Beyond Traditional Structure-Based Drug Design: The Role of Iron Complexation, Strain, and Water in the Binding of Inhibitors for Hypoxia-Inducible Factor Prolyl Hydroxylase 2.

A combination of structure-based drug design and medicinal chemistry efforts led us from benzimidazole-2-carboxamide with modestly active hypoxia-inducible factor prolyl hydroxylase 2 inhibition to certain benzimidazole-2-pyrazole carboxylic acids that were more potent as well as orally efficacious stimulators of erythropoietin secretion in our in vivo mouse model. To better understand the structure-activity relationship, it was necessary to account for (i) the complexation of the ligand with the active site Fe2+, (ii) the strain incurred by the ligand upon binding, and (iii) certain key water interactions identified by a crystal structure analysis. With this more complete computational model, we arrived at an overarching paradigm that accounted for the potency differences between benzimidazole-2-carboxamide and benzimidazole-2-pyrazole carboxylic acid enzyme inhibitors. Moreover, the computational paradigm allowed us to anticipate that the bioisostere replacement strategy (amide → pyrazole), which had shown success in the benzimidazole series, was not generally applicable to other series. This illustrates that to fully reconcile the important ligand-active site interactions for certain targets, one often needs to move beyond traditional structure-based drug design (such as crystallographic analysis, docking, etc.) and appeal to a higher level of computational theory.

Glutamatergic stimulation induces GluN2B translation by the nitric oxide-Heme-Regulated eIF2α kinase in cortical neurons.

The activation of N-Methyl D-Aspartate Receptor (NMDAR) by glutamate is crucial in the nervous system function, particularly in memory and learning. NMDAR is composed by two GluN1 and two GluN2 subunits. GluN2B has been reported to participate in the prevalent NMDAR subtype at synapses, the GluN1/2A/2B. Here we studied the regulation of GluN2B expression in cortical neurons finding that glutamate up-regulates GluN2B translation through the action of nitric oxide (NO), which induces the phosphorylation of the eukaryotic translation initiation factor 2 α (eIF2α). It is a process mediated by the NO-heme-regulated eIF2α kinase (HRI), as the effect was avoided when a specific HRI inhibitor or a HRI small interfering RNA (siHRI) were used. We found that the expressed GluN2B co-localizes with PSD-95 at the postsynaptic ending, which strengthen the physiological relevance of the proposed mechanism. Moreover the receptors bearing GluN2B subunits upon NO stimulation are functional as high Ca2+ entry was measured and increases the co-localization between GluN2B and GluN1 subunits. In addition, the injection of the specific HRI inhibitor in mice produces a decrease in memory retrieval as tested by the Novel Object Recognition performance. Summarizing our data suggests that glutamatergic stimulation induces HRI activation by NO to trigger GluN2B expression and this process would be relevant to maintain postsynaptic activity in cortical neurons.

Prolyl hydroxylase inhibition corrects functional iron deficiency and inflammation-induced anaemia in rats.

BACKGROUND AND PURPOSE: Small-molecule inhibitors of prolyl hydroxylase (PHD) enzymes are a novel target for the treatment of anaemia and functional iron deficiency (FID). Other than being orally bioavailable, the differentiation of PHD inhibitors from recombinant human erythropoietin (rhEPO) has not been demonstrated. EXPERIMENTAL APPROACH: JNJ-42905343 was identified and characterized as a novel inhibitor of PHD and its action was compared with rhEPO in healthy rats and in a rat model of inflammation-induced anaemia and FID [peptidoglycan-polysaccharide (PGPS) model]. KEY RESULTS: Oral administration of JNJ-42905343 to healthy rats increased the gene expression of cytochrome b (DcytB) and divalent metal-ion transporter 1 (DMT1) in the duodenum, and increased plasma EPO. Repeated administration of JNJ-42905343 for 28 days increased blood haemoglobin, mean corpuscular haemoglobin (MCH) and mean corpuscular volume (MCV). The serum iron concentration was increased with low doses (0.3 mg·kg(-1) ) but reduced at high doses (6 mg·kg(-1) ). In PGPS-treated rats, administration of JNJ-42905343 for 28 days corrected FID and anaemia, as reflected by increased blood haemoglobin, MCH and MCV. Increased expression of DcytB and DMT1 genes in the duodenum resulting in increased iron availability was defined as the mechanism for these effects. rhEPO did not affect DcytB and DMT1 and was not effective in PGPS-treated rats. CONCLUSIONS AND IMPLICATIONS: PHD inhibition has a beneficial effect on iron metabolism in addition to stimulating the release of EPO. Small-molecule inhibitors of PHD such as JNJ-42905343 represent a mechanism distinct from rhEPO to treat anaemia and FID.

Physiological Control of Nitric Oxide in Neuronal BACE1 Translation by Heme-Regulated eIF2α Kinase HRI Induces Synaptogenesis.

AIMS: Hippocampus is the brain center for memory formation, a process that requires synaptogenesis. However, hippocampus is dramatically compromised in Alzheimer's disease due to the accumulation of amyloid ß-peptide, whose production is initiated by ß-site APP Cleaving Enzyme 1 (BACE1). It is known that pathological stressors activate BACE1 translation through the phosphorylation of the eukaryotic initiation factor-2α (eIF2α) by GCN2, PERK, or PKR kinases, leading to amyloidogenesis. However, BACE1 physiological regulation is still unclear. Since nitric oxide (NO) participates directly in hippocampal glutamatergic signaling, we investigated the neuronal role of the heme-regulated eukaryotic initiation factor eIF2α kinase (HRI), which can bind NO by a heme group, in BACE1 translation and its physiological consequences. RESULTS: We found that BACE1 is expressed on glutamate activation with NO being the downstream effector by triggering eIF2α phosphorylation, as it was obtained by Western blot and luciferase assay. It is due to the activation of HRI by NO as assayed by Western blot and immunofluorescence with an HRI inhibitor and HRI siRNA. BACE1 expression was early detected at synaptic spines, contributing to spine growth and consolidating the hippocampal memory as assayed with mice treated with HRI or neuronal NO synthase inhibitors. INNOVATION: We provide the first description that HRI and eIF2α are working in physiological conditions in the brain under the control of nitric oxide and glutamate signaling, and also that BACE1 has a physiological role in hippocampal function. CONCLUSION: We conclude that BACE1 translation is controlled by NO through HRI in glutamatergic hippocampal synapses, where it plays physiological functions, allowing the spine growth and memory consolidation.

Inhibition of hypoxia-inducible factor prolyl hydroxylase domain oxygen sensors: tricking the body into mounting orchestrated survival and repair responses.

Hypoxia-inducible factor (HIF) is an oxygen-sensitive dimeric transcription factor that responds to pathophysiologically low O2 tensions via up-regulation, which leads to an orchestrated biological response to hypoxia. The HIF prolyl hydroxylase domain (PHD) enzymes are non-heme, iron-containing dioxygenases requiring for activity both molecular oxygen and 2-oxoglutarate that, under normoxia, selectively hydroxylate proline residues of HIF, initiating proteosomal degradation of the latter. The dependence of HIF protein levels on the concentration of O2 present, mediated by the PHD enzymes, forms the basis for one of the most significant biological sensor systems of tissue oxygenation in response to ischemic and inflammatory events. Consequently, pharmacological inhibition of PHD enzymes, leading to stabilization of HIF, may be of considerable therapeutic potential in treating conditions of tissue stress and injury. This Perspective reviews the PHDs and small molecule drug discovery efforts. A critical view of this challenging field is offered, which addresses potential concerns and highlights exciting possibilities for the future.

JNJ-26070109 [(R)4-bromo-N-[1-(2,4-difluoro-phenyl)-ethyl]-2-(quinoxaline-5-sulfonylamino)-benzamide]: a novel, potent, and selective cholecystokinin 2 receptor antagonist with good oral bioavailability.

JNJ-26070109 [(R)4-bromo-N-[1-(2,4-difluoro-phenyl)-ethyl]-2-(quinoxaline-5-sulfonylamino)-benzamide] is a representative of a new chemical class of competitive antagonists of cholecystokinin 2 (CCK2) receptors. In this study, the primary in vitro pharmacology of JNJ-26070109 was evaluated along with the pharmacokinetic and pharmacodynamic properties of this compound in rat and canine models of gastric acid secretion. JNJ-26070109 expressed high affinity for human (pK(I) = 8.49 ± 0.13), rat (pK(I) = 7.99 ± 0.08), and dog (pK(I) = 7.70 ± 0.14) CCK2 receptors. The selectivity of JNJ-26070109 at the CCK2 receptor versus the CCK1 receptor was species-dependent, with the greatest degree of selectivity (>1200-fold) measured at the human isoforms of the CCK1 receptor (selectivity at CCK2 versus CCK1 receptors: human, ∼1222-fold; rat, ∼324-fold; dog ∼336-fold). JNJ-26070109 behaved as a surmountable, competitive, antagonist of human CCK2 receptors in a calcium mobilization assay (pK(B) = 8.53 ± 0.05) and in pentagastrin-stimulated gastric acid secretion in the isolated, lumen-perfused, mouse stomach assay (pK(B) = 8.19 ± 0.13). The pharmacokinetic profile of this compound was determined in vivo in rats and dogs. JNJ-26070109 was shown to have high oral bioavailability (%F rat = 73 ± 16; %F dog = 92 ± 12) with half lives of 1.8 ± 0.3 and 1.2 ± 0.1 h in rat and dog, respectively. The pharmacodynamic properties of this compound were investigated using two in vivo models. In conscious rat and dog chronic gastric fistula models of pentagastrin-stimulated acid secretion, JNJ-26070109 had oral EC(50) values of 1.5 and 0.26 µM, respectively. Overall, we have demonstrated that JNJ-26070109 is a high-affinity, selective CCK2 receptor antagonist with good pharmacokinetic properties.

Pharmacological characterization of 1-(5-chloro-6-(trifluoromethoxy)-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylic acid (JNJ-42041935), a potent and selective hypoxia-inducible factor prolyl hydroxylase inhibitor.

The hypoxia-inducible factor (HIF) prolyl hydroxylase (PHD) enzymes represent novel targets for the treatment of anemia, ulcerative colitis, and ischemic and metabolic disease inter alia. We have identified a novel small-molecule inhibitor of PHD, 1-(5-chloro-6-(trifluoromethoxy)-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylic acid (JNJ-42041935), through structure-based drug design methods. The pharmacology of JNJ-42041935 was investigated in enzyme, cellular, and whole-animal systems and was compared with other compounds described in the literature as PHD inhibitors. JNJ-42041935, was a potent (pK(I) = 7.3-7.9), 2-oxoglutarate competitive, reversible, and selective inhibitor of PHD enzymes. In addition, JNJ-42041935 was used to compare the effect of selective inhibition of PHD to intermittent, high doses (50 µg/kg i.p.) of an exogenous erythropoietin receptor agonist in an inflammation-induced anemia model in rats. JNJ-42041935 (100 µmol/kg, once a day for 14 days) was effective in reversing inflammation-induced anemia, whereas erythropoietin had no effect. The results demonstrate that JNJ-42041935 is a new pharmacological tool, which can be used to investigate PHD inhibition and demonstrate that PHD inhibitors offer great promise for the treatment of inflammation-induced anemia.

A one-step synthesis of 2,4-unsubstituted quinoline-3-carboxylic acid esters from o-nitrobenzaldehydes.

A straightforward and efficient one-step procedure for the synthesis of 2,4-unsubstituted quinoline-3-carboxylic acid ethyl esters is described. The simple reductive cyclization is carried out by treating various substituted o-nitrobenzaldehydes with inexpensive, commercially available 3,3-diethoxypropionic acid ethyl ester and SnCl(2).2H(2)O in refluxing ethanol.

Benzimidazole-2-pyrazole HIF Prolyl 4-Hydroxylase Inhibitors as Oral Erythropoietin Secretagogues.

HIF prolyl 4-hydroxylases (PHD) are a family of enzymes that mediate key physiological responses to hypoxia by modulating the levels of hypoxia inducible factor 1-α (HIF1α). Certain benzimidazole-2-pyrazole carboxylates were discovered to be PHD2 inhibitors using ligand- and structure-based methods and found to be potent, orally efficacious stimulators of erythropoietin secretion in vivo.

Anthranilic sulfonamide CCK1/CCK2 dual receptor antagonists II: tuning of receptor selectivity and in vivo efficacy.

In the previous article we demonstrated how certain CCK2R-selective anthranilic amides could be structurally modified to afford high-affinity, selective CCK1R activity. We now describe our efforts at modulating and optimizing the CCK1R and CCK2R affinities aimed at producing compounds with good pharmacokinetics properties and in vivo efficacy in rat models of gastric acid and pancreatic amylase secretion.

Anthranilic sulfonamide CCK1/CCK2 dual receptor antagonists I: discovery of CCKR1 selectivity in a previously CCKR2-selective lead series.

A series of CCK2R-selective anthranilic amides is shown to derive CCK1R affinity via selective substitution of the amide side chain. Thus, extending the length of the original benzamide side chain by a single methylene unit imparts CCK1R affinity to the series, and further fine tuning of the affinity results in CCK1R selectivity of greater than 100-fold.

Discovery of the first known small-molecule inhibitors of heme-regulated eukaryotic initiation factor 2alpha (HRI) kinase.

A series of indeno[1,2-c]pyrazoles were discovered to be the first known inhibitors of heme-regulated eukaryotic initiation factor 2alpha (HRI) kinase. The synthesis, structure-activity relationship profile, and in-vitro pharmacological characterization of this inaugural series of HRI kinase inhibitors are detailed.

Characterization of a robust enzymatic assay for inhibitors of 2-oxoglutarate-dependent hydroxylases.

The prolyl-4-hydroxylase proteins regulate the hypoxia-inducible transcription factors (HIFs) by hydroxylation of proline residues targeting HIF-1alpha for proteasomal degradation. Using the purified catalytic domain of prolyl hydroxylase 2 (PHD2(181-417)), an enzymatic assay has been developed to test inhibitors of the enzyme in vitro. Because PHD2 hydroxylates HIF-1alpha, with succinic acid produced as an end product, radiolabeled [5-(14)C]-2-oxoglutaric acid was used and formation of [14C]-succinic acid was measured to quantify PHD2(181-417) enzymatic activity. Comparison of the separation of 2-oxoglutaric acid and succinic acid by either ion exchange chromatography or precipitation with phenylhydrazine showed similar results, but the quantification and throughput were vastly increased using the latter method. The PHD2 reaction was substrate and concentration dependent. The addition of iron to the enzyme reaction mix resulted in an increase in enzymatic activity. The Km value for 2-oxoglutaric acid was determined to be 0.9 microM, and known PHD2 inhibitors were used to validate the assay. In addition, the authors demonstrate that this assay can be applied to other 2-oxoglutaric acid-dependent enzymes, including the asparaginyl hydroxylase, factor-inhibiting HIF-1alpha (FIH). A concentration-dependent increase in succinic acid production using recombinant FIH enzyme with a synthetic peptide substrate was observed. The authors conclude that a by-product enzyme assay measuring the conversion of 2-oxoglutaric acid to succinic acid using the catalytic domain of the human PHD2 provides a convenient method for the biochemical evaluation of inhibitors of the 2-oxoglutaric acid-dependent hydroxylases.

Discovery of potent cholecystokinin-2 receptor antagonists: elucidation of key pharmacophore elements by X-ray crystallographic and NMR conformational analysis.

A novel series of cholecystokinin-2 receptor (CCK-2R) antagonists has been identified, as exemplified by anthranilic sulfonamide 1 (pK(i)=7.6). Pharmacokinetic and stability studies indicated that this series of compounds suffered from metabolic degradation, and that both the benzothiadiazole and piperidine rings were rapidly oxidized by liver enzymes. A combination of synthesis, computational methods, (1)H NMR conformational studies, and X-ray crystallographic analyses were applied to elucidate key pharmacophore elements, and to discover analogs with improved pharmacokinetic profiles, and high receptor binding affinity and selectivity.

Synthesis and solid-phase purification of anthranilic sulfonamides as CCK-2 ligands.

A novel strategy for the synthesis of cholecystokinin-2 receptor ligands was developed. The route employs a solution-phase synthesis of a series of anthranilic sulfonamides followed by a resin capture purification strategy to produce multi-milligram quantities of compounds for bioassay. The synthesis was used to produce >100 compounds containing various functional groups, highlighting the general applicability of this strategy and to address specific metabolism issues in our CCK-2 program.

Identification and optimization of anthranilic sulfonamides as novel, selective cholecystokinin-2 receptor antagonists.

A high throughput screening approach to the identification of selective cholecystokinin-2 receptor (CCK-2R) ligands resulted in the discovery of a novel series of antagonists, represented by 1-[2-[(2,1,3-benzothiadiazol-4-ylsulfonyl)amino]-5-chlorobenzoyl]-piperidine (1; CCK-2R, pK(I) = 6.4). Preliminary exploration of the structure-activity relationships around the anthranilic ring and the amide and sulfonamide moieties led to a nearly 50-fold improvement of receptor affinity and showed a greater than 1000-fold selectivity over the related cholecystokinin-1 receptor. Pharmacokinetic evaluation led to the identification of 4-[4-iodo-2-[(5-quinoxalinylsulfonyl)amino]benzoyl]-morpholine, 26d, a compound that demonstrates promising pharmacokinetic properties in the rat and dog with respect to plasma clearance and oral bioavailability and is a potent inhibitor in vivo of pentagastrin-stimulated acid secretion in the rat when dosed orally.

Substrate specificity and novel selective inhibitors of TNF-alpha converting enzyme (TACE) from two-dimensional substrate mapping.

We report a systematic analysis of the P1' and P2' substrate specificity of TNF-alpha converting enzyme (TACE) using a peptide library and a novel analytical method, and we use the substrate specificity information to design novel reverse hydroxamate inhibitors. Initial truncation studies, using the amino acid sequence around the cleavage site in precursor-TNF-alpha, showed that good turnover was obtained with the peptide DNP-LAQAVRSS-NH2. Based on this result, 1000 different peptide substrates of the form Biotin-LAQA-P1'-P2'-SSK(DNP)-NH2 were prepared, with 50 different natural and unnatural amino acids at P1' in combination with 20 different amino acids at P2'. The peptides were pooled, treated with purified microsomal TACE, and the reaction mixtures were passed over a streptavidin affinity column to remove unreacted substrate and the N-terminal biotinylated product. C-terminal cleavage products not binding to streptavidin were subjected to liquid chromatography/mass spectrometry analysis where individual products were identified and semiquantitated. 25 of the substrates were resynthesized as discrete peptides and assayed with recombinant TACE. The experiments show that recombinant TACE prefers lipophilic amino acids at the P1' position, such as phenylglycine, homophenylalanine, leucine and valine. At the P2' position, TACE can accommodate basic amino acids, such as arginine and lysine, as well as certain non-basic amino acids such as citrulline, methionine sulfoxide and threonine. These substrate preferences were used in the design of novel reverse hydroxamate TACE inhibitors with phenethyl and 5-methyl-thiophene-methyl side-chains at P1', and threonine and nitro-arginine at P2'.

Hantzsch synthesis of pyrazolo[1',2':1,2]pyrazolo[3,4-b]pyridines: partial agonists of the calcitonin receptor.

Small molecule calcitonin receptor agonists are of potential utility in the treatment and prevention of osteoporosis. Bicycloeneamine 1 was a useful intermediate in the synthesis of pyrazolopyridine calcitonin receptor partial agonists 2a-f. Dihydropyridines 10a-c were conveniently prepared by reaction of 1 with Knoevenagel adducts 9a-c, or in the case of 10d, by a three component reaction with 1, beta-ketoester 7b, and aldehyde 8c. Oxidation of 10a-d to pyridines 11a-d and subsequent amide formation afforded the title compounds.