Discovery and Characterization of a Bicyclic Peptide (Bicycle) Binder to Thymic Stromal Lymphopoietin.

Thymic stromal lymphopoietin (TSLP) is an epithelial-derived pro-inflammatory cytokine involved in the development of asthma and other atopic diseases. We used Bicycle Therapeutics' proprietary phage display platform to identify bicyclic peptides (Bicycles) with high affinity for TSLP, a target that is difficult to drug with conventional small molecules due to the extended protein-protein interactions it forms with both receptors. The hit series was shown to bind to TSLP in a hotspot, that is also used by IL-7Rα. Guided by the first X-ray crystal structure of a small peptide binding to TSLP and the identification of key metabolites, we were able to improve the proteolytic stability of this series in lung S9 fractions without sacrificing binding affinity. This resulted in the potent Bicycle 46 with nanomolar affinity to TSLP (KD = 13 nM), low plasma clearance of 6.4 mL/min/kg, and an effective half-life of 46 min after intravenous dosing to rats.

How well does molecular simulation reproduce environment-specific conformations of the intrinsically disordered peptides PLP, TP2 and ONEG?

Understanding the conformational ensembles of intrinsically disordered proteins and peptides (IDPs) in their various biological environments is essential for understanding their mechanisms and functional roles in the proteome, leading to a greater knowledge of, and potential treatments for, a broad range of diseases. To determine whether molecular simulation is able to generate accurate conformational ensembles of IDPs, we explore the structural landscape of the PLP peptide (an intrinsically disordered region of the proteolipid membrane protein) in aqueous and membrane-mimicking solvents, using replica exchange with solute scaling (REST2), and examine the ability of four force fields (ff14SB, ff14IDPSFF, CHARMM36 and CHARMM36m) to reproduce literature circular dichroism (CD) data. Results from variable temperature (VT) 1H and Rotating frame Overhauser Effect SpectroscopY (ROESY) nuclear magnetic resonance (NMR) experiments are also presented and are consistent with the structural observations obtained from the simulations and CD. We also apply the optimum simulation protocol to TP2 and ONEG (a cell-penetrating peptide (CPP) and a negative control peptide, respectively) to gain insight into the structural differences that may account for the observed difference in their membrane-penetrating abilities. Of the tested force fields, we find that CHARMM36 and CHARMM36m are best suited to the study of IDPs, and accurately predict a disordered to helical conformational transition of the PLP peptide accompanying the change from aqueous to membrane-mimicking solvents. We also identify an α-helical structure of TP2 in the membrane-mimicking solvents and provide a discussion of the mechanistic implications of this observation with reference to the previous literature on the peptide. From these results, we recommend the use of CHARMM36m with the REST2 protocol for the study of environment-specific IDP conformations. We believe that the simulation protocol will allow the study of a broad range of IDPs that undergo conformational transitions in different biological environments.

The regulatory role of PGC1α-related coactivator in response to drug-induced liver injury.

PGC1α-Related Coactivator (PRC) is a transcriptional coactivator promoting cytokine expression in vitro in response to mitochondrial injury and oxidative stress, however, its physiological role has remained elusive. Herein we investigate aspects of the immune response function of PRC, first in an in vivo thioacetamide (TAA)-induced mouse model of drug-induced liver injury (DILI), and subsequently in vitro in human monocytes, HepG2, and dendritic (DC) cells. TAA treatment resulted in the dose-dependent induction of PRC mRNA and protein, both of which were shown to correlate with liver injury markers. Conversely, an adenovirus-mediated knockdown of PRC attenuated this response, thereby reducing hepatic cytokine mRNA expression and monocyte infiltration. Subsequent in vitro studies with conditioned media from HepG2 cells overexpressing PRC, activated human monocytes and monocyte-derived DC, demonstrated up to 20% elevated expression of CD86, CD40, and HLA-DR. Similarly, siRNA-mediated knockdown of PRC abolished this response in oligomycin stressed HepG2 cells. A putative mechanism was suggested by the co-immunoprecipitation of Signal Transducer and Activator of Transcription 1 (STAT1) with PRC, and induction of a STAT-dependent reporter. Furthermore, PRC co-activated an NF-κB-dependent reporter, indicating interaction with known major inflammatory factors. In summary, our study indicates PRC as a novel factor modulating inflammation in DILI.

Design and Synthesis of Soluble and Cell-Permeable PI3Kδ Inhibitors for Long-Acting Inhaled Administration.

PI3Kδ is a lipid kinase that is believed to be important in the migration and activation of cells of the immune system. Inhibition is hypothesized to provide a powerful yet selective immunomodulatory effect that may be beneficial for the treatment of conditions such as asthma or rheumatoid arthritis. In this work, we describe the identification of inhibitors based on a thiazolopyridone core structure and their subsequent optimization for inhalation. The initially identified compound (13) had good potency and isoform selectivity but was not suitable for inhalation. Addition of basic substituents to a region of the molecule pointing to solvent was tolerated (enzyme inhibition pIC50 > 9), and by careful manipulation of the pKa and lipophilicity, we were able to discover compounds (20b, 20f) with good lung retention and cell potency that could be taken forward to in vivo studies where significant target engagement could be demonstrated.

Discovery of triazole aminopyrazines as a highly potent and selective series of PI3Kδ inhibitors.

A novel class of potent PI3Kδ inhibitors with >1000-fold selectivity against other class I PI3K isoforms is described. Optimization of the substituents on a triazole aminopyrazine scaffold, emerging from an in-house PI3Kα program, turned moderately selective PI3Kδ compounds into highly potent and selective PI3Kδ inhibitors. These efforts resulted in a series of aminopyrazines with PI3Kδ IC50⩽1nM in the enzyme assay, some of the most selective PI3Kδ inhibitors published to date, with a cell potency in a JeKo-cell assay of 20-120nM.

Design and synthesis of conformationally restricted inhibitors of active thrombin activatable fibrinolysis inhibitor (TAFIa).

A series of 4,5,6,7-tetrahydro-1H-benzimidazole-5-carboxylic acid and 5,6,7,8-tetrahydroimidazo[1,2-a]pyridine-7-carboxylic acid derivatives designed as inhibitors of TAFIa has been prepared via a common hydrogenation-alkylation sequence starting from the appropriate benzimidazole and imidazopyridine system. We present a successful design strategy using a conformational restriction approach resulting in potent and selective inhibitors of TAFIa. The X-ray structure of compound 5 in complex with a H333Y/H335Q double mutant TAFI indicate that the conformational restriction is responsible for the observed potency increase.

Lactam sulfonamides as potent inhibitors of the Kv1.5 potassium ion channel.

A series of lactam sulfonamides has been discovered and optimized as inhibitors of the Kv1.5 potassium ion channel for treatment of atrial fibrillation. In vitro structure-activity relationships from lead structure C to optimized structure 3y are described. Compound 3y was evaluated in a rabbit PD-model and was found to selectively prolong the atrial effective refractory period at submicromolar concentrations.

Intravenous hypertonic NaCl acts via cerebral sodium-sensitive and angiotensinergic mechanisms to improve cardiac function in haemorrhaged conscious sheep.

Acute NaCl loading as resuscitation in haemorrhagic hypovolaemia is known to induce rapid cardiovascular recovery. Besides an osmotically induced increase in plasma volume the physiological mechanisms of action are unknown. We hypothesized that a CNS mechanism, elicited by increased periventricular [Na(+)] and mediated by angiotensin II type 1 receptors (AT(1)), is obligatory for the full effect of hypertonic NaCl. To test this we investigated the cardiovascular responses to haemorrhage and subsequent hypertonic NaCl infusion (7.5% NaCl, 4 ml (kg BW)(-1)) in six conscious sheep subjected to intracerebroventricular (i.c.v.) infusion of artificial cerebrospinal fluid (aCSF; control), mannitol solution (Man; 75 mmol l(-1) [Na(+)], total osmolality 295 mosmol kg(-1)) or losartan (Los; 1 mg ml(-1), AT(1) receptor antagonist) at three different occasions. Man normalized (144 +/- 6 mmol l(-1), mean +/- s.d.) the increase in i.c.v. [Na(+)] seen after aCSF (161 +/- 2 mmol l(-1)). Compared with control, both Man and Los significantly (P < 0.05) attenuated the improvement in mean arterial blood pressure (MAP), cardiac index and mesenteric blood flow (SMBF) in response to intravenous hypertonic NaCl: MAP, rapid response +45 mmHg versus +38 mmHg (Man) and +35 mmHg (Los); after 180 min, +32 mmHg versus +21 mmHg (Man) and +19 mmHg (Los); cardiac index after 180 min, +1.9 l min(-1) (m(2))(-1) versus +0.9 l min(-1) (m(2))(-1) (Man) and +0.9 l min(-1) (m(2))(-1) (Los); SMBF rapid response, +981 ml min(-1) versus +719 ml min(-1) (Man) and +744 ml min(-1) (Los); after 180 min, +602 ml min(-1) versus +372 ml min(-1) (Man) and +314 ml min(-1) (Los). The results suggest that increased periventricular [Na(+)] and cerebral AT(1) receptors contribute, together with plasma volume expansion, to improve systemic haemodynamics after treatment with hypertonic NaCl in haemorrhagic hypovolaemia.

Structure-activity relationships for the linker in a series of pyridinyl-alkynes that are antagonists of the metabotropic glutamate receptor 5 (mGluR5).

Studies of structure-activity relationships for the linker in a new series of metabotropic glutamate receptor 5 antagonists are presented together with in vitro and in vivo pharmacokinetic data.

A new series of pyridinyl-alkynes as antagonists of the metabotropic glutamate receptor 5 (mGluR5).

Synthesis and some structure-activity relationships for a new series of propargyl ethers as mGluR5 antagonists are reported.