Erythropoietin and small molecule agonists of the tissue-protective erythropoietin receptor increase FXN expression in neuronal cells in vitro and in Fxn-deficient KIKO mice in vivo.

Friedreich's ataxia (FA) is a progressive neurodegenerative disease caused by reduced levels of the mitochondrial protein frataxin (FXN). Recombinant human erythropoietin (rhEPO) increased FXN protein in vitro and in early clinical studies, while no published reports evaluate rhEPO in animal models of FA. STS-E412 and STS-E424 are novel small molecule agonists of the tissue-protective, but not the erythropoietic EPO receptor. We find that rhEPO, STS-E412 and STS-E424 increase FXN expression in vitro and in vivo. RhEPO, STS-E412 and STS-E424 increase FXN by up to 2-fold in primary human cortical cells and in retinoic-acid differentiated murine P19 cells. In primary human cortical cells, the increase in FXN protein was accompanied by an increase in FXN mRNA, detectable within 4 h. RhEPO and low nanomolar concentrations of STS-E412 and STS-E424 also increase FXN in normal and FA patient-derived PBMC by 20%-40% within 24 h, an effect that was comparable to that by HDAC inhibitor 4b. In vivo, STS-E412 increased Fxn mRNA and protein in wild-type C57BL6/j mice. RhEPO, STS-E412, and STS-E424 increase FXN expression in the heart of FXN-deficient KIKO mice. In contrast, FXN expression in the brains of KIKO mice increased following treatment with STS-E412 and STS-E424, but not following treatment with rhEPO. Unexpectedly, rhEPO-treated KIKO mice developed severe splenomegaly, while no splenomegaly was observed in STS-E412- or STS-E424-treated mice. RhEPO, STS-E412 and STS-E424 upregulate FXN expression in vitro at equal efficacy, however, the effects of the small molecules on FXN expression in the CNS are superior to rhEPO in vivo.

Discovery and Characterization of Nonpeptidyl Agonists of the Tissue-Protective Erythropoietin Receptor.

Erythropoietin (EPO) and its receptor are expressed in a wide variety of tissues, including the central nervous system. Local expression of both EPO and its receptor is upregulated upon injury or stress and plays a role in tissue homeostasis and cytoprotection. High-dose systemic administration or local injection of recombinant human EPO has demonstrated encouraging results in several models of tissue protection and organ injury, while poor tissue availability of the protein limits its efficacy. Here, we describe the discovery and characterization of the nonpeptidyl compound STS-E412 (2-[2-(4-chlorophenoxy)ethoxy]-5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyrimidine), which selectively activates the tissue-protective EPO receptor, comprising an EPO receptor subunit (EPOR) and the common ß-chain (CD131). STS-E412 triggered EPO receptor phosphorylation in human neuronal cells. STS-E412 also increased phosphorylation of EPOR, CD131, and the EPO-associated signaling molecules JAK2 and AKT in HEK293 transfectants expressing EPOR and CD131. At low nanomolar concentrations, STS-E412 provided EPO-like cytoprotective effects in primary neuronal cells and renal proximal tubular epithelial cells. The receptor selectivity of STS-E412 was confirmed by a lack of phosphorylation of the EPOR/EPOR homodimer, lack of activity in off-target selectivity screening, and lack of functional effects in erythroleukemia cell line TF-1 and CD34(+) progenitor cells. Permeability through artificial membranes and Caco-2 cell monolayers in vitro and penetrance across the blood-brain barrier in vivo suggest potential for central nervous system availability of the compound. To our knowledge, STS-E412 is the first nonpeptidyl, selective activator of the tissue-protective EPOR/CD131 receptor. Further evaluation of the potential of STS-E412 in central nervous system diseases and organ protection is warranted.

Agonists of the tissue-protective erythropoietin receptor in the treatment of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disease affecting more than a million people in the USA alone. While there are effective symptomatic treatments for PD, there is an urgent need for new therapies that slow or halt the progressive death of dopaminergic neurons. Significant progress has been made in understanding the pathophysiology of PD, which has substantially facilitated the discovery efforts to identify novel drugs. The tissue-protective erythropoietin (EPO) receptor, EPOR/CD131, has emerged as one promising target for disease-modifying therapies. Recombinant human EPO (rhEPO), several variants of EPO, EPO-mimetic peptides, cell-based therapies using cells incubated with or expressing EPO, gene therapy vectors encoding EPO, and small molecule EPO mimetic compounds all show potential as therapeutic candidates. Agonists of the EPOR/CD131 receptor demonstrate potent anti-apoptotic, antioxidant, and anti-inflammatory effects and protect neurons, including dopaminergic neurons, from diverse insults in vitro and in vivo. When delivered directly to the striatum, rhEPO protects dopaminergic neurons in animal models of PD. Early-stage clinical trials testing systemic rhEPO have provided encouraging results, while additional controlled studies are required to fully assess the potential of the treatment. Poor CNS availability of proteins and challenges related to invasive delivery limit delivery of EPO protein. Several variants of EPO and small molecule agonists of the EPO receptors are making progress in preclinical studies and may offer solutions to these challenges. While EPO was initially discovered as the primary modulator of erythropoiesis, the discovery and characterization of the tissue-protective EPOR/CD131 receptor offer an opportunity to selectively target the neuroprotective receptor as an approach to identify disease-modifying treatments for PD.

Discovery of 5-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-7-phenyl-(E)-2,3,6,7-tetrahydro-1,4-thiazepines as a new series of apoptosis inducers using a cell- and caspase-based HTS assay.

We report the discovery of 5-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-7-(4-methylphenyl)-(E)-2,3,6,7-tetrahydro-1,4-thiazepine (2a) as an inducer of apoptosis using our proprietary cell- and caspase-based HTS assay. Through structure activity relationship (SAR) studies, 5-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-7-(2-methoxy-4-(methylthio)phenyl)-(E)-2,3,6,7-tetrahydro-1,4-thiazepine (5d) was identified as a potent apoptosis inducer with an EC(50) value of 0.08 microM in T47D cells, which was >15-fold more potent than screening hit 2a. Compound 5d also was found to be highly active in a growth inhibition assay with a GI(50) value of 0.05 microM in T47D cells and to function as an inhibitor of tubulin polymerization.

Evaluation of antitumor properties of novel saframycin analogs in vitro and in vivo.

Novel analogs of (-)-saframycin A are described. The analogs are shown to be potent inhibitors of the in vitro growth of several tumor cells in a broad panel and promising as leads for further optimization. The first in vivo studies in a solid tumor model (HCT-116) reveal potent antitumor activity with associated toxicity of daily administration.

Discovery and structure-activity relationship of 2-phenyl-oxazole-4-carboxamide derivatives as potent apoptosis inducers.

As a continuation of our efforts to discover novel apoptosis inducers as anticancer agents using a cell-based caspase HTS assay, 2-phenyl-oxazole-4-carboxamide derivatives were identified. The structure-activity relationships of this class of molecules were explored. Compound 1k, with EC(50) of 270 nM and GI(50) of 229 nM in human colorectal DLD-1 cells, was selected and demonstrated the ability to cleave PARP and displayed DNA laddering, the hallmarks of apoptosis. Compound 1k showed 63% tumor growth inhibition in human colorectal DLD-1 xenograft mouse model at 50 mpk, bid.

Novel, potent, selective, and orally bioavailable human betaII-tryptase inhibitors.

The synthesis of novel [1,2,4]oxadiazoles and their structure-activity relationship (SAR) for the inhibition of tryptase and related serine proteases is presented. Elaboration of the P'-side afforded potent, selective, and orally bioavailable tryptase inhibitors.

Design of novel, potent, and selective human beta-tryptase inhibitors based on alpha-keto-[1,2,4]-oxadiazoles.

A series of novel alpha-keto-[1,2,4]-oxadiazoles has been synthesized as human tryptase inhibitors for evaluation as a new class of anti-asthmatic agent. The inhibitor design is focused on using a prime-side hydrophobic pocket and the S2 pocket of beta-tryptase to achieve inhibition potency and selectivity over other serine proteases.

Antitumor activity of tetrahydroisoquinoline analogues 3-epi-jorumycin and 3-epi-renieramycin G.

Analogues of the tetrahydroisoquinoline family of antitumor antibiotics, 3-epi-jorumycin (3) and 3-epi-renieramycin G (4), in addition to their respective parent natural products (-)-jorumycin (1) and (-)-renieramycin G (2) were evaluated against both human colon (HCT-116) and human lung (A549) cancer cell lines. (-)-Jorumycin (1) displayed potent growth inhibition with GI50 values in the low nanomolar range (1.9-24.3 nM), while compounds 2-4 were found to be substantially less cytotoxic (GI50 0.6-14.0 microM).

Design and synthesis of selective keto-1,2,4-oxadiazole-based tryptase inhibitors.

Using a scaleable, directed library approach based on orthogonally protected advanced intermediates, we have prepared a series of potent keto-1,2,4-oxadiazoles designed to explore the P(2) binding pocket of human mast cell tryptase, while building in a high degree of selectivity over human trypsin and other serine proteases.

Discovery of novel hydroxy pyrazole based factor IXa inhibitor.

Synthesis and biological data of a novel selective and efficacious factor IXa inhibitor are described along with its crystal structure in factor VIIa.

Elaborate manifold of short hydrogen bond arrays mediating binding of active site-directed serine protease inhibitors.

An extensive structural manifold of short hydrogen bond-mediated, active site-directed, serine protease inhibition motifs is revealed in a set of over 300 crystal structures involving a large suite of small molecule inhibitors (2-(2-phenol)-indoles and 2-(2-phenol)-benzimidazoles) determined over a wide range of pH (3.5-11.4). The active site hydrogen-bonding mode was found to vary markedly with pH, with the steric and electronic properties of the inhibitor, and with the type of protease (trypsin, thrombin or urokinase type plasminogen activator (uPA)). The pH dependence of the active site hydrogen-bonding motif is often intricate, constituting a distinct fingerprint of each complex. Isosteric replacements or minor substitutions within the inhibitor that modulate the pK(a) of the phenol hydroxyl involved in short hydrogen bonding, or that affect steric interactions distal to the active site, can significantly shift the pH-dependent structural profile characteristic of the parent scaffold, or produce active site-binding motifs unique to the bound analog. Ionization equilibria at the active site associated with inhibitor binding are probed in a series of the protease-inhibitor complexes through analysis of the pH dependence of the structure and environment of the active site-binding groups involved in short hydrogen bond arrays. Structures determined at high pH (>11), suggest that the pK(a) of His57 is dramatically elevated, to a value as high as approximately 11 in certain complexes. K(i) values involving uPA and trypsin determined as a function of pH for a set of inhibitors show pronounced parabolic pH dependence, the pH for optimal inhibition governed by the pK(a) of the inhibitor phenol involved in short hydrogen bonds. Comparison of structures of trypsin, thrombin and uPA, each bound by the same inhibitor, highlights important structural variations in the S1 and active sites accessible for engineering notable selectivity into remarkably small molecules with low nanomolar K(i) values.

Highly potent non-peptidic inhibitors of the HCV NS3/NS4A serine protease.

Screening of a diverse set of bisbenzimidazoles for inhibition of the hepatitis C virus (HCV) serine protease NS3/NS4A led to the identification of a potent Zn(2+)-dependent inhibitor (1). Optimization of this screening hit afforded a 10-fold more potent inhibitor (46) under Zn(2+) conditions (K(i)=27nM). This compound (46) binds also to NS3/NS4A in a Zn(2+) independent fashion (K(i)=1microM). The SAR of this class of compounds under Zn(2+) conditions is highly divergent compared to the SAR in the absence of Zn(2+), suggesting two distinct binding modes.

Contribution of multicentered short hydrogen bond arrays to potency of active site-directed serine protease inhibitors.

We describe and compare the pH dependencies of the potencies and of the bound structures of two inhibitor isosteres that form multicentered short hydrogen bond arrays at the active sites of trypsin, thrombin, and urokinase type plasminogen activator (urokinase or uPA) over certain ranges of pH. Depending on the pH, short hydrogen bond arrays at the active site are mediated by two waters, one in the oxyanion hole (H(2)O(oxy)) and one on the other (S2) side of the inhibitor (H(2)O(S2)), by one water (H(2)O(oxy)), or by no water. The dramatic variation in the length of the active site hydrogen bonds as a function of pH, of inhibitor, and of enzyme, along with the involvement or absence of ordered water, produces a large structural manifold of active site hydrogen bond motifs. Diverse examples of multicentered and two-centered short hydrogen bond arrays, both at and away from the active site, recently discovered in several protein crystal systems, suggest that short hydrogen bonds in proteins may be more common than has been recognized. The short hydrogen bond arrays resemble one another with respect to ionic nature, highly polar environment, multitude of associated ordinary hydrogen bonds, and disparate pK(a) values of participating groups. Comparison of structures and K(i) values of trypsin complexes at pH values where the multicentered short hydrogen bond arrays mediating inhibitor binding are present or absent indicate that these arrays have a minor effect on inhibitor potency. These features suggest little covalent nature within the short hydrogen bonds, despite their extraordinary shortness (as short as 2.0 A).

2-(2-Hydroxy-3-alkoxyphenyl)-1H-benzimidazole-5-carboxamidine derivatives as potent and selective urokinase-type plasminogen activator inhibitors.

The development of potent and selective urokinase-type plasminogen activator (uPA) inhibitors based on the lead molecule 2-(2-hydroxy-3-ethoxyphenyl)-1H-benzimidazole-5-carboxamidine (3a) is described.

4-Aminoarylguanidine and 4-aminobenzamidine derivatives as potent and selective urokinase-type plasminogen activator inhibitors.

The structure-based design of potent and selective urokinase-type plasminogen activator (uPA) inhibitors with 4-aminoarylamidine or 4-aminoarylguanidine S1 binding groups, is described.