BET-KAc binding has emerged as an attractive therapeutic strategy with a number of small molecule inhibitors now under investigation in the clinic. However, until the utility of these advanced candidates is fully assessed by these trials, there remains scope for the discovery of inhibitors from new chemotypes with alternative physicochemical, pharmacokinetic, and pharmacodynamic profiles. Herein, we describe the discovery of a candidate-quality dimethylpyridone benzimidazole compound which originated from the hybridization of a dimethylphenol benzimidazole series, identified using encoded library technology, with an N-methyl pyridone series identified through fragment screening. Optimization via structure- and property-based design led to I-BET469, which possesses favorable oral pharmacokinetic properties, displays activity in vivo, and is projected to have a low human efficacious dose.

Optimisation of a novel series of potent and orally bioavailable azanaphthyridine SYK inhibitors.

The optimisation of the azanaphthyridine series of Spleen Tyrosine Kinase inhibitors is described. The medicinal chemistry strategy was focused on optimising the human whole blood activity whilst achieving a sufficient margin over hERG activity. A good pharmacokinetic profile was achieved by modification of the pKa. Morpholine compound 32 is a potent SYK inhibitor showing moderate selectivity, good oral bioavailability and good efficacy in the rat Arthus model but demonstrated a genotoxic potential in the Ames assay.

Syk tyrosine kinase is critical for B cell antibody responses and memory B cell survival.

Signals from the BCR are required for Ag-specific B cell recruitment into the immune response. Binding of Ag to the BCR induces phosphorylation of immune receptor tyrosine-based activation motifs in the cytoplasmic domains of the CD79a and CD79b signaling subunits, which subsequently bind and activate the Syk protein tyrosine kinase. Earlier work with the DT40 chicken B cell leukemia cell line showed that Syk was required to transduce BCR signals to proximal activation events, suggesting that Syk also plays an important role in the activation and differentiation of primary B cells during an immune response. In this study, we show that Syk-deficient primary mouse B cells have a severe defect in BCR-induced activation, proliferation, and survival. Furthermore, we demonstrate that Syk is required for both T-dependent and T-independent Ab responses, and that this requirement is B cell intrinsic. In the absence of Syk, Ag fails to induce differentiation of naive B cells into germinal center B cells and plasma cells. Finally, we show that the survival of existing memory B cells is dependent on Syk. These experiments demonstrate that Syk plays a critical role in multiple aspects of B cell Ab responses.

The BAFF receptor transduces survival signals by co-opting the B cell receptor signaling pathway.

Follicular B cell survival requires signaling from BAFFR, a receptor for BAFF and the B cell antigen receptor (BCR). This "tonic" BCR survival signal is distinct from that induced by antigen binding and may be ligand-independent. We show that inducible inactivation of the Syk tyrosine kinase, a key signal transducer from the BCR following antigen binding, resulted in the death of most follicular B cells because Syk-deficient cells were unable to survive in response to BAFF. Genetic rescue studies demonstrated that Syk transduces BAFFR survival signals via ERK and PI3 kinase. Surprisingly, BAFFR signaling directly induced phosphorylation of both Syk and the BCR-associated Igα signaling subunit, and this Syk phosphorylation required the BCR. We conclude that the BCR and Igα may be required for B cell survival because they function as adaptor proteins in a BAFFR signaling pathway leading to activation of Syk, demonstrating previously unrecognized crosstalk between the two receptors.

Identification of a novel series of BET family bromodomain inhibitors: binding mode and profile of I-BET151 (GSK1210151A).

A novel series of quinoline isoxazole BET family bromodomain inhibitors are discussed. Crystallography is used to illustrate binding modes and rationalize their SAR. One member, I-BET151 (GSK1210151A), shows good oral bioavailability in both the rat and minipig as well as demonstrating efficient suppression of bacterial induced inflammation and sepsis in a murine in vivo endotoxaemia model.

Identification of 5-HT(2C) mediated mechanisms involved in urethral sphincter reflexes in a guinea-pig model of urethral function.

UNLABELLED: Preclinically, studies investigating urethral function have shown that the clinical benefit of agents such as duloxetine may partly reflect increases in urethral striated muscle activity via a central mode of action. Duloxetine has been shown to inhibit the presynaptic reuptake of 5-HT and norepinephrine in Onuf's nucleus, leading to an increased activity of pudendal motor neurones and a subsequent increase in the strength of urethral sphincter contractions. Preclinical studies have postulated a role for both 5-HT(2C) receptors and 5-HT(2A) receptors in external urethral sphincter (EUS) function, with differences between species that may reflect the differing physiological roles of the EUS in different preclinical species. The present study therefore aimed to investigate the 5-HT receptor subtype involved in EUS function in the guinea-pig. The in vivo data reported in the present study suggest that the effects of clinical agents used to treat stress urinary incontinence, which enhance serotonergic drive, may be mediated, at least in part, via 5-HT(2C) receptors. OBJECTIVE: To elucidate the subtype of 5-HT receptor involved in urethral function using a preclinical model of urethral function. MATERIALS AND METHODS: The effects of the 5-HT(2C) agonist Ro 600-175 were investigated by measuring the urethral pressure profile in anaesthetized guinea-pigs together with antagonists at 5-HT(2A) , 5-HT(2B) and 5-HT(2C) receptor subtypes. RESULTS: Ro 600-175 increased peak urethral pressure in a dose-dependent manner. This effect was reversed by the selective 5-HT(2C) antagonist SB 242084. Neither the 5-HT(2A) antagonist MDL 100907, nor the 5-HT(2B) antagonist SB 240741 had any significant effect on the response. CONCLUSIONS: The clinical benefit of drugs used to treat stress urinary incontinence through enhanced serotonergic and adrenergic drive may be mediated, at least in part, via 5-HT(2C) receptors. Selective 5-HT(2C) agonism increases urethral tone, and hence provides an opportunity for developing new pharmacotherapies for stress urinary incontinence with reduced side-effects.

Discovery of GSK143, a highly potent, selective and orally efficacious spleen tyrosine kinase inhibitor.

The lead optimisation of the diaminopyrimidine carboxamide series of spleen tyrosine kinase inhibitors is described. The medicinal chemistry strategy was focused on optimising the human whole blood activity whilst achieving a sufficient margin over liability kinases and hERG activity. GSK143 is a potent and highly selective SYK inhibitor showing good efficacy in the rat Arthus model.

Progression and variability of TNBS colitis-associated inflammation in rats assessed by contrast-enhanced and T2-weighted MRI.

BACKGROUND: A common feature of preclinical models of colitis is that the time-course, magnitude, and persistence of inflammation vary considerably within the experimental animal group. Accordingly, noninvasive, serial quantification of colonic inflammation could advantageously guide dosing regimens and assess drug efficacy, thus enhancing the value of colitis models in research. This investigation using magnetic resonance imaging (MRI) was therefore undertaken to objectively determine inflammatory progression, variability, and response to therapy associated with trinitrobenzene sulfonic acid (TNBS)-induced colitis in Wistar rats. METHODS: Rats underwent TNBS treatment on Day 0 and received sulfasalazine or vehicle (methylcellulose) orally, daily, from Day -1 (prophylactically) or Day 2 (therapeutically). T2-weighted and semidynamic T1-weighted contrast-enhanced MRI (CE-MRI) was repeated over 7-10 days to measure colon wall thickness and perfusion-related aspects of inflammation. Rectal bleeding, stool consistency, and disease activity were scored throughout and colon pathology determined terminally. RESULTS: Principal component analysis of the CE-MRI time-series highlighted colon wall and mesenteric inflammation, which increased by 6-8x naïve values. Peristaltic artifacts were distinguished from perfusion changes using the normalized temporal standard deviation. MRI correlated strongly with terminal colon weight (mean correlation r = 0.8), well with body weight change (r = -0.7), but little with conventional clinical scores. Sulfasalazine reduced inflammation administered prophylactically and therapeutically. CONCLUSIONS: Inflammation and therapeutic efficacy can be sensitively quantified noninvasively using MRI in TNBS-treated rats. This methodology provides unique and objective in vivo measures of inflammation that can guide dosing strategies, enhancing colitis research effectiveness and the assessment of potential IBD therapeutics.

Ovariohysterectomy in the rat: a model of surgical pain for evaluation of pre-emptive analgesia?

Ovariohysterectomy in the rat led to the induction of abdominal postures and referred mechanical allodynia in the hind paws. The latter was differentiated into static and dynamic subtypes. The abdominal postures were present up to 4-5 h, whilst the two types of allodynia lasted for at least 2 days. A single administration of morphine 30 min before surgery dose-dependently (0.1-3 mg/kg, s.c.) blocked the development of abdominal postures and the two types of mechanical allodynia. The highest dose of morphine almost completely blocked these responses. The duration of action of 3 mg/kg morphine was short and similar (1.5-2 h) when administered either before or after surgery. However, multiple administrations of morphine (0.5 h before, and 0.5 and 2 h after surgery) blocked the development of abdominal postures and both allodynias for up to 2 days. In contrast, administration of three doses of morphine (3 mg/kg) in a similar dosing regime but starting 24 h after surgery, only blocked the two types of allodynia for 4 h. These data indicate the importance of blocking the induction phase of surgical pain and support the concept of pre-emptive analgesia. It is suggested that the ovariohysterectomy model should prove to be useful for studying mechanisms and designing novel therapeutic strategies for the treatment of post-operative pain.