Autoinducer-fluorophore conjugates enable FRET in LuxR proteins in vitro and in cells.

Cell-to-cell signaling, or quorum sensing (QS), in many Gram-negative bacteria is governed by small molecule signals (N-acyl-L-homoserine lactones, AHLs) and their cognate receptors (LuxR-type proteins). The mechanistic underpinnings of QS in these bacteria are severely limited due to the challenges of isolating and manipulating most LuxR-type proteins. Reports of quantitative direct-binding experiments on LuxR-type proteins are scarce, and robust and generalizable methods that provide such data are largely nonexistent. We report herein a Förster resonance energy transfer (FRET) assay that leverages (1) conserved tryptophans located in the LuxR-type protein ligand-binding site and synthetic fluorophore-AHL conjugates, and (2) isolation of the proteins bound to weak agonists. The FRET assay permits straightforward measurement of ligand-binding affinities with receptor-either in vitro or in cells-and was shown to be compatible with six LuxR-type proteins. These methods will advance fundamental investigations of LuxR-type protein mechanism and the development of small molecule QS modulators.

An integrated chemical biology approach reveals the mechanism of action of HIV replication inhibitors.

Continuous flow (microfluidic) chemistry was employed to prepare a small focused library of dihydropyrimidinone (DHPM) derivatives. Compounds in this class have been reported to exhibit activity against the human immunodeficiency virus (HIV), but their molecular target had not been identified. We tested the initial set of DHPMs in phenotypic assays providing a hit (1i) that inhibited the replication of the human immunodeficiency virus HIV in cells. Flow chemistry-driven optimization of 1i led to the identification of HIV replication inhibitors such as 1l with cellular potency comparable with the clinical drug nevirapine (NVP). Mechanism of action (MOA) studies using cellular and biochemical assays coupled with 3D fingerprinting and in silico modeling demonstrated that these drug-like probe compounds exert their effects by inhibiting the viral reverse transcriptase polymerase (RT). This led to the design and synthesis of the novel DHPM 1at that inhibits the replication of drug resistant strains of HIV. Our work demonstrates that combining flow chemistry-driven analogue refinement with phenotypic assays, in silico modeling and MOA studies is a highly effective strategy for hit-to-lead optimization applicable to the discovery of future therapeutic agents.

Potent and Selective Modulation of the RhlR Quorum Sensing Receptor by Using Non-native Ligands: An Emerging Target for Virulence Control in Pseudomonas aeruginosa.

Pseudomonas aeruginosa uses N-acylated L-homoserine lactone signals and a triumvirate of LuxR-type receptor proteins--LasR, RhlR, and QscR--for quorum sensing (QS). Each of these receptors can contribute to QS activation or repression and, thereby, the control of myriad virulence phenotypes in this pathogen. LasR has traditionally been considered to be at the top of the QS receptor hierarchy in P. aeruginosa; however, recent reports suggest that RhlR plays a more prominent role in infection than originally predicted, in some circumstances superseding that of LasR. Herein, we report the characterization of a set of synthetic, small-molecule agonists and antagonists of RhlR. Using E. coli reporter strains, we demonstrated that many of these compounds can selectively activate or inhibit RhlR instead of LasR and QscR. Moreover, several molecules maintain their activities in P. aeruginosa at concentrations analogous to native RhlR signal levels. These compounds represent useful chemical probes to study the role of RhlR in the complex QS circuitry of P. aeruginosa, its direct (and indirect) effects on virulence, and its overall merit as a target for anti-infective therapy.

Identification of positive allosteric modulators VU0155094 (ML397) and VU0422288 (ML396) reveals new insights into the biology of metabotropic glutamate receptor 7.

Metabotropic glutamate receptor 7 (mGlu7) is a member of the group III mGlu receptors (mGlus), encompassed by mGlu4, mGlu6, mGlu7, and mGlu8. mGlu7 is highly expressed in the presynaptic active zones of both excitatory and inhibitory synapses, and activation of the receptor regulates the release of both glutamate and GABA. mGlu7 is thought to be a relevant therapeutic target for a number of neurological and psychiatric disorders, and polymorphisms in the GRM7 gene have been linked to autism, depression, ADHD, and schizophrenia. Here we report two new pan-group III mGlu positive allosteric modulators, VU0155094 and VU0422288, which show differential activity at the various group III mGlus. Additionally, both compounds show probe dependence when assessed in the presence of distinct orthosteric agonists. By pairing studies of these nonselective compounds with a synapse in the hippocampus that expresses only mGlu7, we have validated activity of these compounds in a native tissue setting. These studies provide proof-of-concept evidence that mGlu7 activity can be modulated by positive allosteric modulation, paving the way for future therapeutics development.

Heterotropic activation of the midazolam hydroxylase activity of CYP3A by a positive allosteric modulator of mGlu5: in vitro to in vivo translation and potential impact on clinically relevant drug-drug interactions.

Allosteric modulation of G protein-coupled receptors has gained considerable attention in the drug discovery arena because it opens avenues to achieve greater selectivity over orthosteric ligands. We recently identified a series of positive allosteric modulators (PAMs) of metabotropic glutamate receptor 5 (mGlu(5)) for the treatment of schizophrenia that exhibited robust heterotropic activation of CYP3A4 enzymatic activity. The prototypical compound from this series, 5-(4-fluorobenzyl)-2-((3-fluorophenoxy)methyl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazine (VU0448187), was found to activate CYP3A4 to >100% of its baseline intrinsic midazolam (MDZ) hydroxylase activity in vitro; activation was CYP3A substrate specific and mGlu(5) PAM dependent. Additional studies revealed the concentration-dependence of CYP3A activation by VU0448187 in multispecies hepatic and intestinal microsomes and hepatocytes, as well as a diminished effect observed in the presence of ketoconazole. Kinetic analyses of the effect of VU0448187 on MDZ metabolism in recombinant P450 or human liver microsomes resulted in a significant increase in V(max) (minimal change in K(m)) and required the presence of cytochrome b5. The atypical kinetics translated in vivo, as rats receiving an intraperitoneal administration of VU0448187 prior to MDZ treatment demonstrated a significant increase in circulating 1- and 4-hydroxy- midazolam (1-OH-MDZ, 4-OH-MDZ) levels compared with rats administered MDZ alone. The discovery of a potent substrate-selective activator of rodent CYP3A with an in vitro to in vivo translation serves to illuminate the impact of increasing intrinsic enzymatic activity of hepatic and extrahepatic CYP3A in rodents, and presents the basis to build models capable of framing the clinical relevance of substrate-dependent heterotropic activation.

Recent progress in the discovery of small molecules for the treatment of amyotrophic lateral sclerosis (ALS).

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder with few therapeutic options. While several gene mutations have been implicated in ALS, the exact cause of neuronal dysfunction is unknown and motor neurons of affected individuals display numerous cellular abnormalities. Ongoing efforts to develop novel ALS treatments involve the identification of small molecules targeting specific mechanisms of neuronal pathology, including glutamate excitotoxicity, mutant protein aggregation, endoplasmic reticulum (ER) stress, loss of trophic factors, oxidative stress, or neuroinflammation. Herein, we review recent advances in the discovery and preclinical characterization of lead compounds that may ultimately provide novel drugs to treat patients suffering from ALS.

Dynamic subunit stoichiometry confers a progressive continuum of pharmacological sensitivity by KCNQ potassium channels.

Voltage-gated KCNQ1 (Kv7.1) potassium channels are expressed abundantly in heart but they are also found in multiple other tissues. Differential coassembly with single transmembrane KCNE beta subunits in different cell types gives rise to a variety of biophysical properties, hence endowing distinct physiological roles for KCNQ1-KCNEx complexes. Mutations in either KCNQ1 or KCNE1 genes result in diseases in brain, heart, and the respiratory system. In addition to complexities arising from existence of five KCNE subunits, KCNE1 to KCNE5, recent studies in heterologous systems suggest unorthodox stoichiometric dynamics in subunit assembly is dependent on KCNE expression levels. The resultant KCNQ1-KCNE channel complexes may have a range of zero to two or even up to four KCNE subunits coassembling per KCNQ1 tetramer. These findings underscore the need to assess the selectivity of small-molecule KCNQ1 modulators on these different assemblies. Here we report a unique small-molecule gating modulator, ML277, that potentiates both homomultimeric KCNQ1 channels and unsaturated heteromultimeric (KCNQ1)4(KCNE1)n (n < 4) channels. Progressive increase of KCNE1 or KCNE3 expression reduces efficacy of ML277 and eventually abolishes ML277-mediated augmentation. In cardiomyocytes, the slowly activating delayed rectifier potassium current, or IKs, is believed to be a heteromultimeric combination of KCNQ1 and KCNE1, but it is not entirely clear whether IKs is mediated by KCNE-saturated KCNQ1 channels or by channels with intermediate stoichiometries. We found ML277 effectively augments IKs current of cultured human cardiomyocytes and shortens action potential duration. These data indicate that unsaturated heteromultimeric (KCNQ1)4(KCNE1)n channels are present as components of IKs and are pharmacologically distinct from KCNE-saturated KCNQ1-KCNE1 channels.

Isatin replacements applied to the highly selective, muscarinic M1 PAM ML137: continued optimization of an MLPCN probe molecule.

This Letter describes the continued optimization of an MLPCN probe molecule (ML137) with a focused effort on the replacement/modification of the isatin moiety present in this highly selective M(1) PAM. A diverse range of structures were validated as viable replacements for the isatin, many of which engendered sizeable improvements in their ability to enhance the potency and efficacy of acetylcholine when compared to ML137. Muscarinic receptor subtype selectivity for the M(1) receptor was also maintained.

Identification of (R)-N-(4-(4-methoxyphenyl)thiazol-2-yl)-1-tosylpiperidine-2-carboxamide, ML277, as a novel, potent and selective K(v)7.1 (KCNQ1) potassium channel activator.

A high-throughput screen utilizing a depolarization-triggered thallium influx through KCNQ1 channels was developed and used to screen the MLSMR collection of over 300,000 compounds. An iterative medicinal chemistry approach was initiated and from this effort, ML277 was identified as a potent activator of KCNQ1 channels (EC(50)=260 nM). ML277 was shown to be highly selective against other KCNQ channels (>100-fold selectivity versus KCNQ2 and KCNQ4) as well as against the distantly related hERG potassium channel.

Development of novel M1 antagonist scaffolds through the continued optimization of the MLPCN probe ML012.

This Paper describes the continued optimization of an MLPCN probe molecule M(1) antagonist (ML012) through an iterative parallel synthesis approach. After several rounds of modifications of the parent compound, we arrived at a new azetidine scaffold that displayed improved potency while maintaining a desirable level of selectivity over other muscarinic receptor subtypes. Data for representative molecules 7w (VU0452865) and 12a (VU0455691) are presented.

Isovaleryl-homoserine lactone, an unusual branched-chain quorum-sensing signal from the soybean symbiont Bradyrhizobium japonicum.

Many species of Proteobacteria communicate by using LuxI-LuxR-type quorum-sensing systems that produce and detect acyl-homoserine lactone (acyl-HSL) signals. Most of the known signals are straight-chain fatty acyl-HSLs, and evidence indicates that LuxI homologs prefer fatty acid-acyl carrier protein (ACP) over fatty acyl-CoA as the acyl substrate for signal synthesis. Two related LuxI homologs, RpaI and BtaI from Rhodopseudomonas palustris and photosynthetic stem-nodulating bradyrhizobia, direct production of the aryl-HSLs p-coumaroyl-HSL and cinnamoyl-HSL, respectively. Here we report that BjaI from the soybean symbiont Bradyrhizobium japonicum USDA110 is closely related to RpaI and BtaI and catalyzes the synthesis of isovaleryl-HSL (IV-HSL), a branched-chain fatty acyl-HSL. We show that IV-HSL induces expression of bjaI, and in this way IV-HSL functions like many other acyl-HSL quorum-sensing signals. Purified histidine-tagged BjaI was an IV-HSL synthase, which was active with isovaleryl-CoA but not detectably so with isovaleryl-ACP. This suggests that the RpaI-BtaI-BjaI subfamily of acyl-HSL synthases may use CoA- rather than ACP-linked substrates for acyl-HSL synthesis. The bjaI-linked bjaR(1) gene is involved in the response to IV-HSL, and BjaR(1) is sensitive to IV-HSL at concentrations as low as 10 pM. Low but sufficient levels of IV-HSL (about 5 nM) accumulate in B. japonicum culture fluid. The low levels of IV-HSL synthesis have likely contributed to the fact that the quorum-sensing signal from this bacterium has not been described elsewhere.

Inhibition of Akt with small molecules and biologics: historical perspective and current status of the patent landscape.

INTRODUCTION: Akt plays a pivotal role in cell survival and proliferation through a number of downstream effectors; unregulated activation of the PI3K/PTEN/Akt pathway is a prominent feature of many human cancers. Akt is considered an attractive target for cancer therapy by the inhibition of Akt alone or in combination with standard cancer chemotherapeutics. Both preclinical animal studies and clinical trials in humans have validated Akt as an important target of cancer drug discovery. AREA COVERED: A historical perspective of Akt inhibitors, including PI analogs, ATP-competitive and allosteric Akt inhibitors, along with other inhibitory mechanisms are reviewed in this paper with a focus on issued patents, patent applications and a summary of clinical trial updates since the last review in 2007. EXPERT OPINION: A vast diversity of inhibitors of Akt, both small molecule and biologic, have been developed in the past 5 years, with over a dozen in various phases of clinical development, and several displaying efficacy in humans. While it is not yet clear which mechanism of Akt inhibition will be optimal in humans, or which Akt isoforms to inhibit, or whether a small molecule or biologic agent will be best, data to all of these points will be available in the near future.

Potent and selective synthetic modulators of a quorum sensing repressor in Pseudomonas aeruginosa identified from second-generation libraries of N-acylated L-homoserine lactones.

Bacteria can coordinate group behavior using chemical signals in a process called quorum sensing (QS). The QS system in the opportunistic pathogen Pseudomonas aeruginosa is largely governed by the LasR receptor and its cognate chemical signal, N-(3-oxo)-dodecanoyl L-homoserine lactone (OdDHL). LasR also appears to share this signal with an orphan LuxR-type receptor in P. aeruginosa, termed QscR, which represses LasR activity. Non-native molecules that modulate QscR would represent valuable tools to study the role of this novel QS repressor protein in P. aeruginosa. We performed a critical analysis of previously identified, non-native N-acylated L-homoserine lactone (AHL) activators and inhibitors of QscR to determine a set of structure-activity relationships (SARs). Based on these SAR data, we designed, synthesized, and screened several second-generation libraries of AHLs for new ligands that could target QscR. These studies revealed the most active AHL agonists and antagonists of QscR reported to date, with activities ranging from nanomolar to low micromolar in a QscR bacterial reporter strain. Several of these AHLs were highly selective for QscR over LasR and other LuxR-type receptors. A small subset of the new QscR activators, however, were also found to inhibit LasR; this demonstrates the exciting potential for the synergistic modulation of these integral P. aeruginosa QS receptors by using a single synthetic compound.

Small molecules that modulate quorum sensing and control virulence in Pseudomonas aeruginosa.

Bacteria use small molecule signals to access their local population densities in a process called quorum sensing (QS). Once a threshold signal concentration is reached, and therefore a certain number of bacteria have assembled, bacteria use QS to change gene expression levels and initiate behaviors that benefit the group. These group processes play central roles in both bacterial virulence and symbiosis and can have significant impacts on human health, agriculture, and the environment. The dependence of QS on small molecule signals has inspired organic chemists to design non-native molecules that can intercept these signals and thereby perturb bacterial group behaviors. The opportunistic pathogen Pseudomonas aeruginosa has been the target of many of these efforts due to its prevalence in human infections. P. aeruginosa uses at least two N-acyl l-homoserine lactone signals and three homologous LuxR-type receptors to initiate a range of pathogenic behaviors at high cell densities, including biofilm formation and the production of an arsenal of virulence factors. This perspective highlights recent chemical efforts to modulate LuxR-type receptor activity in P. aeruginosa and offers insight into the development of receptor-specific ligands as potential antivirulence strategies.

Evaluation of a focused library of N-aryl L-homoserine lactones reveals a new set of potent quorum sensing modulators.

A focused library of N-aryl l-homoserine lactones was designed around known lactone leads and evaluated for antagonistic and agonistic activity against quorum-sensing receptors in Agrobacterium tumefaciens, Pseudomonas aeruginosa, and Vibrio fischeri. Several compounds were identified with significantly heightened activities relative to the lead compounds, and new structure-activity relationships (SARs) were delineated. Notably, 4-substituted N-phenoxyacetyl and 3-substituted N-phenylpropionyl l-homoserine lactones were identified as potent antagonists of TraR and LuxR, respectively.

Comparative analyses of N-acylated homoserine lactones reveal unique structural features that dictate their ability to activate or inhibit quorum sensing.

Bacterial quorum sensing is mediated by low molecular-weight signals and plays a critical role in both the pathogenesis of infectious disease and beneficial symbioses. There is significant interest in the development of synthetic ligands that can intercept bacterial quorum sensing signals and modulate these outcomes. Here, we report the design and comparative analysis of the effects of approximately 90 synthetic N-acylated homoserine lactones (AHLs) on quorum sensing in three Gram negative bacterial species and a critical examination of the structural features of these ligands that dictate agonistic and antagonistic activity, and selectivity for different R protein targets. These studies have revealed the most comprehensive set of structure-activity relationships to date that direct AHL-mediated quorum sensing and a new set of chemical probes with which to study this complex signaling process. Furthermore, this work provides a foundation on which to design next-generation quorum sensing modulators with improved activities and selectivities.

Synthetic ligands that activate and inhibit a quorum-sensing regulator in Pseudomonas aeruginosa.

The transcription factor QscR is a regulator of quorum sensing in Pseudomonas aeruginosa and plays a role in controlling virulence in this prevalent opportunistic pathogen. This study outlines the discovery of a set of synthetic N-acylated homoserine lactones that are capable of either activating or strongly inhibiting QscR in a cell-based reporter gene assay. We demonstrate that the synthetic antagonists inhibit ligand-dependent QscR binding to DNA. Several of these ligands can selectively modulate QscR instead of LasR, or modulate the activity of both receptors, and represent new chemical tools to study the hierarchy of quorum-sensing signaling in P. aeruginosa.

Modulation of bacterial quorum sensing with synthetic ligands: systematic evaluation of N-acylated homoserine lactones in multiple species and new insights into their mechanisms of action.

Bacteria use a language of low molecular weight ligands to assess their population densities in a process called quorum sensing. This chemical signaling process plays a pivotal role both in the pathogenesis of infectious disease and in beneficial symbioses. There is intense interest in the development of synthetic ligands that can intercept quorum-sensing signals and attenuate these divergent outcomes. Both broad-spectrum and species-selective modulators of quorum sensing hold significant value as small-molecule tools for fundamental studies of this complex cell-cell signaling process and for future biomedical and environmental applications. Here, we report the design and synthesis of focused collections of non-native N-acylated homoserine lactones and the systematic evaluation of these approximately 90 ligands across three Gram-negative bacterial species: the pathogens Agrobacterium tumefaciens and Pseudomonas aeruginosa; the model symbiont Vibrio fischeri. This study is the first to report and compare the activities of a set of ligands across multiple species and has revealed some of the most potent synthetic modulators of quorum sensing to date. Moreover, several of these ligands exhibit agonistic or antagonistic activity in all three species, while other ligands are only active in one or two species. Analysis of the screening data revealed that at least a subset of these ligands modulate quorum sensing via a partial agonism mechanism. We also demonstrate that selected ligands can either inhibit or promote the production of elastase B, a key virulence factor in wild-type P. aeruginosa, depending on their concentrations. Overall, this work provides broad insights into the molecular features required for small-molecule inhibition or activation of quorum sensing in Gram-negative bacteria. In addition, this study has supplied an expansive set of chemical tools for the further investigation of quorum-sensing pathways and responses.

Control of quorum sensing by a Burkholderia pseudomallei multidrug efflux pump.

The Burkholderia pseudomallei KHW quorum-sensing systems produced N-octanoyl-homoserine lactone, N-decanoyl-homoserine lactone, N-(3-hydroxy)-octanoyl-homoserine lactone, N-(3-hydroxy)-decanoyl-homoserine lactone, N-(3-oxo)-decanoyl-homoserine lactone, and N-(3-oxo)-tetradecanoyl-homoserine lactone. The extracellular secretion of these acyl-homoserine lactones is dependent absolutely on the function of the B. pseudomallei BpeAB-OprB efflux pump.