Exploration of the P1 residue in 3CL protease inhibitors leading to the discovery of a 2-tetrahydrofuran P1 replacement.

The virally encoded 3C-like protease (3CLpro) is a well-validated drug target for the inhibition of coronaviruses including Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). Most inhibitors of 3CLpro are peptidomimetic, with a γ-lactam in place of Gln at the P1 position of the pseudopeptide chain. An effort was pursued to identify a viable alternative to the γ-lactam P1 mimetic which would improve physicochemical properties while retaining affinity for the target. Discovery of a 2-tetrahydrofuran as a suitable P1 replacement that is a potent enzymatic inhibitor of 3CLpro in SARS-CoV-2 virus is described herein.

NRF2 Activation Reprograms Defects in Oxidative Metabolism to Restore Macrophage Function in Chronic Obstructive Pulmonary Disease.

Rationale: Chronic obstructive pulmonary disease (COPD) is a disease characterized by persistent airway inflammation and disordered macrophage function. The extent to which alterations in macrophage bioenergetics contribute to impaired antioxidant responses and disease pathogenesis has yet to be fully delineated. Objectives: Through the study of COPD alveolar macrophages (AMs) and peripheral monocyte-derived macrophages (MDMs), we sought to establish if intrinsic defects in core metabolic processes drive macrophage dysfunction and redox imbalance. Methods: AMs and MDMs from donors with COPD and healthy donors underwent functional, metabolic, and transcriptional profiling. Measurements and Main Results: We observed that AMs and MDMs from donors with COPD display a critical depletion in glycolytic- and mitochondrial respiration-derived energy reserves and an overreliance on glycolysis as a source for ATP, resulting in reduced energy status. Defects in oxidative metabolism extend to an impaired redox balance associated with defective expression of the NADPH-generating enzyme, ME1 (malic enzyme 1), a known target of the antioxidant transcription factor NRF2 (nuclear factor erythroid 2-related factor 2). Consequently, selective activation of NRF2 resets the COPD transcriptome, resulting in increased generation of TCA cycle intermediaries, improved energetic status, favorable redox balance, and recovery of macrophage function. Conclusions: In COPD, an inherent loss of metabolic plasticity leads to metabolic exhaustion and reduced redox capacity, which can be rescued by activation of the NRF2 pathway. Targeting these defects, via NRF2 augmentation, may therefore present an attractive therapeutic strategy for the treatment of the aberrant airway inflammation described in COPD.

Fragment-Guided Discovery of Pyrazole Carboxylic Acid Inhibitors of the Kelch-like ECH-Associated Protein 1: Nuclear Factor Erythroid 2 Related Factor 2 (KEAP1:NRF2) Protein-Protein Interaction.

The NRF2-mediated cytoprotective response is central to cellular homoeostasis, and there is increasing interest in developing small-molecule activators of this pathway as therapeutics for diseases involving chronic oxidative stress. The protein KEAP1, which regulates NRF2, is a key point for pharmacological intervention, and we recently described the use of fragment-based drug discovery to develop a tool compound that directly disrupts the protein-protein interaction between NRF2 and KEAP1. We now present the identification of a second, chemically distinct series of KEAP1 inhibitors, which provided an alternative chemotype for lead optimization. Pharmacophoric information from our original fragment screen was used to identify new hit matter through database searching and to evolve this into a new lead with high target affinity and cell-based activity. We highlight how knowledge obtained from fragment-based approaches can be used to focus additional screening campaigns in order to de-risk projects through the rapid identification of novel chemical series.

Structure-Activity and Structure-Conformation Relationships of Aryl Propionic Acid Inhibitors of the Kelch-like ECH-Associated Protein 1/Nuclear Factor Erythroid 2-Related Factor 2 (KEAP1/NRF2) Protein-Protein Interaction.

The KEAP1-NRF2-mediated cytoprotective response plays a key role in cellular homoeostasis. Insufficient NRF2 signaling during chronic oxidative stress may be associated with the pathophysiology of several diseases with an inflammatory component, and pathway activation through direct modulation of the KEAP1-NRF2 protein-protein interaction is being increasingly explored as a potential therapeutic strategy. Nevertheless, the physicochemical nature of the KEAP1-NRF2 interface suggests that achieving high affinity for a cell-penetrant druglike inhibitor might be challenging. We recently reported the discovery of a highly potent tool compound which was used to probe the biology associated with directly disrupting the interaction of NRF2 with the KEAP1 Kelch domain. We now present a detailed account of the medicinal chemistry campaign leading to this molecule, which included exploration and optimization of protein-ligand interactions in three energetic "hot spots" identified by fragment screening. In particular, we also discuss how consideration of ligand conformational stabilization was important to its development and present evidence for preorganization of the lead compound which may contribute to its high affinity and cellular activity.

Discovery of a crystalline sulforaphane analog with good solid-state stability and engagement of the Nrf2 pathway in vitro and in vivo.

The antioxidant natural product sulforaphane (SFN) is an oil with poor aqueous and thermal stability. Recent work with SFN has sought to optimize methods of formulation for oral and topical administration. Herein we report the design of new analogs of SFN with the goal of improving stability and drug-like properties. Lead compounds were selected based on potency in a cellular screen and physicochemical properties. Among these, 12 had good aqueous solubility, permeability and long-term solid-state stability at 23 °C. Compound 12 also displayed comparable or better efficacy in cellular assays relative to SFN and had in vivo activity in a mouse cigarette smoke challenge model of acute oxidative stress.

Characterization of the Potent, Selective Nrf2 Activator, 3-(Pyridin-3-Ylsulfonyl)-5-(Trifluoromethyl)-2H-Chromen-2-One, in Cellular and In Vivo Models of Pulmonary Oxidative Stress.

Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a key regulator of oxidative stress and cellular repair and can be activated through inhibition of its cytoplasmic repressor, Kelch-like ECH-associated protein 1 (Keap1). Several small molecule disrupters of the Nrf2-Keap1 complex have recently been tested and/or approved for human therapeutic use but lack either potency or selectivity. The main goal of our work was to develop a potent, selective activator of NRF2 as protection against oxidative stress. In human bronchial epithelial cells, our Nrf2 activator, 3-(pyridin-3-ylsulfonyl)-5-(trifluoromethyl)-2H-chromen-2-one (PSTC), induced Nrf2 nuclear translocation, Nrf2-regulated gene expression, and downstream signaling events, including induction of NAD(P)H:quinone oxidoreductase 1 (NQO1) enzyme activity and heme oxygenase-1 protein expression, in an Nrf2-dependent manner. As a marker of subsequent functional activity, PSTC restored oxidant (tert-butyl hydroperoxide)-induced glutathione depletion. The compound's engagement of the Nrf2 signaling pathway translated to an in vivo setting, with induction of Nrf2-regulated gene expression and NQO1 enzyme activity, as well as restoration of oxidant (ozone)-induced glutathione depletion, occurring in the lungs of PSTC-treated rodents. Under disease conditions, PSTC engaged its target, inducing the expression of Nrf2-regulated genes in human bronchial epithelial cells derived from patients with chronic obstructive pulmonary disease, as well as in the lungs of cigarette smoke-exposed mice. Subsequent to the latter, a dose-dependent inhibition of cigarette smoke-induced pulmonary inflammation was observed. Finally, in contrast with bardoxolone methyl and sulforaphane, PSTC did not inhibit interleukin-1ß-induced nuclear factor-κB translocation or insulin-induced S6 phosphorylation in human cells, emphasizing the on-target activity of this compound. In summary, we characterize a potent, selective Nrf2 activator that offers protection against pulmonary oxidative stress in several cellular and in vivo models.

Discovering Drugs with DNA-Encoded Library Technology: From Concept to Clinic with an Inhibitor of Soluble Epoxide Hydrolase.

DNA-encoded chemical library technology was developed with the vision of its becoming a transformational platform for drug discovery. The hope was that a new paradigm for the discovery of low-molecular-weight drugs would be enabled by combining the vast molecular diversity achievable with combinatorial chemistry, the information-encoding attributes of DNA, the power of molecular biology, and a streamlined selection-based discovery process. Here, we describe the discovery and early clinical development of GSK2256294, an inhibitor of soluble epoxide hydrolase (sEH, EPHX2), by using encoded-library technology (ELT). GSK2256294 is an orally bioavailable, potent and selective inhibitor of sEH that has a long half life and produced no serious adverse events in a first-time-in-human clinical study. To our knowledge, GSK2256294 is the first molecule discovered from this technology to enter human clinical testing and represents a realization of the vision that DNA-encoded chemical library technology can efficiently yield molecules with favorable properties that can be readily progressed into high-quality drugs.

Monoacidic Inhibitors of the Kelch-like ECH-Associated Protein 1: Nuclear Factor Erythroid 2-Related Factor 2 (KEAP1:NRF2) Protein-Protein Interaction with High Cell Potency Identified by Fragment-Based Discovery.

KEAP1 is the key regulator of the NRF2-mediated cytoprotective response, and increasingly recognized as a target for diseases involving oxidative stress. Pharmacological intervention has focused on molecules that decrease NRF2-ubiquitination through covalent modification of KEAP1 cysteine residues, but such electrophilic compounds lack selectivity and may be associated with off-target toxicity. We report here the first use of a fragment-based approach to directly target the KEAP1 Kelch-NRF2 interaction. X-ray crystallographic screening identified three distinct "hot-spots" for fragment binding within the NRF2 binding pocket of KEAP1, allowing progression of a weak fragment hit to molecules with nanomolar affinity for KEAP1 while maintaining drug-like properties. This work resulted in a promising lead compound which exhibits tight and selective binding to KEAP1, and activates the NRF2 antioxidant response in cellular and in vivo models, thereby providing a high quality chemical probe to explore the therapeutic potential of disrupting the KEAP1-NRF2 interaction.

Structure of the BTB domain of Keap1 and its interaction with the triterpenoid antagonist CDDO.

The protein Keap1 is central to the regulation of the Nrf2-mediated cytoprotective response, and is increasingly recognized as an important target for therapeutic intervention in a range of diseases involving excessive oxidative stress and inflammation. The BTB domain of Keap1 plays key roles in sensing environmental electrophiles and in mediating interactions with the Cul3/Rbx1 E3 ubiquitin ligase system, and is believed to be the target for several small molecule covalent activators of the Nrf2 pathway. However, despite structural information being available for several BTB domains from related proteins, there have been no reported crystal structures of Keap1 BTB, and this has precluded a detailed understanding of its mechanism of action and interaction with antagonists. We report here the first structure of the BTB domain of Keap1, which is thought to contain the key cysteine residue responsible for interaction with electrophiles, as well as structures of the covalent complex with the antagonist CDDO/bardoxolone, and of the constitutively inactive C151W BTB mutant. In addition to providing the first structural confirmation of antagonist binding to Keap1 BTB, we also present biochemical evidence that adduction of Cys 151 by CDDO is capable of inhibiting the binding of Cul3 to Keap1, and discuss how this class of compound might exert Nrf2 activation through disruption of the BTB-Cul3 interface.

Analytical approaches for quantification of a Nrf2 pathway activator: overcoming bioanalytical challenges to support a toxicity study.

Activation of the Nrf2 stress pathway is known to play an important role in the defense mechanism against electrophilic and oxidative damage to biological macromolecules (DNA, lipids, and proteins). Chemical inducers of Nrf2 such as sulforaphane, dimethyl fumarate (Tecfidera®), CDDO-Me (bardoxolone-methyl), and 3-(dimethylamino)-4-((3-isothiocyanatopropyl)(methyl)amino)cyclobut-3-ene-1,2-dione (a synthetic sulforaphane analogue; will be referred to as ) have the ability to react with Keap1 cysteine residues, leading to activation of the Antioxidant Response Element (ARE). Due to their electrophilic nature and poor matrix stability, these compounds represent great challenges when developing bioanalytical methods to evaluate in vivo exposure. like SFN reacts rapidly with glutathione (GSH) and nucleophilic groups in proteins to form covalent adducts. In this work, three procedures were developed to estimate the exposure of in a non-GLP 7 day safety study in rats: (1) protein precipitation of blood samples with methanol containing the free thiol trapping reagent 4-fluoro-7-aminosulfonylbenzofurazan (ABD-F) to measure GSH- and N-acetylcysteine conjugated metabolites of ; (2) an Edman degradation procedure to cleave and analyze N-terminal adducts of at the valine moiety; and (3) treatment with ammonium hydroxide to measure circulating free- and all sulfhydryl bound .

Keap calm, and carry on covalently.

The Nrf2-Keap1 system plays a major role in cellular defense against oxidative stress. Upon exposure to electrophiles, the cysteine-rich protein Keap1 is covalently modified, and it is this modification of Keap1 that allows the accumulation and subsequent nuclear translocation of Nrf2 where it induces the transcription of over 100 protective genes. This mechanism can be exploited in drug discovery approaches to diseases such as chronic kidney disease (CKD), chronic obstructive pulmonary disease (COPD), asthma, and neurodegenerative diseases like multiple sclerosis (MS) and Parkinson's, utilizing the modification of Keap1 by electrophiles, compounds that would not normally be considered useful in drug discovery programs. This Perspective discusses the development of potential therapies based on potent electrophiles, such as isothiocyanates and Michael acceptors, that, far from being associated with toxic events, can actually initiate a range of beneficial protective pathways.

In vitro and in vivo characterization of a novel soluble epoxide hydrolase inhibitor.

Soluble epoxide hydrolase (sEH, EPHX2) metabolizes eicosanoid epoxides, including epoxyeicosatrienoic acids (EETs) to the corresponding dihydroxyeicosatrienoic acids (DHETs), and leukotoxin (LTX) to leukotoxin diol (LTX diol). EETs, endothelium-derived hyperpolarizing factors, exhibit potentially beneficial properties, including anti-inflammatory effects and vasodilation. A novel, potent, selective inhibitor of recombinant human, rat and mouse sEH, GSK2256294A, exhibited potent cell-based activity, a concentration-dependent inhibition of the conversion of 14,15-EET to 14,15-DHET in human, rat and mouse whole blood in vitro, and a dose-dependent increase in the LTX/LTX diol ratio in rat plasma following oral administration. Mice receiving 10 days of cigarette smoke exposure concomitant with oral administration of GSK2256294A exhibited significant, dose-dependent reductions in pulmonary leukocytes and keratinocyte chemoattractant (KC, CXCL1) levels. Mice receiving oral administration of GSK2256294A following 10 days of cigarette smoke exposure exhibited significant reductions in pulmonary leukocytes compared to vehicle-treated mice. These data indicate that GSK2256294A attenuates cigarette smoke-induced inflammation by both inhibiting its initiation and/or maintenance and promoting its resolution. Collectively, these data indicate that GSK2256294A would be an appropriate agent to evaluate the role of sEH in clinical studies, for example in diseases where cigarette smoke is a risk factor, such as chronic obstructive pulmonary disease (COPD) and cardiovascular disease.

Development of a semi-automated LC/MS/MS method for the simultaneous quantitation of 14,15-epoxyeicosatrienoic acid, 14,15-dihydroxyeicosatrienoic acid, leukotoxin and leukotoxin diol in human plasma as biomarkers of soluble epoxide hydrolase activity in vivo.

Substrates and products of soluble epoxide hydrolase (sEH) such as 14,15-epoxyeicosatrienoic acid (14,15-EET), 14,15-dihydroxyeicosatrienoic acid (14,15-DHET), leukotoxin, and leukotoxin diol are potential biomarkers for assessing sEH activity in clinical trial subjects. To quantify them, we have developed and validated a semi-automated and relatively high-throughput assay in a 96-well plate format using liquid chromatography-mass spectrometry. 14,15-EET, 14,15-DHET, leukotoxin and leukotoxin diol, as well as their deuterium labeled internal standards were extracted from human plasma by liquid-liquid extraction using ethyl acetate. The four analytes were separated from other endogenous lipid isomers using liquid chromatography coupled with tandem mass spectrometry. The method was validated over a concentration range of 0.05-50 ng/mL. The validation results show that the method is precise, accurate and well-suited for analysis of clinical samples. The turn-around rate of the assay is approximately 200 samples per day.

Specialized training on addictions for physicians in the United States.

In the United States accredited residency programs in addiction exist only for psychiatrists specializing in addiction psychiatry (ADP); nonpsychiatrists seeking training in addiction medicine (ADM) can train in nonaccredited "fellowships," or can receive training in some ADP programs, only to not be granted a certificate of completion of accredited training. Information about ADP residency programs has been tabulated, but it is not available for ADM fellowships. The authors conducted a national survey to compile information about the location, structure, curriculum, and other characteristics of active ADM fellowships. Of the 40 accredited ADP residency programs, 7 offered training in addiction to nonpsychiatrists. The authors identified 14 nonaccredited ADM fellowships. In 2009 and 2010, there were approximately 15 nonpsychiatrists in ADP programs and 25 in ADM fellowships. Clinical experiences included inpatient services, outpatient treatment services such as methadone maintenance or buprenorphine maintenance, and providing addiction consult services. The most common academic activities included weekly lectures and the teaching of medical students.

An orally active calcium-sensing receptor antagonist that transiently increases plasma concentrations of PTH and stimulates bone formation.

Daily subcutaneous administration of exogenous parathyroid hormone (PTH) promotes bone formation in patients with osteoporosis. Here we describe two novel, short-acting calcium-sensing receptor antagonists (SB-423562 and its orally bioavailable precursor, SB-423557) that elicit transient PTH release from the parathyroid gland in several preclinical species and in humans. In an ovariectomized rat model of bone loss, daily oral administration of SB-423557 promoted bone formation and improved parameters of bone strength at lumbar spine, proximal tibia and midshaft femur. Chronic administration of SB-423557 did not increase parathyroid cell proliferation in rats. In healthy human volunteers, single doses of intravenous SB-423562 and oral SB-423557 elicited transient elevations of endogenous PTH concentrations in a profile similar to that observed with subcutaneously administered PTH. Both agents were well tolerated in humans. Transient increases in serum calcium, an expected effect of increased parathyroid hormone concentrations, were observed post-dose at the higher doses of SB-423557 studied. These data constitute an early proof of principle in humans and provide the basis for further development of this class of compound as a novel, orally administered bone-forming treatment for osteoporosis.

Antagonists of the calcium receptor. 2. Amino alcohol-based parathyroid hormone secretagogues.

When administered as a single agent to rats, the previously reported calcium receptor antagonist 3 elicited a sustained elevation of plasma PTH resulting in no increase in overall bone mineral density. The lack of a bone building effect for analogue 3 was attributed to the large volume of distribution (V(dss)(rat) = 11 L/kg), producing a protracted plasma PTH profile. Incorporation of a carboxylic acid functionality into the amino alcohol template led to the identification of 12 with a lower volume of distribution (V(dss)(12) = 1.18 L/kg) and a shorter half-life. The zwitterionic nature of antagonist 12 necessitated the utility of an ester prodrug approach to increase overall permeability. Antagonist 12 elicited a rapid and transient increase in circulating levels of PTH following oral dosing of the ester prodrug 11 in the dog. The magnitude and duration of the increases in plasma levels of PTH would be expected to stimulate new bone formation.

Progress towards the development of anti-inflammatory inhibitors of IKKbeta.

The IkappaB kinases (IKKs) are essential components of the signaling pathway by which the NF-kappaB p50/RelA transcription factor is activated in response to pro-inflammatory stimuli such as lipopolysaccharide (LPS) and tumor necrosis factor (TNFalpha). NF-kappaB signaling results in the expression of numerous genes involved in innate and adaptive immune responses. The pathway is also implicated in chronic inflammatory disorders including rheumatoid arthritis (RA), chronic obstructive pulmonary disorder (COPD), and asthma. Inhibition of the kinase activity of the IKKs is therefore a promising mechanism for intervention in these diseases. Here, we will review the literature describing small molecule inhibitors of IKKbeta (IKK2), the most widely studied of the IKKs.

Antagonists of the calcium receptor I. Amino alcohol-based parathyroid hormone secretagogues.

Functional screening of the former SmithKline Beecham compound collection against the human calcium receptor (CaR) resulted in the identification of the amino alcohol-based hit 2 (IC(50) = 11 microM). Structure-activity studies of 2 focused on the optimization of the right- and left-hand side aromatic moieties as well as the amino alcohol linker region. Critical to the optimization of this antagonist template was the discovery that the chirality of the C-2 secondary alcohol played a key role in enhancing both CaR potency as well as selectivity over the beta-adrenergic receptor subtypes. These SAR studies ultimately led to the identification of 38 (NPS 2143; SB-262470A), a potent and orally active CaR antagonist. Pharmacokinetic characterization of 38 in the rat revealed that this molecule had a large volume of distribution (11 L/kg), which resulted in a prolonged systemic exposure, protracted increases in the plasma levels of PTH, and an overall lack of net bone formation effect in a rodent model of osteoporosis.

Attenuation of murine collagen-induced arthritis by a novel, potent, selective small molecule inhibitor of IkappaB Kinase 2, TPCA-1 (2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide), occurs via reduction of proinflammatory cytokines and antigen-induced T cell Proliferation.

Demonstration that IkappaB kinase 2 (IKK-2) plays a pivotal role in the nuclear factor-kappaB-regulated production of proinflammatory molecules by stimuli such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 suggests that inhibition of IKK-2 may be beneficial in the treatment of rheumatoid arthritis. In the present study, we demonstrate that a novel, potent (IC(50) = 17.9 nM), and selective inhibitor of human IKK-2, 2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide (TPCA-1), inhibits lipopolysaccharide-induced human monocyte production of TNF-alpha, IL-6, and IL-8 with an IC(50) = 170 to 320 nM. Prophylactic administration of TPCA-1 at 3, 10, or 20 mg/kg, i.p., b.i.d., resulted in a dose-dependent reduction in the severity of murine collagen-induced arthritis (CIA). The significantly reduced disease severity and delay of disease onset resulting from administration of TPCA-1 at 10 mg/kg, i.p., b.i.d. were comparable to the effects of the antirheumatic drug, etanercept, when administered prophylactically at 4 mg/kg, i.p., every other day. Nuclear localization of p65, as well as levels of IL-1beta, IL-6, TNF-alpha, and interferon-gamma, were significantly reduced in the paw tissue of TPCA-1- and etanercept-treated mice. In addition, administration of TPCA-1 in vivo resulted in significantly decreased collagen-induced T cell proliferation ex vivo. Therapeutic administration of TPCA-1 at 20 mg/kg, but not at 3 or 10 mg/kg, i.p., b.i.d., significantly reduced the severity of CIA, as did etanercept administration at 12.5 mg/kg, i.p., every other day. These results suggest that reduction of proinflammatory mediators and inhibition of antigen-induced T cell proliferation are mechanisms underlying the attenuation of CIA by the IKK-2 inhibitor, TPCA-1.

SB-431542 is a potent and specific inhibitor of transforming growth factor-beta superfamily type I activin receptor-like kinase (ALK) receptors ALK4, ALK5, and ALK7.

Small molecule inhibitors have proven extremely useful for investigating signal transduction pathways and have the potential for development into therapeutics for inhibiting signal transduction pathways whose activities contribute to human diseases. Transforming growth factor beta (TGF-beta) is a member of a large family of pleiotropic cytokines that are involved in many biological processes, including growth control, differentiation, migration, cell survival, adhesion, and specification of developmental fate, in both normal and diseased states. TGF-beta superfamily members signal through a receptor complex comprising a type II and type I receptor, both serine/threonine kinases. Here, we characterize a small molecule inhibitor (SB-431542) that was identified as an inhibitor of activin receptor-like kinase (ALK)5 (the TGF-beta type I receptor). We demonstrate that it inhibits ALK5 and also the activin type I receptor ALK4 and the nodal type I receptor ALK7, which are very highly related to ALK5 in their kinase domains. It has no effect on the other, more divergent ALK family members that recognize bone morphogenetic proteins (BMPs). Consistent with this, we demonstrate that SB-431542 is a selective inhibitor of endogenous activin and TGF-beta signaling but has no effect on BMP signaling. To demonstrate the specificity of SB-431542, we tested its effect on several other signal transduction pathways whose activities depend on the concerted activation of multiple kinases. SB-431542 has no effect on components of the ERK, JNK, or p38 MAP kinase pathways or on components of the signaling pathways activated in response to serum.