3D printer platform and conductance feedback loop for automated imaging of uneven surfaces by liquid microjunction-surface sampling probe mass spectrometry.

RATIONALE: Molecular imaging of samples using mass spectrometric techniques, such as matrix-assisted laser desorption ionization or desorption electrospray ionization, requires the sample surface to be even/flat and sliced into thin sections (c. 10 µm). Furthermore, sample preparation steps can alter the analyte composition of the sample. The liquid microjunction-surface sampling probe (LMJ-SSP) is a robust sampling interface that enables surface profiling with minimal sample preparation. In conjunction with a conductance feedback system, the LMJ-SSP can be used to automatically sample uneven specimens. METHODS: A sampling stage was built with a modified 3D printer where the LMJ-SSP is attached to the printing head. This setup can scan across flat and even surfaces in a predefined pattern ("static sampling mode"). Uneven samples are automatically probed in "conductance sampling mode" where an electric potential is applied and measured at the probe. When the probe contacts the electrically grounded sample, the potential at the probe drops, which is used as a feedback signal to determine the optimal position of the probe for sampling each location. RESULTS: The applicability of the probe/sensing system was demonstrated by first examining the strawberry tissue using the "static sampling mode." Second, porcine tissue samples were profiled using the "conductance sampling mode." With minimal sample preparation, an area of 11 × 15 mm was profiled in less than 2 h. From the obtained results, adipose areas could be distinguished from non-adipose parts. The versatility of the approach was further demonstrated by directly sampling the bacteria colonies on agar and resected human kidney (intratumoral hemorrhage) specimens with thicknesses ranging from 1 to 4 mm. CONCLUSION: The LMJ-SSP in conjunction with a conductive feedback system is a powerful tool that allows for fast, reproducible, and automated assessment of uneven surfaces with minimal sample preparation. This setup could be used for perioperative assessment of tissue samples, food screening, and natural product discovery, among others.

GNE-064: A Potent, Selective, and Orally Bioavailable Chemical Probe for the Bromodomains of SMARCA2 and SMARCA4 and the Fifth Bromodomain of PBRM1.

Bromodomains are acetyllysine recognition domains present in a variety of human proteins. Bromodomains also bind small molecules that compete with acetyllysine, and therefore bromodomains have been targets for drug discovery efforts. Highly potent and selective ligands with good cellular permeability have been proposed as chemical probes for use in exploring the functions of many of the bromodomain proteins. We report here the discovery of a class of such inhibitors targeting the family VIII bromodomains of SMARCA2 (BRM) and SMARCA4 (BRG1), and PBRM1 (polybromo-1) bromodomain 5. We propose one example from this series, GNE-064, as a chemical probe for the bromodomains SMARCA2, SMARCA4, and PBRM1(5) with the potential for in vivo use.

Risk-Stratified Pathways for Cancer Survivorship Care: Insights from a Deliberative Multi-Stakeholder Consultation.

Risk-stratified pathways of survivorship care seek to optimize coordination between cancer specialists and primary care physicians based on the whole person needs of the individual. While the principle is supported by leading cancer institutions, translating knowledge to practice confronts a lack of clarity about the meaning of risk stratification, uncertainties around the expectations the model holds for different actors, and health system structures that impede communication and coordination across the care continuum. These barriers must be better understood and addressed to pave the way for future implementation. Recognizing that an innovation is more likely to be adopted when user experience is incorporated into the planning process, a deliberative consultation was held as a preliminary step to developing a pilot project of risk-stratified pathways for patients transitioning from specialized oncology teams to primary care providers. This article presents findings from the deliberative consultation that sought to understand the perspectives of cancer specialists, primary care physicians, oncology nurses, allied professionals, cancer survivors and researchers regarding the following questions: what does a risk stratified model of cancer survivorship care mean to care providers and users? What are the prerequisites for translating risk stratification into practice? What challenges are involved in establishing these prerequisites? The multi-stakeholder consultation provides empirical data to guide actions that support the development of risk-stratified pathways to coordinate survivorship care.

Detection of Opioids on Mail/Packages Using Open Port Interface Mass Spectrometry (OPI-MS).

Opioids (and their more potent synthetic analogues) are used therapeutically as effective pain killers; however, recreational use and consequent overdoses are implicated in the deaths of thousands of people across the world annually. Trafficking of opioids and other illegal drugs through international mail has become a significant challenge for law enforcement personnel. Hundreds of millions of letters are sorted by the U.S. and Canadian postal services every day. Chemical analysis of this immense volume of mail requires a very fast sampling/detection method. This work explores the use of real-time mass spectrometry analysis with the recently developed Open Port Interface (OPI) for acoustically dispensed nanoliter volume sample droplets, a type of liquid microjunction surface sampling probe, for rapid and easy non-intrusive detection of fentanyl, heroin, and oxycodone. The OPI coupled to mass spectrometry is a novel sample introduction method that allows the rapid analysis of sample surfaces without preparation or modification. Opioids on different packaging materials (e.g., paper, bubble wrap, Ziploc bags) were rapidly (<10 s) interrogated by the OPI, and the sensitivities of the method compared. Furthermore, an opioid surrogate (caffeine) could be facilely detected on envelopes after processing through postal services.

Scout-multiple reaction monitoring: A liquid chromatography tandem mass spectrometry approach for multi-residue pesticide analysis without time scheduling.

In this work, an innovative method is described for multi-residue pesticide analysis by liquid chromatography coupled to targeted mass spectrometry, called "Scout-MRM, this new acquisition mode relies on the monitoring by either endogenous or spiked Scout compounds, hence fully releasing the monitoring of target molecules from time scheduling. As a proof of concept, a Scout-MRM method was built where 5 transitions groups tracking a total of 191 pesticides where successively triggered under the control of 5 spiked-in deuterated pesticides. As expected from its retention time independency, Scout-MRM demonstrates strong detection robustness towards modifications of gradient parameters, as well as easy method transfer between distinct analytical platforms with nearly 100% recovery after a single run. Finally, Scout-MRM was used for the multi-residue screening and quantification of pesticides in real surface water samples, by applying an external calibration procedure and comparing it with classical scheduled reaction monitoring methods.

The Integrated Psychological Treatment for Individuals With Schizophrenia in Correctional and Forensic Psychiatric Settings: An Implementation Study.

This study aims to identify the factors that need to be considered in the implementation of the integrated psychological treatment (IPT), a cognitive-behavioral group approach for individuals with schizophrenia, in correctional and forensic psychiatric settings. To meet this objective, a multiple case study (n = 2) was conducted. Stakeholders involved in the implementation were interviewed. Findings showed that IPT is relevant and can be delivered in correctional and forensic psychiatric settings. However, several issues impeded its implementation and sustainability, some of which were more specific to secure settings (i.e., the legal and clinical picture of the clientele, security requirements, interdisciplinary collaboration, and recognition of IPT in national correctional programming). Adaptations and additional considerations for implementation of IPT in correctional and forensic psychiatric settings are discussed further.

Identification of potent, selective KDM5 inhibitors.

This communication describes the identification and optimization of a series of pan-KDM5 inhibitors derived from compound 1, a hit initially identified against KDM4C. Compound 1 was optimized to afford compound 20, a 10nM inhibitor of KDM5A. Compound 20 is highly selective for the KDM5 enzymes versus other histone lysine demethylases and demonstrates activity in a cellular assay measuring the increase in global histone 3 lysine 4 tri-methylation (H3K4me3). In addition compound 20 has good ADME properties, excellent mouse PK, and is a suitable starting point for further optimization.

Fragment-Based Discovery of a Selective and Cell-Active Benzodiazepinone CBP/EP300 Bromodomain Inhibitor (CPI-637).

CBP and EP300 are highly homologous, bromodomain-containing transcription coactivators involved in numerous cellular pathways relevant to oncology. As part of our effort to explore the potential therapeutic implications of selectively targeting bromodomains, we set out to identify a CBP/EP300 bromodomain inhibitor that was potent both in vitro and in cellular target engagement assays and was selective over the other members of the bromodomain family. Reported here is a series of cell-potent and selective probes of the CBP/EP300 bromodomains, derived from the fragment screening hit 4-methyl-1,3,4,5-tetrahydro-2H-benzo[b][1,4]diazepin-2-one.

Discovery of Benzotriazolo[4,3-d][1,4]diazepines as Orally Active Inhibitors of BET Bromodomains.

Inhibition of the bromodomains of the BET family, of which BRD4 is a member, has been shown to decrease myc and interleukin (IL) 6 in vivo, markers that are of therapeutic relevance to cancer and inflammatory disease, respectively. Herein we report substituted benzo[b]isoxazolo[4,5-d]azepines and benzotriazolo[4,3-d][1,4]diazepines as fragment-derived novel inhibitors of the bromodomain of BRD4. Compounds from these series were potent and selective in cells, and subsequent optimization of microsomal stability yielded representatives that demonstrated dose- and time-dependent reduction of plasma IL-6 in mice.

Identification of a Benzoisoxazoloazepine Inhibitor (CPI-0610) of the Bromodomain and Extra-Terminal (BET) Family as a Candidate for Human Clinical Trials.

In recent years, inhibition of the interaction between the bromodomain and extra-terminal domain (BET) family of chromatin adaptors and acetyl-lysine residues on chromatin has emerged as a promising approach to regulate the expression of important disease-relevant genes, including MYC, BCL-2, and NF-κB. Here we describe the identification and characterization of a potent and selective benzoisoxazoloazepine BET bromodomain inhibitor that attenuates BET-dependent gene expression in vivo, demonstrates antitumor efficacy in an MV-4-11 mouse xenograft model, and is currently undergoing human clinical trials for hematological malignancies (CPI-0610).

Development of methyl isoxazoleazepines as inhibitors of BET.

In this report we detail the evolution of our previously reported thiophene isoxazole BET inhibitor chemotype exemplified by CPI-3 to a novel bromodomain selective chemotype (the methyl isoxazoleazepine chemotype) exemplified by carboxamide 23. The methyl isoxazoleazepine chemotype provides potent inhibition of the bromodomains of the BET family, excellent in vivo PK across species, low unbound clearance, and target engagement in a MYC PK-PD model.

A practical synthesis of indoles via a Pd-catalyzed C-N ring formation.

A method for the synthesis of N-functionalized C2-/C3-substituted indoles via Pd-catalyzed C-N bond coupling of halo-aryl enamines is described. The general strategy utilizes a variety of amines and ß-keto esters which are elaborated into halo-aryl enamines as latent precursors to indoles. The preferred conditions comprising the RuPhos precatalyst and RuPhos in the presence of NaOMe in 1,4-dioxane tolerate a variety of substituents and are scalable for the construction of indoles in multigram quantities.

Discovery, Design, and Optimization of Isoxazole Azepine BET Inhibitors.

The identification of a novel series of small molecule BET inhibitors is described. Using crystallographic binding modes of an amino-isoxazole fragment and known BET inhibitors, a structure-based drug design effort lead to a novel isoxazole azepine scaffold. This scaffold showed good potency in biochemical and cellular assays and oral activity in an in vivo model of BET inhibition.

Nicotinic acids: liver-targeted SCD inhibitors with preclinical anti-diabetic efficacy.

An in vitro screening protocol was used to transform a systemically-distributed SCD inhibitor into a liver-targeted compound. Incorporation of a key nicotinic acid moiety enables molecular recognition by OATP transporters, as demonstrated by uptake studies in transfected cell lines, and likely serves as a critical component of the observed liver-targeted tissue distribution profile. Preclinical anti-diabetic oGTT efficacy is demonstrated with nicotinic acid-based, liver-targeting SCD inhibitor 10, and studies with a close-structural analog devoid of SCD1 activity, suggest this efficacy is a result of on-target activity.

Chemical noise reduction via mass spectrometry and ion/ion charge inversion: amino acids.

Charge inversion ion/ion reactions can provide a significant reduction in chemical noise associated with mass spectra derived from complex mixtures for species composed of both acidic and basic sites, provided the ions derived from the matrix largely undergo neutralization. Amino acids constitute an important class of amphoteric compounds that undergo relatively efficient charge inversion. Precipitated plasma constitutes a relatively complex biological matrix that yields detectable signals at essentially every mass-to-charge value over a wide range. This chemical noise can be dramatically reduced using multiply charged reagent ions that can invert the charge of species amenable to the transfer of multiple charges upon a single interaction and by detecting product ions of opposite polarity. The principle is illustrated here with amino acids present in precipitated plasma subjected to ionization in the positive mode, reaction with anions derived from negative nanoelectrospray ionization of poly (amido amine) dendrimer generation 3.5, and mass analysis in the negative ion mode.

New indole amide derivatives as potent CRTH2 receptor antagonists.

A new series of indole amide acting as hCRTH2 receptor ligands had been explored and are described herein. Several amide derivatives displaying low nanomolar activity in hCRTH2 binding and whole blood assays were identified. They were found to behave as a full antagonists, exhibiting good selectivity over related prostaglandin receptors. Also, prototypical compounds in this novel series which displayed acceptable CYP profiles and were orally bioavailable in rats were identified.

Charge inversion via concurrent cation and anion transfer: application to corticosteroids.

A novel charge inversion process that involves the removal of an excess cation from an analyte ion and the transfer of an anion to the neutral analyte in a single ion/ion encounter is described. Polyamidoamine (PAMAM) half-generation dendrimer anions that contain small anions, such as the chloride ion, were used as charge inversion reagents. Several competing processes can occur that include removal of the cation to neutralize the analyte, the removal of the excess cation and an additional proton to yield the deprotonated molecule, or removal of the excess cation and transfer of a small anion to the analyte. For the latter process to dominate, several requirements for both the reagent anion and the analyte cation must be met. The reagent anion must form multiply charged anions and must be able to incorporate one or more small anions for transfer. The analyte must have no strongly acidic sites as well as a relatively high affinity for small anion attachment. The PAMAM dendrimer anions must meet the conditions for the reagent anions and the cations of the corticosteroids meet the conditions for the analyte. The estrogenic steroid estrone, on the other hand, does not meet the requirements and, as a result, is largely neutralized when reacted with the reagent anions. This reaction, therefore, is highly selective and might serve as a useful reaction for the screening of appropriate analytes.

Azaindoles as potent CRTH2 receptor antagonists.

A new class of 7-azaindole analogs of MK-7246 as potent and selective CRTH2 antagonists is reported. The SAR leading to the identification of the optimal azaindole regioisomer as well as the pharmacokinetics and off-target activities of the most potent antagonists are disclosed.

Discovery of MK-7246, a selective CRTH2 antagonist for the treatment of respiratory diseases.

In this manuscript we wish to report the discovery of MK-7246 (4), a potent and selective CRTH2 (DP2) antagonist. SAR studies leading to MK-7246 along with two synthetic sequences enabling the preparation of this novel class of CRTH2 antagonist are reported. Finally, the pharmacokinetic and metabolic profile of MK-7246 is disclosed.

Synthesis and biological activity of a potent and orally bioavailable SCD inhibitor (MF-438).

A series of stearoyl-CoA desaturase 1 (SCD1) inhibitors were developed. Investigations of enzyme potency and metabolism led to the identification of the thiadiazole-pyridazine derivative MF-438 as a potent SCD1 inhibitor. MF-438 exhibits good pharmacokinetics and metabolic stability, thereby serving as a valuable tool for further understanding the role of SCD inhibition in biological and pharmacological models of diseases related to metabolic disorders.