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Mucosal-associated invariant T (MAIT) cells are innate-like lymphocytes that recognize microbial vitamin B metabolites and have emerging roles in infectious disease, autoimmunity, and cancer. Although MAIT cells are identified by a semi-invariant TCR, their phenotypic and functional heterogeneity is not well understood. Here we present an integrated single cell transcriptomic analysis of over 76,000 human MAIT cells during early and prolonged Ag-specific activation with the MR1 ligand 5-OP-RU and nonspecific TCR stimulation. We show that MAIT cells span a broad range of homeostatic, effector, helper, tissue-infiltrating, regulatory, and exhausted phenotypes, with distinct gene expression programs associated with CD4+ or CD8+ coexpression. During early activation, MAIT cells rapidly adopt a cytotoxic phenotype characterized by high expression of GZMB, IFNG and TNF In contrast, prolonged stimulation induces heterogeneous states defined by proliferation, cytotoxicity, immune modulation, and exhaustion. We further demonstrate a FOXP3 expressing MAIT cell subset that phenotypically resembles conventional regulatory T cells. Moreover, scRNAseq-defined MAIT cell subpopulations were also detected in individuals recently exposed to Mycobacterium tuberculosis, confirming their presence during human infection. To our knowledge, our study provides the first comprehensive atlas of human MAIT cells in activation conditions and defines substantial functional heterogeneity, suggesting complex roles in health and disease.

A newly validated target for tuberculosis treatment is phosphopantetheinyl transferase, an essential enzyme that plays a critical role in the biosynthesis of cellular lipids and virulence factors in Mycobacterium tuberculosis. The structure-activity relationships of a recently disclosed inhibitor, amidinourea (AU) 8918 (1), were explored, focusing on the biochemical potency, determination of whole-cell on-target activity for active compounds, and profiling of selective active congeners. These studies show that the AU moiety in AU 8918 is largely optimized and that potency enhancements are obtained in analogues containing a para-substituted aromatic ring. Preliminary data reveal that while some analogues, including 1, have demonstrated cardiotoxicity (e.g., changes in cardiomyocyte beat rate, amplitude, and peak width) and inhibit Cav1.2 and Nav1.5 ion channels (although not hERG channels), inhibition of the ion channels is largely diminished for some of the para-substituted analogues, such as 5k (p-benzamide) and 5n (p-phenylsulfonamide).

Select triazole-based small molecules possess potent and selective kappa opioid receptor (KOR) agonism. Here, we designed twenty new analogs to investigate the structure–activity relationship effects for all three functional groups attached to the triazole core. We identified specific groups that are critical for KOR potency and further extended the range of moieties explored. These efforts revealed analogs with potency on par with our lead triazole probe molecule, Triazole 1.1, in addition to analogs possessing a spectrum of less potent KOR agonist activity.

Homodimers are the most abundant type of enzyme in cells, and as such, they represent the most elemental system for studying the phenomenon of allostery. In these systems, in which the allosteric features are manifest by the effect of the first binding event on a similar event at the second site, the most informative state is the asymmetric singly bound (lig1) form, yet it tends to be thermodynamically elusive. Here we obtain milligram quantities of lig1 of the allosteric homodimer, chorismate mutase, in the form of a mixed isotopically labeled dimer stabilized by Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) between the subunits. Below, we outline several critical steps required to generate high yields of both types of unnatural amino acid-containing proteins and overcome multiple pitfalls intrinsic to CuAAC to obtain high yields of a highly purified, fully intact, active mixed labeled dimer, which provides the first glimpse of the lig1 intermediate. These data not only will make possible NMR-based investigations of allostery envisioned by us but also should facilitate other structural applications in which specific linkage of proteins is helpful.

MLS1082 is a structurally novel pyrimidone-based D1-like dopamine receptor positive allosteric modulator. Potentiation of D1 dopamine receptor (D1R) signaling is a therapeutic strategy for treating neurocognitive disorders. Here, we investigate the relationship between D1R potentiation and two prominent structural features of MLS1082, namely the pendant N-aryl and C-alkyl groups on the pyrimidone ring. To this end, we synthesized 24 new analogues and characterized their ability to potentiate dopamine signaling at the D1R and the closely related D5R. We identified structure–activity relationship trends for both aryl and alkyl modifications and our efforts afforded several analogues with improvements in activity. The most effective analogues demonstrated an approximately 8-fold amplification of dopamine-mediated D1R signaling. These findings advance the understanding of structural moieties underlying the activity of pyrimidone-based D1R positive allosteric modulators.

To identify novel D3 dopamine receptor (D3R) agonists, we conducted a high-throughput screen using a β-arrestin recruitment assay. Counterscreening of the hit compounds provided an assessment of their selectivity, efficacy, and potency. The most promising scaffold was optimized through medicinal chemistry resulting in enhanced potency and selectivity. The optimized compound, ML417 (20), potently promotes D3R-mediated β-arrestin translocation, G protein activation, and ERK1/2 phosphorylation (pERK) while lacking activity at other dopamine receptors. Screening of ML417 against multiple G protein-coupled receptors revealed exceptional global selectivity. Molecular modeling suggests that ML417 interacts with the D3R in a unique manner, possibly explaining its remarkable selectivity. ML417 was also found to protect against neurodegeneration of dopaminergic neurons derived from iPSCs. Together with promising pharmacokinetics and toxicology profiles, these results suggest that ML417 is a novel and uniquely selective D3R agonist that may serve as both a research tool and a therapeutic lead for the treatment of neuropsychiatric disorders.

The aberrant protein–protein interaction between calmodulin and mutant huntingtin protein in Huntington’s disease patients has been found to contribute to Huntington’s disease progression. A high-throughput screen for small molecules capable of disrupting this interaction revealed a sultam series as potent small-molecule disruptors. Diversification of the sultam scaffold afforded a set of 24 analogs or further evaluation. Several structure–activity trends within the analog set were found, most notably a negligible effect of absolute stereochemistry and a strong beneficial correlation with electron-withdrawing aromatic substituents. The most promising analogs were profiled for off-target effects at relevant kinases and, ultimately, one candidate molecule was evaluated for neuroprotection in a neuronal cell model of Huntington’s disease.

Mu opioid receptor (MOR)-targeting analgesics are efficacious pain treatments, but notorious for their abuse potential. In preclinical animal models, coadministration of traditional kappa opioid receptor (KOR)-targeting agonists with MOR-targeting analgesics can decrease reward and potentiate analgesia. However, traditional KOR-targeting agonists are well known for inducing antitherapeutic side effects (psychotomimesis, depression, anxiety, dysphoria). Recent data suggest that some functionally selective, or biased, KOR-targeting agonists might retain the therapeutic effects of KOR activation without inducing undesirable side effects. Nalfurafine, used safely in Japan since 2009 for uremic pruritus, is one such functionally selective KOR-targeting agonist. Here, we quantify the bias of nalfurafine and several other KOR agonists relative to an unbiased reference standard (U50,488) and show that nalfurafine and EOM-salvinorin-B demonstrate marked G protein-signaling bias. While nalfurafine (0.015 mg/kg) and EOM-salvinorin-B (1 mg/kg) produced spinal antinociception equivalent to 5 mg/kg U50,488, only nalfurafine significantly enhanced the supraspinal analgesic effect of 5 mg/kg morphine. In addition, 0.015 mg/kg nalfurafine did not produce significant conditioned place aversion, yet retained the ability to reduce morphine-induced conditioned place preference in C57BL/6J mice. Nalfurafine and EOM-salvinorin-B each produced robust inhibition of both spontaneous and morphine-stimulated locomotor behavior, suggesting a persistence of sedative effects when coadministered with morphine. Taken together, these findings suggest that nalfurafine produces analgesic augmentation, while also reducing opioid-induced reward with less risk of dysphoria. Thus, adjuvant administration of G protein-biased KOR agonists like nalfurafine may be beneficial in enhancing the therapeutic potential of MOR-targeting analgesics, such as morphine.

A systematic study of the stability of a set of cephalosporins in mouse plasma reveals that cephalosporins lacking an acidic moiety at C-2 may be vulnerable to β-lactam cleavage in mouse plasma.

Concerns about the extent to which graduate programs adequately prepare students for the workplace have prompted numerous calls for reform. Understanding what employers look for in doctoral graduates can help schools better align graduate training with workplace needs. Twelve pharmaceutical scientists across diverse specialties and career pathways described the skills considered requisite for success in today’s science economy. Depth and breadth of knowledge, communication, collaboration, adaptability, research productivity, experiential training, and motivation and drive were among the themes identified. These results can be used to inform the development of doctoral curricula in the biomedical sciences.

As part of a program to explore the chemistry of β-lactams and their derivatives, we prepared a focused set of benzazetidine, indoline, and indole heterocycles, as well as flexible unconstrained variations of the four-membered heterocyclic compounds. These analogues mimic the three-dimensional shape of cephalosporins but are not prone to covalent binding via ring opening. Although these analogues were inactive against the ESKAPE pathogens and Mtb, they represent unique and underexplored chemotypes for future biological screening.

We report a catalytic hydroarylation method to convert phenols to dihydrocoumarins in hexafluoroisopropanol (HFIP) using acid generated from sub-stoichiometric amounts of acetyl chloride as catalyst. Attractive elements include easy set-up and isolation, and applicability to a range of phenols including natural product substrates.

The D 1 dopamine receptor is linked to a variety of neuropsychiatric disorders and represents an attractive drug target for the enhancement of cognition in schizophrenia, Alzheimer disease, and other disorders. Positive allosteric modulators (PAMs), with their potential for greater selectivity and larger therapeutic windows, may represent a viable drug development strategy, as orthosteric D 1 receptor agonists possess known clinical liabilities. We discovered two structurally distinct D 1 receptor PAMs, MLS6585 and MLS1082, via a high-throughput screen of the NIH Molecular Libraries program small-molecule library. Both compounds potentiate dopamine-stimulated G protein- and b-arrestin-mediated signaling and increase the affinity of dopamine for the D 1 receptor with low micromolar potencies. Neither compound displayed any intrinsic agonist activity. Both compounds were also found to potentiate the efficacy of partial agonists. We tested maximally effective concentrations of each PAM in combination to determine if the compounds might act at separate or similar sites. In combination, MLS1082 1 MLS6585 produced an additive potentiation of dopamine potency beyond that caused by either PAM alone for both b-arrestin recruitment and cAMP accumulation, suggesting diverse sites of action. In addition, MLS6585, but not MLS1082, had additive activity with the previously described D 1 receptor PAM “Compound B,” suggesting that MLS1082 and Compound B may share a common binding site. A point mutation (R130Q) in the D 1 receptor was found to abrogate MLS1082 activity without affecting that of MLS6585, suggesting this residue may be involved in the binding/activity of MLS1082 but not that of MLS6585. Together, MLS1082 and MLS6585 may serve as important tool compounds for the characterization of diverse allosteric sites on the D 1 receptor as well as the development of optimized lead compounds for therapeutic use.

A. 4-(3,4-Dimethoxyphenyl)butanoyl chloride (2). An oven-dried, 250-mL, three-necked, 14/20 round-bottomed flask is equipped with an egg-shaped magnetic stir bar (2 cm), a pressure-equalizing addition funnel (25 mL), a rubber septum, and an argon inlet (Figure 1). The flask is charged with 4- (3,4-dimethoxyphenyl)butanoic acid (1) (7.25 g, 32.3 mmol, 1.0 equiv) (Note 2), anhydrous DCM (40 mL) (Note 3) and DMF (50. μL, 0.65 mmol, 0.02 equiv) (Note 4). Oxalyl chloride (5.5 mL, 64 mmol, 2.0 equiv) (Note 5) is added via syringe to the addition funnel, and the stopcock is opened such that the oxalyl chloride is added over 4 min, resulting in effervescence. The reaction mixture is stirred at 23 °C for 30 min from the start of the addition of oxalyl chloride (Note 6). The argon inlet is replaced with a rubber septum, the addition funnel is removed, and the flask placed on a rotary evaporator. Concentration under reduced pressure (35 °C, 30 mm Hg) afforded 8.35 g of crude acid chloride (2) as a yellow oil (Note 7 and 8), which is used in the next step without purification.

The dopamine D2 receptor (D2R) is known to elicit effects through activating two major signaling pathways mediated by either G proteins (Gi/o) or β-arrestins. However, the specific role of each pathway in physiological or therapeutic activities is not known with certainty. One approach to the dissection of these pathways is through the use of drugs that can selectively modulate one pathway vs. the other through a mechanism known as functional selectivity or biased signaling. Our laboratory has previously described a G protein signaling-biased agonist, MLS1547, for the D2R using a variety of in vitro functional assays. To further evaluate the biased signaling activity of this compound, we investigated its ability to promote D2R internalization, a process known to be mediated by β-arrestin. Using multiple cellular systems and techniques, we found that MLS1547 promotes little D2R internalization, which is consistent with its inability to recruit β-arrestin. Importantly, we validated these results in primary striatal neurons where the D2R is most highly expressed suggesting that MLS1547 will exhibit biased signaling activity in vivo. In an effort to optimize and further explore structure-activity relationships (SAR) for this scaffold, we conducted an iterative chemistry campaign to synthesize and characterize novel analogs of MLS1547. The resulting analysis confirmed previously described SAR requirements for G protein-biased agonist activity and, importantly, elucidated new structural features that are critical for agonist efficacy and signaling bias of the MLS1547 scaffold. One of the most important determinants for G protein-biased signaling is the interaction of a hydrophobic moiety of the compound with a defined pocket formed by residues within transmembrane five and extracellular loop two of the D2R. These results shed new light on the mechanism of biased signaling of the D2R and may lead to improved functionally-selective molecules.

Mucosal-associated invariant T (MAIT) cells are an abundant class of innate T cells restricted by the MHC I-related molecule MR1. MAIT cells can recognize bacterially-derived metabolic intermediates from the riboflavin pathway presented by MR1 and are postulated to play a role in innate antibacterial immunity through production of cytokines and direct bacterial killing. MR1 tetramers, typically stabilized by the adduct of 5-amino-6-D-ribitylaminour-acil (5-A-RU) and methylglyoxal (MeG), are important tools for the study of MAIT cells. A long-standing problem with 5-A-RU is that it is unstable upon storage. Herein we report an efficient synthetic approach to the HCl salt of this ligand, which has improved stability during storage. We also show that synthetic 5-A-RU•HCl produced by this method may be used in protocols for the stimulation of human MAIT cells and production of both human and mouse MR1 tetramers for MAIT cell identification.

Biased agonists of G protein-coupled receptors may present a means to refine receptor signaling in a way that separates side effects from therapeutic properties. Several studies have shown that agonists that activate the k-opioid receptor (KOR) in a manner that favors G protein coupling over b-Arrestin2 recruitment in cell culture may represent a means to treat pain and itch while avoiding sedation and dysphoria. Although it is attractive to speculate that the bias between G protein signaling and b-Arrestin2 recruitment is the reason for these divergent behaviors, little evidence has emerged to show that these signaling pathways diverge in the neuronal environment. We further explored the influence of cellular context on biased agonism at KOR ligand-directed signaling toward G protein pathways over b-Arrestin-dependent pathways and found that this bias persists in striatal neurons. These findings advance our understanding of how a G protein-biased agonist signal differs between cell lines and primary neurons, demonstrate that measuring [35S]GTPgS binding and the regulation of adenylyl cyclase activity are not necessarily orthogonal assays in cell lines, and emphasize the contributions of the environment to assessing biased agonism .

The rerouting of the nitrilium ion formed in the Schmidt reaction of ketones and TMSN 3 to encompass C-C bond formation with an electron-rich aromatic group is reported. Thus, when the reaction is carried out in HFIP using AlCl 3 or AlBr 3 as the promoter, imines, iminium ions, or enamide derivatives are obtained through one-pot procedures. The scope and possible mechanisms of these new transformations are considered.

Kinetoplastid-based infections are neglected diseases that represent a significant human health issue. Chemotherapeutic options are limited due to toxicity, parasite susceptibility, and poor patient compliance. In response, we studied a molecular-target-directed approach involving intervention of hexokinase activity—a pivotal enzyme in parasite metabolism. A benzamidobenzoic acid hit with modest biochemical inhibition of Trypanosoma brucei hexokinase 1 (TbHK1, IC50=9.1 μm), low mammalian cytotoxicity (IMR90 cells, EC50>25 μm), and no appreciable activity on whole bloodstream-form (BSF) parasites was optimized to afford a probe with improved TbHK1 potency and, significantly, efficacy against whole BSF parasites (TbHK1, IC50=0.28 μm; BSF, ED50=1.9 μm). Compounds in this series also inhibited the hexokinase enzyme from Leishmania major (LmHK1), albeit with less potency than toward TbHK1, suggesting that inhibition of the glycolytic pathway may be a promising opportunity to target multiple disease-causing trypanosomatid protozoa.

Small-molecule probes can illuminate biological processes and aid in the assessment of emerging therapeutic targets by perturbing biological systems in a manner distinct from other experimental approaches. Despite the tremendous promise of chemical tools for investigating biology and disease, small-molecule probes were unavailable for most targets and pathways as recently as a decade ago. In 2005, the NIH launched the decade-long Molecular Libraries Program with the intent of innovating in and broadening access to small-molecule science. This Perspective describes how novel small-molecule probes identified through the program are enabling the exploration of biological pathways and therapeutic hypotheses not otherwise testable. These experiences illustrate how small-molecule probes can help bridge the chasm between biological research and the development of medicines but also highlight the need to innovate the science of therapeutic discovery.

Differential modulation of kappa opioid receptor (KOR) signaling has been a proposed strategy for developing therapies for drug addiction and depression by either activating or blocking this receptor. Hence, there have been significant efforts to generate ligands with diverse pharmacological properties including partial agonists, antagonists, allosteric modulators as well as ligands that selectively activate some pathways while not engaging others (biased agonists). It is becoming increasingly evident that G protein coupled receptor signaling events are context dependent and that what may occur in cell based assays may not be fully indicative of signaling events that occur in the naturally occurring environment. As new ligands are developed, it is important to assess their signaling capacity in relevant endogenous systems in comparison to the performance of endogenous agonists. Since KOR is considered the cognate receptor for dynorphin peptides we have evaluated the selectivity profiles of dynorphin peptides in wild-type (WT), KOR knockout (KOR-KO), and mu opioid receptor knockout (MOR-KO) mice using [35S]GTPγS binding assay in striatal membrane preparations. We find that while the small molecule KOR agonist U69,593, is very selective for KOR, dynorphin peptides promiscuously stimulate G protein signaling in striatum. Furthermore, our studies demonstrate that norBNI and 5′GNTI are highly nonselective antagonists as they maintain full potency and efficacy against dynorphin signaling in the absence of KOR. Characterization of a new KOR antagonist, which may be more selective than NorBNI and 5′GNTI, is presented using this approach.

The intramolecular Schmidt reaction of ketones and tethered azides is an efficient method for the generation of amides and lactams. This reaction is catalyzed by Lewis acids, which tightly bind the strongly basic amide product and result in product inhibition. We report herein conditions to achieve a catalytic Schmidt reaction using substoichiometric amounts of the heat-stable Lewis acid Sc(OTf)3. This species was shown to effectively release products of the Schmidt reaction in a temperature-dependent fashion. Thus, heat was able to promote catalyst turnover. A brief substrate scope was conducted using these conditions.

Optimization of the sulfonamide-based kappa opioid receptor (KOR) antagonist probe molecule ML140 through constraint of the sulfonamide nitrogen within a tetrahydroisoquinoline moiety afforded a marked increase in potency. This strategy, when combined with additional structure-activity relationship exploration, has led to a compound only six-fold less potent than norBNI, a widely utilized KOR antagonist tool compound, but significantly more synthetically accessible. The new optimized probe is suitably potent for use as an in vivo tool to investigate the therapeutic potential of KOR antagonists.

Electrochemistry provides a powerful tool for the late-stage functionalization of complex lactams. A two-stage protocol for converting lactams, many of which can be prepared through the intramolecular Schmidt reaction of keto azides, is presented. In the first step, anodic oxidation in MeOH using a repurposed power source provides a convenient route to lactams bearing a methoxy group adjacent to nitrogen. Treatment of these intermediates with a Lewis acid in dichloromethane permits the regeneration of a reactive acyliminium ion that is then reacted with a range of nucleophilic species.

A means of generating an N-alkyl-N-arylaminodiazonium ion, which then loses nitrogen to form a reactive aryl nitrenium intermediate, is described. In this sequence, a set of triazenyl acetonitriles was synthesized by nucleophilic addition of a nitrile anion to an aryl azide followed by temperature-dependent alkylation at either of two nucleophilic triazenyl nitrogen atoms. An α,α-disubstituted acetonitrile and N-arylaminodiazonium moiety (or aryl nitrenium ion upon loss of N2) are embedded in the resulting 1,3,3-trisubsituted triazene, which undergoes liberation and recombination of these two components under acidic conditions to yield an α-arylated acetonitrile containing an all-carbon-quaternary center. We propose that the N-alkyl-N-arylaminodiazonium ion loses nitrogen to generate an aryl nitrenium species, which then reacts with an α,α-disubstituted acetonitrile either at the para- or meta-position of the aromatic ring to afford p-alkylaminoaryl acetonitrile derivatives or 3,3-substituted 1-methylindolin-2-imines, depending on the substrates and conditions.

Screening of small-molecule libraries is an important aspect of probe and drug discovery science. Numerous authors have suggested that bioactive natural products are attractive starting points for such libraries because of their structural complexity and sp 3 -rich character. Here, we describe the construction of a screening library based on representative members of four families of biologically active alkaloids (Stemonaceae, the structurally related cyclindricine and lepadiformine families, lupin and Amaryllidaceae). In each case, scaffolds were based on structures of the naturally occurring compounds or a close derivative. Scaffold preparation was pursued following the development of appropriate enabling chemical methods. Diversification provided 686 new compounds suitable for screening. The libraries thus prepared had structural characteristics, including sp 3 content, comparable to a basis set of representative natural products and were highly rule-of-five compliant.

TASK-1 is a two-pore domain potassium channel that is important to modulating cell excitability, most notably in the context of neuronal pathways. In order to leverage TASK-1 for therapeutic benefit, its physiological role needs better characterization; however, designing selective inhibitors that avoid the closely related TASK-3 channel has been challenging. In this study, a series of bis-amide derived compounds were found to demonstrate improved TASK-1 selectivity over TASK-3 compared to reported inhibitors. Optimization of a marginally selective hit led to analog 35 which displays a TASK-1 IC 50 = 16 nM with 62-fold selectivity over TASK-3 in an orthogonal electrophysiology assay.

Substituted pyrimidine inhibitors of the Clk and Dyrk kinases have been developed, exploring structure-activity relationships around four different chemotypes. The most potent compounds have low-nanomolar inhibitory activity against Clk1, Clk2, Clk4, Dyrk1A and Dyrk1B. Kinome scans with 442 kinases using agents representing three of the chemotypes show these inhibitors to be highly selective for the Clk and Dyrk families. Further off-target pharmacological evaluation with ML315, the most selective agent, supports this conclusion.

The kappa opioid receptor (KOR) is widely expressed in the CNS and can serve as a means to modulate pain perception, stress responses, and affective reward states. Therefore, the KOR has become a prominent drug discovery target toward treating pain, depression, and drug addiction. Agonists at KOR can promote G protein coupling and βarrestin2 recruitment as well as multiple downstream signaling pathways, including ERK1/2 MAPK activation. It has been suggested that the physiological effects ofKORactivation result from different signaling cascades, with analgesia being G protein-mediated and dysphoria being mediated through βarrestin2 recruitment. Dysphoria associated with KOR activation limits the therapeutic potential in the use of KOR agonists as analgesics; therefore, it may be beneficial to develop KOR agonists that are biased toward G protein coupling and away from βarrestin2 recruitment. Here, we describe two classes of biased KOR agonists that potently activateGprotein coupling but weakly recruitβarrestin2. These potent and functionally selective small molecule compounds may prove to be useful tools for refining the therapeutic potential of KOR-directed signaling in vivo.

Purpose: 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), one of the most prevalent and procarcinogenic compounds in tobacco, is bioactivated by respiratory cytochrome P450 (CYP) 2A13, forming DNA adducts and initiating lung cancer. CYP2A13 inhibition offers a novel strategy for chemoprevention of tobacco-associated lung cancer. Methods: Twenty-four analogs of a 4-benzylmorpholine scaffold identified by high throughput screening were evaluated for binding and inhibition of both functional human CYP2A enzymes, CYP2A13 and the 94%-identical hepatic CYP2A6, whose inhibition is undesirable. Thus, selectivity is a major challenge in compound design. Results: A key feature resulting in CYP2A13-selective binding and inhibition was substitution at the benzyl ortho position, with three analogs being >25-fold selective for CYP2A13 over CYP2A6. Conclusions: Two such analogs were negative for genetic and hERG toxicities and metabolically stable in human lung microsomes, but displayed rapid metabolism in human liver and in mouse and rat lung and liver microsomes, likely due to CYP2B-mediated degradation. A specialized knockout mouse mimicking the human lung demonstrates compound persistence in lung and provides an appropriate test model. Compound delivered by inhalation may be effective in the lung but rapidly cleared otherwise, limiting systemic exposure.

[No abstract available]

The authors conducted a high-throughput screening campaign for inhibitors of SV40 large T antigen ATPase activity to identify candidate antivirals that target the replication of polyomaviruses. The primary assay was adapted to 1536-well microplates and used to screen the National Institutes of Health Molecular Libraries Probe Centers Network library of 306 015 compounds. The primary screen had an Z value of ∼0.68, signal/background = 3, and a high (5%) DMSO tolerance. Two counterscreens and two secondary assays were used to prioritize hits by EC50, cytotoxicity, target specificity, and off-target effects. Hits that inhibited ATPase activity by >44% in the primary screen were tested in dose-response efficacy and eukaryotic cytotoxicity assays. After evaluation of hit cytotoxicity, drug likeness, promiscuity, and target specificity, three compounds were chosen for chemical optimization. Chemical optimization identified a class of bisphenols as the most effective biochemical inhibitors. Bisphenol A inhibited SV40 large T antigen ATPase activity with an IC50 of 41 μM in the primary assay and 6.2 μM in a cytoprotection assay. This compound class is suitable as probes for biochemical investigation of large T antigen ATPase activity, but because of their cytotoxicity, further optimization is necessary for their use in studying polyomavirus replication in vivo.

Go with the flow! A method for conversion of keto halides to lactams by means of sequential azidation and intramolecular Schmidt reaction in a combined flow format is described (see scheme; MWI=microwave irradiation, TFA=trifluoroacetic acid).

Flipping diversity: Minimization of 1,3-allylic strain (A1, 3 strain) is a recurring element in the design of a stereochemically and spatially diverse collection of 2-arylpiperidines. A1, 3 strain guides the regioselective addition of nucleophiles and N-substituents leverage A 1, 3 strain to direct each stereoisomer to two different conformer populations, thus doubling the number of library members.

A tandem Diels-Alder/azido-Schmidt reaction sequence provides rapid access to the core skeleton shared by several Stemona alkaloids including stenine, neostenine, tuberostemonine, and neotuberostemonine. The discovery and evolution of inter- and intramolecular variations of this process and their applications to total syntheses of (±)-stenine and (±)-neostenine are described. The stereochemical outcome of the reaction depends on both substrate type and reaction conditions, enabling the preparation of both (±)-stenine and (±)-neostenine from the same diene/dienophile combination.

Gaining control: An attractive electrostatic force stabilizes the conformation of an intermediate (see scheme; n=nonbonded electron), thus controlling the leaving group configuration to give superior diastereoselectivity in an asymmetric ring expansion.

Four stereocenters and three rings are established in one step in a new formal synthesis of (±)-stenine (see scheme). This key reaction cascade consists of an intramolecular Diels-Alder reaction followed by an intramolecular Schmidt reaction. The resulting tricyclic intermediate was readily transformed into a known intermediate en route to stenine, thus completing the formal synthesis.