Discovery of ITX 4520: a highly potent orally bioavailable hepatitis C virus entry inhibitor.

The manuscript reports an identification of a highly potent, orally bioavailable hepatitis C virus entry inhibitor through optimization of a previously reported class of molecules (1) that were not stable in the rat plasma. Compound 39 (ITX 4520) exhibited an excellent PK profile in both rats and dogs with good oral exposure, half-life and oral bioavailability. The compound is also well-tolerated in the preliminary in vivo toxicity studies and has been selected as a pre-clinical candidate for our HCV clinical pipeline.

Treatment and prevention of pathological mitochondrial dysfunction in retinal degeneration and in photoreceptor injury.

Pathological deterioration of mitochondrial function is increasingly linked with multiple degenerative illnesses as a mediator of a wide range of neurologic and age-related chronic diseases, including those of genetic origin. Several of these diseases are rare, typically defined in the United States as an illness affecting fewer than 200,000 people in the U.S. population, or about one in 1600 individuals. Vision impairment due to mitochondrial dysfunction in the eye is a prominent feature evident in numerous primary mitochondrial diseases and is common to the pathophysiology of many of the familiar ophthalmic disorders, including age-related macular degeneration, diabetic retinopathy, glaucoma and retinopathy of prematurity - a collection of syndromes, diseases and disorders with significant unmet medical needs. Focusing on metabolic mitochondrial pathway mechanisms, including the possible roles of cuproptosis and ferroptosis in retinal mitochondrial dysfunction, we shed light on the potential of α-lipoyl-L-carnitine in treating eye diseases. α-Lipoyl-L-carnitine is a bioavailable mitochondria-targeting lipoic acid prodrug that has shown potential in protecting against retinal degeneration and photoreceptor cell loss in ophthalmic indications.

Difluoroethylamines as an amide isostere in inhibitors of cathepsin K.

The trifluoroethylamine group found in cathepsin K inhibitors like odanacatib can be replaced by a difluoroethylamine group. This change increased the basicity of the nitrogen which positively impacted the log D. This translated into an improved oral bioavailability in pre-clinical species. Difluoroethylamine compounds exhibit a similar potency against cathepsin K and selectivity profile against other cathepsins when compared to trifluoroethylamine analogs.

Pathogenic mitochondrial dysfunction and metabolic abnormalities.

Herein we trace links between biochemical pathways, pathogenesis, and metabolic diseases to set the stage for new therapeutic advances. Cellular and acellular microorganisms including bacteria and viruses are primary pathogenic drivers that cause disease. Missing from this statement are subcellular compartments, importantly mitochondria, which can be pathogenic by themselves, also serving as key metabolic disease intermediaries. The breakdown of food molecules provides chemical energy to power cellular processes, with mitochondria as powerhouses and ATP as the principal energy carrying molecule. Most animal cell ATP is produced by mitochondrial synthase; its central role in metabolism has been known for >80 years. Metabolic disorders involving many organ systems are prevalent in all age groups. Progressive pathogenic mitochondrial dysfunction is a hallmark of genetic mitochondrial diseases, the most common phenotypic expression of inherited metabolic disorders. Confluent genetic, metabolic, and mitochondrial axes surface in diabetes, heart failure, neurodegenerative disease, and even in the ongoing coronavirus pandemic.

Differential efficacy of caspase inhibitors on apoptosis markers during sepsis in rats and implication for fractional inhibition requirements for therapeutics.

A rodent model of sepsis was used to establish the relationship between caspase inhibition and inhibition of apoptotic cell death in vivo. In this model, thymocyte cell death was blocked by Bcl-2 transgene, indicating that apoptosis was predominantly dependent on the mitochondrial pathway that culminates in caspase-3 activation. Caspase inhibitors, including the selective caspase-3 inhibitor M867, were able to block apoptotic manifestations both in vitro and in vivo but with strikingly different efficacy for different cell death markers. Inhibition of DNA fragmentation required substantially higher levels of caspase-3 attenuation than that required for blockade of other apoptotic events such as spectrin proteolysis and phosphatidylserine externalization. These data indicate a direct relationship between caspase inhibition and some apoptotic manifestations but that small quantities of uninhibited caspase-3 suffice to initiate genomic DNA breakdown, presumably through the escape of catalytic quantities of caspase-activated DNase. These findings suggest that putative caspase-independent apoptosis may be overestimated in some systems since blockade of spectrin proteolysis and other cell death markers does not accurately reflect the high degrees of caspase-3 inhibition needed to prevent DNA fragmentation. Furthermore, this requirement presents substantial therapeutic challenges owing to the need for persistent and complete caspase blockade.

Potent and highly selective DP1 antagonists with 2,3,4,9-tetrahydro-1H-carbazole as pharmacophore.

We discovered that the introduction of a methyl group to the benzylic position of the N-benzyl group in lead compound 1a has a dramatic effect on improving the binding selectivity of this ligand for the prostanoid receptors DP1 (receptor for prostaglandin D(2)) as compared to TP (receptor for thromboxane A(2)). Based on this discovery, we have synthesized a series of potent and highly selective DP1 antagonists. Among them, compound 1h was identified as a highly selective DP1 antagonist with excellent overall properties. It has a K(i) of 0.43 nM to DP1 in binding assay and an IC(50) of 2.5 nM in the DP1 functional assay. Its selectivity for DP1 over TP (the most potent receptor after DP1) exceeds 750-fold based on both binding and functional assays. These properties make 1h a very potent and highly selective DP1 receptor antagonist suitable for investigating the biological functions of DP1 in normal physiology and models of disease.

The discovery of MK-0674, an orally bioavailable cathepsin K inhibitor.

MK-0674 is a potent and selective cathepsin K inhibitor from the same structural class as odanacatib with a comparable inhibitory potency profile against Cat K. It is orally bioavailable and exhibits long half-life in pre-clinical species. In vivo studies using deuterated MK-0674 show stereoselective epimerization of the alcohol stereocenter via an oxidation/reduction cycle. From in vitro incubations, two metabolites could be identified: the hydroxyleucine and the glucuronide conjugate which were confirmed using authentic synthetic standards.

Klotho Pathways, Myelination Disorders, Neurodegenerative Diseases, and Epigenetic Drugs.

In this review we outline a rationale for identifying neuroprotectants aimed at inducing endogenous Klotho activity and expression, which is epigenetic action, by definition. Such an approach should promote remyelination and/or stimulate myelin repair by acting on mitochondrial function, thereby heralding a life-saving path forward for patients suffering from neuroinflammatory diseases. Disorders of myelin in the nervous system damage the transmission of signals, resulting in loss of vision, motion, sensation, and other functions depending on the affected nerves, currently with no effective treatment. Klotho genes and their single-pass transmembrane Klotho proteins are powerful governors of the threads of life and death, true to the origin of their name, Fates, in Greek mythology. Among its many important functions, Klotho is an obligatory co-receptor that binds, activates, and/or potentiates critical fibroblast growth factor activity. Since the discovery of Klotho a little over two decades ago, it has become ever more apparent that when Klotho pathways go awry, oxidative stress and mitochondrial dysfunction take over, and age-related chronic disorders are likely to follow. The physiological consequences can be wide ranging, potentially wreaking havoc on the brain, eye, kidney, muscle, and more. Central nervous system disorders, neurodegenerative in nature, and especially those affecting the myelin sheath, represent worthy targets for advancing therapies that act upon Klotho pathways. Current drugs for these diseases, even therapeutics that are disease modifying rather than treating only the symptoms, leave much room for improvement. It is thus no wonder that this topic has caught the attention of biomedical researchers around the world.

Design and synthesis of dipeptidyl nitriles as potent, selective, and reversible inhibitors of cathepsin C.

A series of dipeptide nitriles with a thienyl alanine in P2 were identified as potent and selective cathepsin C inhibitors. Incorporation of a substituted cyclopropyl moiety in P1 effectively protects these derivatives against hydrolase activity in whole blood.

Photo-crosslinking of proteins in intact cells reveals a dimeric structure of cyclooxygenase-2 and an inhibitor-sensitive oligomeric structure of microsomal prostaglandin E2 synthase-1.

We have characterized the structures of cyclooxygenase-2 (COX-2) and microsomal prostaglandin E(2) synthase-1 (mPGES-1) in intact cells using bifunctional and photo-activatable crosslinking agents. A dimeric complex was detected for COX-2 by both crosslinking approaches, consistent with the crystal structure of the enzyme. For mPGES-1, treatment of A549 cells with disuccinimidyl suberate yielded immunoreactive protein bands corresponding to a dimer (33 kDa) and a trimer (45 kDa), as observed for the isolated enzyme. Photo-crosslinking with photoactivatable methionine in intact cells generated complexes with molecular weights corresponding to the dimer (33 kDa) and two putative trimer forms (50 and 55 kDa). Treatment with the selective mPGES-1 inhibitor MF63 prevented the formation of the 50 and 55 kDa crosslinked complexes, while an inactive structural analogue had no effect. Our data indicate that COX-2 forms a dimer in intact cells and that mPGES-1 has an oligomeric structure that can be disrupted by a selective inhibitor.

Identification of prostaglandin D2 receptor antagonists based on a tetrahydropyridoindole scaffold.

A new series of indole-based antagonists of the PGD(2) receptor subtype 1 (DP1 receptor) was identified and the progress of the structure-activity relationship study to the identification of potent and selective antagonists is presented. Selective DP1 antagonists with high potency and selectivity were prepared. Of particular interest is the DP1 antagonist 26 with a K(i) value of 1 nM for the DP1 receptor and an IC(50) value of 4.6 nM in a DP1 functional assay for the inhibition of the PGD(2) induced cAMP production in platelet rich plasma (PRP).

The discovery of odanacatib (MK-0822), a selective inhibitor of cathepsin K.

Odanacatib is a potent, selective, and neutral cathepsin K inhibitor which was developed to address the metabolic liabilities of the Cat K inhibitor L-873724. Substituting P1 and modifying the P2 side chain led to a metabolically robust inhibitor with a long half-life in preclinical species. Odanacatib was more selective in whole cell assays than the published Cat K inhibitors balicatib and relacatib. Evaluation in dermal fibroblast culture showed minimal intracellular collagen accumulation relative to less selective Cat K inhibitors.

Enthalpy-Driven Stabilization of Transthyretin by AG10 Mimics a Naturally Occurring Genetic Variant That Protects from Transthyretin Amyloidosis.

Transthyretin (TTR) amyloid cardiomyopathy (ATTR-CM) is a fatal disease with no available disease-modifying therapies. While pathogenic TTR mutations (TTRm) destabilize TTR tetramers, the T119M variant stabilizes TTRm and prevents disease. A comparison of potency for leading TTR stabilizers in clinic and structural features important for effective TTR stabilization is lacking. Here, we found that molecular interactions reflected in better binding enthalpy may be critical for development of TTR stabilizers with improved potency and selectivity. Our studies provide mechanistic insights into the unique binding mode of the TTR stabilizer, AG10, which could be attributed to mimicking the stabilizing T119M variant. Because of the lack of animal models for ATTR-CM, we developed an in vivo system in dogs which proved appropriate for assessing the pharmacokinetics-pharmacodynamics profile of TTR stabilizers. In addition to stabilizing TTR, we hypothesize that optimizing the binding enthalpy could have implications for designing therapeutic agents for other amyloid diseases.

Discovery of a potent and selective prostaglandin D2 receptor antagonist, [(3R)-4-(4-chloro-benzyl)-7-fluoro-5-(methylsulfonyl)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl]-acetic acid (MK-0524).

The discovery of the potent and selective prostaglandin D2 (PGD2) receptor (DP) antagonist [(3R)-4-(4-chlorobenzyl)-7-fluoro-5-(methylsulfonyl)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl]-acetic acid (13) is presented. Initial lead antagonists 6 and 7 were found to be potent and selective DP antagonists (DP Ki = 2.0 nM for each); however, they both suffered from poor pharmacokinetic profiles, short half-lives and high clearance rates in rats. Rat bile duct cannulation studies revealed that high concentrations of parent drug were present in the biliary fluid (Cmax = 1100 microM for 6 and 3900 microM for 7). This pharmacokinetic liability was circumvented by replacing the 7-methylsulfone substituent present in 6 and 7 with a fluorine atom resulting in antagonists with diminished propensity for biliary excretion and with superior pharmacokinetic profiles. Further optimization led to the discovery of the potent and selective DP antagonist 13.

Suppression of CYP1 members of the AHR response by pathogen-associated molecular patterns.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that triggers a broad response, which includes the regulation of proinflammatory cytokine production by monocytes and macrophages. AHR is negatively regulated by a set of genes that it transcriptionally activates, including the AHR repressor (Ahrr) and the cytochrome P450 1 (Cyp1) family, which are critical for preventing exacerbated AHR activity. An imbalance in these regulatory mechanisms has been shown to cause severe defects in lymphoid cells. Therefore, we wanted to assess how AHR activation is regulated in monocytes and macrophages in the context of innate immune responses induced by pathogen-associated molecular patterns (PAMPs). We found that concomitant stimulation of primary human monocytes with PAMPs and the AHR agonist 6-formylindolo(3,2-b)carbazole (FICZ) led to a selective dose-dependent inhibition of Cyp1 family members induction. Two other AHR-dependent genes [Ahrr and NADPH quinone dehydrogenase 1 (Nqo1)] were not affected under these conditions, suggesting a split in the AHR regulation by PAMPs. This down-regulation of Cyp1 family members did not require de novo protein production nor signaling through p38, ERK, or PI3K-Akt-mammalian target of rapamycin (mTOR) pathways. Furthermore, such a split regulation of the AHR response was more apparent in GM-CSF-derived macrophages, a finding corroborated at the functional level by decreased CYP1 activity and decreased proinflammatory cytokine production in response to FICZ and LPS. Collectively, our findings identify a role for pattern recognition receptor (PRR) signaling in regulating the AHR response through selective down-regulation of Cyp1 expression in human monocytes and macrophages.

Reducing the peptidyl features of caspase-3 inhibitors: a structural analysis.

Caspases are cysteine proteases that specifically cleave Asp-Xxx bonds. They are key agents in inflammation and apoptosis and are attractive targets for therapy against inflammation, neurodegeneration, ischemia, and cancer. Many caspase structures are known, but most involve either peptide or protein inhibitors, unattractive candidates for drug development. We present seven crystal structures of inhibited caspase-3 that illustrate several approaches to reducing the peptidyl characteristics of the inhibitors while maintaining their potency and selectivity. The inhibitors reduce the peptidyl nature of inhibitors while preserving binding potency by (1). exploiting a hydrophobic binding site C-terminal to the cleavage site, (2). replacing the negatively charged aspartyl residue at P4 with neutral groups, and (3). using a peptidomimetic 5,6,7-tricyclic system or a pyrazinone at P2-P3. In addition, we have found that two nicotinic acid aldehydes induce a significant conformational change in the S2 and S3 subsites of caspase-3, revealing an unexpected binding mode. These results advance the search for caspase-directed drugs by revealing how unacceptable molecular features can be removed without loss of potency.

Neuroprotective effects of M826, a reversible caspase-3 inhibitor, in the rat malonate model of Huntington's disease.

1. Caspases, key enzymes in the apoptosis pathway, have been detected in the brain of HD patients and in animal models of the disease. In the present study, we investigated the neuroprotective properties of a new, reversible, caspase-3-specific inhibitor, M826 (3-([(2S)-2-[5-tert-butyl-3-[[(4-methyl-1,2,5-oxadiazol-3-yl)methyl]amino]-2-oxopyrazin-1(2H)-yl]butanoyl]amino)-5-[hexyl(methyl)amino]-4-oxopentanoic acid), in a rat malonate model of HD. 2. Pharmacokinetic and autoradiography studies after intrastriatal (i.str.) injection of 1.5 nmol of M826 or its tritiated analogue [(3)H]M826 indicated that the compound diffused within the entire striatum. The elimination half-life (T(1/2)) of M826 in the rat striatum was 3 h. 3. I.str. injection of 1.5 nmol of M826 10 min after malonate infusion induced a significant reduction (66%) in the number of neurones expressing active caspase-3 in the ipsilateral striatum. 4. Inhibition of active caspase-3 translated into a significant but moderate reduction (39%) of the lesion volume, and of cell death (24%), 24 h after injury. The efficacy of M826 at inhibiting cell death was comparable to that of the noncompetitive NMDA receptor antagonist MK801. 5. These data provide in vivo proof-of-concept of the neuroprotective effects of reversible caspase-3 inhibitors in a model of malonate-induced striatal injury in the adult rat.

Metabolic activation of indole-containing prostaglandin D2 receptor 1 antagonists: impacts of glutathione trapping and glucuronide conjugation on covalent binding.

Some DP1 receptor antagonists from an indole-containing series were shown to cause in vitro covalent binding to protein in rat and human liver microsomes. Glutathione trapping experiments along with in vitro labeling assays confirmed that the presence of a strong electron withdrawing group was necessary to abrogate in vitro covalent binding, leading to the discovery of MK-0524. Hepatocyte incubations and in vivo studies showed that acyl-glucuronide formation did not translate into covalent binding.

Solid-phase analogue synthesis of caspase-3 inhibitors via palladium-catalyzed amination of 3-bromopyrazinones.

Caspase-3 is a cysteinyl protease that mediates apoptotic cell death. Its inhibition may have an important impact on the treatment of several degenerative diseases. Here we report the synthesis of reversible inhibitors via a solid-support palladium-catalyzed amination of 3-bromopyrazinones and the discovery of a pan-caspase reversible inhibitor.

In vitro biotransformations of the prostaglandin D2 (DP) antagonist MK-0524 and synthesis of metabolites.

Metabolites of the potent DP antagonist, MK-0524, were generated using in vitro systems including hepatic microsomes and hepatocytes. Four metabolites (two hydroxylated diastereomers, a ketone and an acyl glucuronide) were characterized by LC-MS/MS and 1H NMR. Larger quantities of these metabolites were prepared by either organic synthesis or biosynthetically to be used as standards in other studies. The propensity for covalent binding was assessed and was found to be acceptable (<50 pmol-equiv/mg protein).