A Process for the Design and Development of Novel Bone Morphogenetic Protein-7 (BMP-7) Mimetics With an Example: THR-184.

Growth Factors have been evaluated as therapeutic targets for the treatment of a broad spectrum of diseases. Because they are proteins with pleiotropic effects, the quest to harness their beneficial effects has presented challenges. Most Growth Factors operate at the extracellular-receptor level and have natural feedback mechanisms that modulate their effects. As proteins, they are difficult and expensive to manufacture. Frequently proteins must be administered parenterally, may invoke an immune response, and may be neutralized by naturally occurring inhibitors. To circumvent these limitations, we have undertaken an effort to develop mimetics for the Bone Morphogenetic Protein (BMP) signaling pathway effects that incorporate the beneficial effects, eliminate the deleterious effects, and thereby create effective drug-like compounds.To this end, we have designed and tested a family of small peptide BMP mimetics. The design used the three-dimensional structure of BMP-7 to identify likely active surface regions. Lead sequences were then optimized based on in vitro assays that examine the selective binding to BMP receptors, demonstrate the phosphorylation of Smad-1,5,8, detect anti-apoptosis and anti-inflammation, and block the epithelial to mesenchymal transition (EMT) in renal tubular epithelial cells. These sequences were further optimized using in vivo assays of the attenuation of acute kidney injury in a rat-model of unilateral clamp ischemic reperfusion. This process uses a Structure Variance Analysis algorithm (SVA) to identify structure/activity relationships. One member of this family, THR-184, is an agonist of BMP signaling and a potent antagonist of TGFß signaling. This small peptide mimetic inhibits inflammation, apoptosis, fibrosis and reverses epithelial to mesenchymal transition (EMT) by regulating multiple signaling pathways involved in the cellular injury of multiple organs. Its effects have been shown to control Acute Kidney Injury (AKI). THR-184 has progressed through phase I and II clinical trials for the prevention of Cardio-Vascular Surgery (CVS) associated AKI. This work provides a roadmap for the development of other growth factor mimetics and demonstrates how we might harness their therapeutic potential.

2-(3-{(3R,4R)-4-Methyl-3-[meth-yl(7H-pyrrolo-[2,3-d]pyrimidin-4-yl)amino]-piperidin-1-yl}oxetan-3-yl)aceto-nitrile monohydrate.

In the title compound, C18H24N6O·H2O, the piperidine ring adopts a chair conformation with an N-C-C-C torsion angle of 39.5 (5)° between the cis-related substituents. The pyrrole N-H group forms a water-mediated inter-molecular hydrogen bond to one of the N atoms of the annelated pyrimidine ring. The water mol-ecule connects two organic mol-ecules and is disorderd over two positions (occupancies of 0.48 and 0.52). The crystal packing shows zigzag chains of alternating organic and water mol-ecules running parallel to the a axis.

A p38 substrate-specific MK2-EGFP translocation assay for identification and validation of new p38 inhibitors in living cells: a comprising alternative for acquisition of cellular p38 inhibition data.

The fundamental role of p38 mitogen-activated protein kinases (MAPKs) in inflammation underlines their importance as therapeutic targets for various inflammatory medical conditions, including infectious, vascular, neurobiological and autoimmune disease. Although decades of research have yielded several p38 inhibitors, most clinical trials have failed, due to lack of selectivity and efficacy in vivo. This underlines the continuous need to screen for novel structures and chemotypes of p38 inhibitors. Here we report an optimized MK2-EGFP translocation assay in a semi-automated image based High Content Analysis (HCA) system to screen a combinatorial library of 3362 proprietary compounds with extensive variations of chemotypes. By determining the levels of redistribution of MK2-EGFP upon activation of the Rac/p38 pathway in combination with compound treatment, new candidates were identified, which modulate p38 activity in living cells. Based on integrated analysis of TNFα release from human whole blood, biochemical kinase activity assays and JNK3 selectivity testing, we show that this cell based assay reveals a high overlap and predictability for cellular efficacy, selectivity and potency of tested compounds. As a result we disclose a new comprehensive short-list of subtype inhibitors which are functional in the low nanomolar range and might provide the basis for further lead-optimization. In accordance to previous reports, we demonstrate that the MK2-EGFP translocation assay is a suitable primary screening approach for p38-MAPK drug development and provide an attractive labor- and cost saving alternative to other cell based methods including determination of cytokine release from hPBMCs or whole blood.

Activin-like kinase 3 is important for kidney regeneration and reversal of fibrosis.

Molecules associated with the transforming growth factor ß (TGF-ß) superfamily, such as bone morphogenic proteins (BMPs) and TGF-ß, are key regulators of inflammation, apoptosis and cellular transitions. Here we show that the BMP receptor activin-like kinase 3 (Alk3) is elevated early in diseased kidneys after injury. We also found that its deletion in the tubular epithelium leads to enhanced TGF-ß1-Smad family member 3 (Smad3) signaling, epithelial damage and fibrosis, suggesting a protective role for Alk3-mediated signaling in the kidney. A structure-function analysis of the BMP-Alk3-BMP receptor, type 2 (BMPR2) ligand-receptor complex, along with synthetic organic chemistry, led us to construct a library of small peptide agonists of BMP signaling that function through the Alk3 receptor. One such peptide agonist, THR-123, suppressed inflammation, apoptosis and the epithelial-to-mesenchymal transition program and reversed established fibrosis in five mouse models of acute and chronic renal injury. THR-123 acts specifically through Alk3 signaling, as mice with a targeted deletion for Alk3 in their tubular epithelium did not respond to therapy with THR-123. Combining THR-123 and the angiotensin-converting enzyme inhibitor captopril had an additive therapeutic benefit in controlling renal fibrosis. Our studies show that BMP signaling agonists constitute a new line of therapeutic agents with potential utility in the clinic to induce regeneration, repair and reverse established fibrosis.

2-[5-Bromo-1-(3-chloro-benz-yl)-2-methyl-1H-indol-3-yl]acetic acid.

In the title compound, C18H15BrClNO2, the indole ring system forms a dihedral angle of 86.9 (2)° with the 3-chloro-benzyl ring. In the crystal, mol-ecules form inversion dimers connected via pairs of O-H⋯O hydrogen bonds.

3-(4-Bromo-phen-yl)-1-phenyl-1H-pyrazole-4-carbaldehyde.

The asymmetric unit of the title compound, C16H11BrN2O, contains two independent mol-ecules with slightly different geometries. The 4-bromo-benzene ring forms dihedral angles of 26.0 (2) and 39.9 (7)° with the pyrazole ring in the two mol-ecules while the phenyl ring is oriented at 19.7 (5) and 7.3 (0)° with respect to the pyrazole ring.

6-(4-Chloro-phen-yl)-7-phenyl-2,3-dihydro-1H-pyrrolizine-5-carbaldehyde.

The 4-chloro-phenyl residue in the title compound, C(20)H(16)ClNO, is oriented at a dihedral angle of 53.6 (3)° towards the phenyl ring and 42.0 (9)° towards the pyrrole ring of the pyrrolizine template. The phenyl ring is oriented at a dihedral angle of 45.4 (4)° towards the pyrrole ring.

6-(4-Meth-oxy-phen-yl)-7-phenyl-2,3-dihydro-1H-pyrrolizine-5-carbaldehyde.

The 4-meth-oxy-phenyl residue in the title compound, C(21)H(19)NO(2), is oriented at a dihedral angle of 54.6 (5)° with respect to the phenyl ring and at a dihedral angle of 52.5 (8)° with respect to the pyrrole ring of the pyrrolizine system. The phenyl ring is oriented at a dihedral angle of 36.2 (5)° with respect to the pyrrole ring. The meth-oxy group makes a C-C-O-C torsion angle of 3.8 (9)° with the attached benzene ring.

Arylpyrrolizines as inhibitors of microsomal prostaglandin E2 synthase-1 (mPGES-1) or as dual inhibitors of mPGES-1 and 5-lipoxygenase (5-LOX).

We synthesized and evaluated inhibitors for the microsomal prostaglandin E2 synthase-1 (mPGES-1), based on the arylpyrrolizine scaffold. In a cell free mPGES-1 assay several "sulfonimides" exceeded our lead ML3000 (3) in potency. The most promising compound, the tolylsulfonimide 11f, revealed an IC50 of 2.1 microM and is equipotent to the literature reference molecule MK886 (1). Selected compounds also potently reduced 5-LOX product formation in intact cells. Inhibition of isolated COX was occasionally remarkably cut down.