Understanding olfactory and behavioural responses to dietary cues in age-1 lake sturgeon Acipenser fulvescens.

Detection of environmental cues is essential for all vertebrates and is typically established by the olfactory epithelium and olfactory sensory neurons (OSNs). In fishes, microvillous and ciliated OSNs are the principal types, typically detecting amino acids and bile salts, respectively. Activation of OSN receptors by specific ligands initiate downstream signal processing often leading to behavioural responses. In this study we used electrophysiological and behavioural techniques to evaluate olfactory detection and behaviour in juvenile lake sturgeon Acipenser fulvescens in response to hatchery- and natural dietary cues. We hypothesized that electro-olfactogram (EOG) and behavioural responses would be dependent on diet type. We predicted that inhibition of the phospholipase C/inositol 1,4,5-triphosphate (PLC/IP3) secondary transduction pathway would reduce EOG responses to dietary cues and, inhibition of the adenylyl cyclase/adenosine 3,5-cyclic monophosphate (cAMP) pathway, would have no effect. Furthermore, we predicted a strong EOG response would be manifested in a change in behaviour. We observed that both the PLC/IP3 and cAMP pathways were significantly involved in the detection of dietary cues. However, EOG responses did not manifest to behavioural responses, although the foraging activity to the hatchery cue was significantly greater compared to the control. Our results support the notion that lake sturgeon raised in a hatchery and fed a commercial pelleted diet may become accustomed to it prior to release into the wild. Further, this study suggests that, in conservation aquaculture settings, lake sturgeon should be exposed to natural dietary cues prior to release as one strategy to promote food recognition.

Novel bacterial topoisomerase inhibitors: unique targeting activities of amide enzyme-binding motifs for tricyclic analogs.

Antimicrobial resistance has made a sizeable impact on public health and continues to threaten the effectiveness of antibacterial therapies. Novel bacterial topoisomerase inhibitors (NBTIs) are a promising class of antibacterial agents with a unique binding mode and distinct pharmacology that enables them to evade existing resistance mechanisms. The clinical development of NBTIs has been plagued by several issues, including cardiovascular safety. Herein, we report a sub-series of tricyclic NBTIs bearing an amide linkage that displays promising antibacterial activity, potent dual-target inhibition of DNA gyrase and topoisomerase IV (TopoIV), as well as improved cardiovascular safety and metabolic profiles. These amide NBTIs induced both single- and double-strand breaks in pBR322 DNA mediated by Staphylococcus aureus DNA gyrase, in contrast to prototypical NBTIs that cause only single-strand breaks. Unexpectedly, amides 1a and 1b targeted human topoisomerase IIα (TOP2α) causing both single- and double-strand breaks in pBR322 DNA, and induced DNA strand breaks in intact human leukemia K562 cells. In addition, anticancer drug-resistant K/VP.5 cells containing decreased levels of TOP2α were cross-resistant to amides 1a and 1b. Together, these results demonstrate broad spectrum antibacterial properties of selected tricyclic NBTIs, desirable safety profiles, an unusual ability to induce DNA double-stranded breaks, and activity against human TOP2α. Future work will be directed toward optimization and development of tricyclic NBTIs with potent and selective activity against bacteria. Finally, the current results may provide an additional avenue for development of selective anticancer agents.

Actions of a Novel Bacterial Topoisomerase Inhibitor against Neisseria gonorrhoeae Gyrase and Topoisomerase IV: Enhancement of Double-Stranded DNA Breaks.

Novel bacterial topoisomerase inhibitors (NBTIs) are an emerging class of antibacterials that target gyrase and topoisomerase IV. A hallmark of NBTIs is their ability to induce gyrase/topoisomerase IV-mediated single-stranded DNA breaks and suppress the generation of double-stranded breaks. However, a previous study reported that some dioxane-linked amide NBTIs induced double-stranded DNA breaks mediated by Staphylococcus aureus gyrase. To further explore the ability of this NBTI subclass to increase double-stranded DNA breaks, we examined the effects of OSUAB-185 on DNA cleavage mediated by Neisseria gonorrhoeae gyrase and topoisomerase IV. OSUAB-185 induced single-stranded and suppressed double-stranded DNA breaks mediated by N. gonorrhoeae gyrase. However, the compound stabilized both single- and double-stranded DNA breaks mediated by topoisomerase IV. The induction of double-stranded breaks does not appear to correlate with the binding of a second OSUAB-185 molecule and extends to fluoroquinolone-resistant N. gonorrhoeae topoisomerase IV, as well as type II enzymes from other bacteria and humans. The double-stranded DNA cleavage activity of OSUAB-185 and other dioxane-linked NBTIs represents a paradigm shift in a hallmark characteristic of NBTIs and suggests that some members of this subclass may have alternative binding motifs in the cleavage complex.

Antibacterial activity of novel bacterial topoisomerase inhibitors against key veterinary pathogens.

Multidrug-resistant bacteria infect companion animals and livestock in addition to their devastating impact on human health. Novel Bacterial Topoisomerase Inhibitors (NBTIs) with excellent activity against Gram-positive bacteria have previously been identified as promising new antibacterial agents. Herein, we evaluate the antibacterial activity of these NBTIs against a variety of important veterinary pathogens and demonstrate outstanding in vitro activity, especially against staphylococci.

Extending the data collection from a clinical trial: The Extended Salford Lung Study research cohort.

The Extended Salford Lung Study (Ext-SLS) is an extension of the Salford Lung Studies (SLS) in asthma and chronic obstructive pulmonary disease (COPD) through retrospective and prospective collection of patient-level electronic health record (EHR) data. We compared the Ext-SLS cohort with the SLS intention-to-treat populations using descriptive analyses to determine if the strengths (e.g. randomization) of the clinical trial were maintained in the new cohort. Historical and patient-reported outcome data were captured from asthma-/COPD-specific questionnaires (e.g., Asthma Control Test [ACT]/COPD Assessment Test [CAT]). The Ext-SLS included 1147 participants (n = 798, SLS asthma; n = 349, SLS COPD). Of participants answering the ACT, 39% scored <20, suggesting poorly controlled asthma. For COPD, 61% of participants answering the CAT scored ≥21, demonstrating a high disease burden. Demographic/clinical characteristics of the cohorts were similar at SLS baseline. EHR data provided a long-term view of participants' disease, and questionnaires provided information not typically captured. The Ext-SLS cohort is a valuable resource for respiratory research, and ongoing prospective data collection will add further value and ensure the Ext-SLS is an important source of patient-level information on obstructive airways disease.

Dioxane-Linked Novel Bacterial Topoisomerase Inhibitors Exhibit Bactericidal Activity against Planktonic and Biofilm Staphylococcus aureus In Vitro.

The development of novel treatments for Staphylococcus aureus infections remains a high priority worldwide. We previously reported compounds 0147 and 0186, novel bacterial topoisomerase inhibitors (NBTIs) with potent antibacterial activity against S. aureus, including methicillin-resistant S. aureus. Here, we further investigated the in vitro activity of 0147 and 0186 against S. aureus ATCC 29213. Both compounds demonstrated bactericidal activity against planktonic and biofilm S. aureus, which then translated into significant inhibition of biofilm formation. Combinations of NBTIs and glycopeptides yielded indifferent interactions against planktonic S. aureus, but several had synergistic effects against S. aureus biofilms. This work reinforces the potential of NBTIs as future therapeutics for S. aureus infections. IMPORTANCE The pathogen Staphylococcus aureus contributes substantially to infection-related mortality. Biofilms render bacteria more recalcitrant to antibacterial therapy. The manuscript describes the potent activity of a new class of antibacterial agents against both planktonic and biofilm populations of Staphylococcus aureus.

Serendipitous Discovery of a Competitive Inhibitor of FraB, a Salmonella Deglycase and Drug Target.

Although salmonellosis, an infectious disease, is a significant global healthcare burden, there are no Salmonella-specific vaccines or therapeutics for humans. Motivated by our finding that FraB, a Salmonella deglycase responsible for fructose-asparagine catabolism, is a viable drug target, we initiated experimental and computational efforts to identify inhibitors of FraB. To this end, our recent high-throughput screening initiative yielded almost exclusively uncompetitive inhibitors of FraB. In parallel with this advance, we report here how a separate structural and computational biology investigation of FrlB, a FraB paralog, led to the serendipitous discovery that 2-deoxy-6-phosphogluconate is a competitive inhibitor of FraB (KI ~ 3 µM). However, this compound was ineffective in inhibiting the growth of Salmonella in a liquid culture. In addition to poor uptake, cellular metabolic transformations by a Salmonella dehydrogenase and different phosphatases likely undermined the efficacy of 2-deoxy-6-phosphogluconate in live-cell assays. These insights inform our ongoing efforts to synthesize non-hydrolyzable/-metabolizable analogs of 2-deoxy-6-phosphogluconate. We showcase our findings largely to (re)emphasize the role of serendipity and the importance of multi-pronged approaches in drug discovery.

Supporting successful recruitment in a randomized control trial comparing clinic and home-based exercise among adults with multiple sclerosis.

BACKGROUND: The Tele- Exercise and Multiple Sclerosis (TEAMS) study, funded by the Patient Centered Outcome Research Institute (PCORI), is a pragmatic, cluster randomized controlled trial aimed at comparing the effectiveness of a 12-week complementary and alternative medicine (CAM) program for people with multiple sclerosis (MS) delivered by a therapist at a clinic and the same program initiated by the participant at home using a tablet and pre-recorded videos. The 20-session CAM program consists of yoga, Pilates and dual tasking exercises. The study aimed to enroll 820 participants with MS living in Alabama, Mississippi and Tennessee. MAIN BODY: The information provided in this paper describes the strategies that led to the largest randomized controlled exercise trial ever conducted for people with multiple sclerosis. Specifically, the paper presents the result of incorporating stakeholder engagement, a novel participant recruitment method, to produce a successful recruitment outcome for a comparative effectiveness randomized controlled trial. This study used three tiers of engagement: panel members (9 members), clinical partners (88 occupational and physical therapists), and community organizations (6 non-profits). CONCLUSION: Engagement of the stakeholder panel, clinical partners and community organizations led to interest of over 1700 people with MS across three states in the Deep South (final enrollment was n = 837). The diversity of our stakeholder groups and their extensive reach into various communities were a critical aspect for achieving our target sample size. The recruitment numbers reflect the importance of involving multiple stakeholder groups at project inception, developing relationships over time, utilizing member strengths, and monitoring their engagement on a regular basis to ensure a meaningful experience for all involved. TRIAL REGISTRATION: NCT03117881. Registered 18 April 2017, https://clinicaltrials.gov/ct2/show/NCT03117881?term=tele+rehabilitation&cond=Multiple+Sclerosis&cntry=US&state=US%3AAL&draw=2&rank=1 .

The Tele Exercise and Multiple Sclerosis (TEAMS) study has been able to successfully screen over 1700 people with multiple sclerosis (MS) across three southern states (Alabama, Mississippi, Tenessee) largely due to the advice and input that the research team received from a stakeholder panel, clinic partners and community organizations. These groups met before the study was submitted for funding to the Patient-Centered Outcome Research Institute (PCORI), and was awarded in 2017. These engagement stages and framework established early in the study process were instrumental in generating strong enthusiasm for the study among various MS constituency groups. The feedback from our stakeholders, clinic partners, and community organizations led to the creation of a variety of recruitment methods (print material, email, social media, attendance at events, and health fairs) to connect with potential participants in a setting convenient within each location. In approximately 26 months, the study enrolled 837 participants with MS and baseline tested 759 individuals who participated in a rehabilitative exercise program at either a clinic site or in their home using a tablet that they were given (and kept) which included a set of preloaded videos. All milestones established by PCORI and research staff were met, leading to the largest exercise trail ever conducted with people with MS.

1,3-Dioxane-Linked Novel Bacterial Topoisomerase Inhibitors: Expanding Structural Diversity and the Antibacterial Spectrum.

Antibacterial resistance continues its devastation of available therapies. Novel bacterial topoisomerase inhibitors (NBTIs) offer one solution to this critical issue. Two series of amine NBTIs bearing tricyclic DNA-binding moieties as well as amide NBTIs with a bicyclic DNA-binding moiety were synthesized and evaluated against methicillin-resistant Staphylococcus aureus (MRSA). Additionally, these compounds and a series of bicyclic amine analogues displayed high activity against susceptible and drug-resistant Neisseria gonorrhoeae, expanding the spectrum of these dioxane-linked NBTIs.

Aryl Methyl Ketones: Versatile Synthons in the Synthesis of Heterocyclic Compounds.

The synthesis of aromatic heterocycles has attracted substantial attention due to the abundance of these heterocycles in drug molecules, natural products, and other compounds of biological interest. Accordingly, there is a demand for straightforward synthetic protocols toward such compounds using readily available starting materials. In the past decade, there have been substantial developments in heterocycle synthesis, especially in metal-catalyzed and iodine-assisted approaches. This graphical review focuses on notable reactions from the past decade using aryl and heteroaryl methyl ketones as starting materials, including representative reaction mechanisms.

Optimization of TopoIV Potency, ADMET Properties, and hERG Inhibition of 5-Amino-1,3-dioxane-Linked Novel Bacterial Topoisomerase Inhibitors: Identification of a Lead with In Vivo Efficacy against MRSA.

Novel bacterial topoisomerase inhibitors (NBTIs) are among the most promising new antibiotics in preclinical/clinical development. We previously reported dioxane-linked NBTIs with potent antistaphylococcal activity and reduced hERG inhibition, a key safety liability. Herein, polarity-focused optimization enabled the delineation of clear structure-property relationships for both microsomal metabolic stability and hERG inhibition, resulting in the identification of lead compound 79. This molecule demonstrates potent antibacterial activity against diverse Gram-positive pathogens, inhibition of both DNA gyrase and topoisomerase IV, a low frequency of resistance, a favorable in vitro cardiovascular safety profile, and in vivo efficacy in a murine model of methicillin-resistant Staphylococcus aureus infection.

Key Learnings from Running an Extension Study to a Real-World Effectiveness Trial: The Extended Salford Lung Study.

INTRODUCTION: The Salford Lung Studies (SLS) were real-world randomised controlled trials set within UK primary care that assessed the effectiveness and safety of initiating once-daily fluticasone furoate/vilanterol versus continuing usual care in patients with chronic obstructive pulmonary disease or asthma. Data were collected for a relatively short period, limiting the study of long-term outcomes. To broaden the capture of SLS patients' data, we undertook the Extended SLS (Ext-SLS), aiming to better understand the patient disease journey and the effects of treatment in a real-world setting, through collection of patient-level data. Here, we present study design information and the challenges and learnings gathered in creating the Ext-SLS. METHODS: The Ext-SLS was intended to augment the SLS by collecting retrospective and prospective (up to 10 years from consent) primary and secondary care electronic health record (EHR) data and patient questionnaires. After ethics approval, general practitioners (GPs) obtained consent from SLS patients remotely (mean 3.2 years post-SLS completion). To facilitate GPs identifying eligible patients, a novel EHR-based approach flagged SLS patients who were alive and registered with their original GP. An automated system sent consent forms/questionnaires to patients. Medical data were collected via EHRs; primary care data were extracted from GPs' systems whilst secondary care data were sourced from the UK NHS. RESULTS: Of the 75 GP sites from the SLS, 35 (47%) declined Ext-SLS participation leaving 4158 potentially eligible patients; 1169 (28%) patients were excluded as GPs could not confirm them as SLS participants or due to incapacity. Of 2989 patients invited, 1189 (40%) consented. CONCLUSIONS: Developing an EHR-based trial extension was achieved, with reasonable consent rates amongst invited patients. The resulting Ext-SLS is a unique and valuable research resource. Leveraging EHRs and technology reduced GP burden, facilitating participation. Initiation of extension studies prior to study close-out may help increase GP and patient participation.

A review of 1,2-dibromo-4-(1,2-dibromoethyl)cyclohexane in the environment and assessment of its persistence, bioaccumulation and toxicity.

Following the ban of many historically-used flame retardants (FRs), numerous replacement chemicals have been produced and used in products, with some being identified as environmental contaminants. One of these replacement flame retardants is 1,2-dibromo-4-(1,2-dibromoethyl)-cyclohexane (DBE-DBCH; formerly abbreviated as TBECH), which to date has not been identified for risk assessment and potential regulation. DBE-DBCH technical mixtures consist largely of α- and ß-diastereomers with trace amounts of γ- and δ-DBE-DBCH. The α- and ß-isomers are known contaminants in various environmental media. While current global use and production volumes of DBE-DBCH are unknown, recent studies identified that DBE-DBCH concentrations were among the highest of the measured bromine-based FRs in indoor and urban air in Europe. Yet our mass balance fugacity model and modeling of the physical-chemical properties of DBE-DBCH estimated only 1% partitioning to air with a half-life of 2.2 d atmospherically. In contrast, our modeling characterized DBE-DBCH adsorbing strongly to suspended particulates in the water column (~12%), settling onto sediment (2.5%) with minimal volatilization, but with most partitioning and adsorbing strongly to soil (~85%) with negligible volatilization and slow biodegradation. Our modeling further predicted that organisms would be exposed to DBE-DBCH through partitioning from the dissolved aquatic phase, soil, and by diet, and given its estimated logKow (5.24) and a half-life of 1.7 d in fish, DBE-DBCH is expected to bioaccumulate into lipophilic tissues. Low concentrations of DBE-DBCH are commonly measured in biota and humans, possibly because evidence suggests rapid metabolism. Yet toxicological effects are evident at low exposure concentrations: DBE-DBCH is a proven endocrine disruptor of sex and thyroid hormone pathways, with in vivo toxic effects on reproductive, metabolic, and other endpoints. The objectives of this review are to identify the current state of knowledge concerning DBE-DBCH through an evaluation of its persistence, potential for bioaccumulation, and characterization of its toxicity, while identifying areas for future research.

Dioxane-Linked Amide Derivatives as Novel Bacterial Topoisomerase Inhibitors against Gram-Positive Staphylococcus aureus.

In recent years, novel bacterial topoisomerase inhibitors (NBTIs) have been developed as future antibacterials for treating multidrug-resistant bacterial infections. A series of dioxane-linked NBTIs with an amide moiety has been synthesized and evaluated. Compound 3 inhibits DNA gyrase, induces the formation of single strand breaks to bacterial DNA, and achieves potent antibacterial activity against a variety of Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). Optimization of this series of analogues led to the discovery of a subseries of compounds (22-25) with more potent anti-MRSA activity, dual inhibition of DNA gyrase and topoisomerase IV, and the ability to induce double strand breaks through inhibition of S. aureus DNA gyrase.

Novel bacterial topoisomerase inhibitors derived from isomannide.

A series of Novel Bacterial Topoisomerase Inhibitors (NBTIs) employing a linker derived from isomannide were synthesized and evaluated. Reduced hERG inhibition was observed compared to structure-matched analogues with different linkers, and compound 6 showed minimal proarrhythmic potential using an in vitro panel of cardiac ion channels. Compound 6 also displayed excellent activity against fluoroquinolone-resistant MRSA (MIC90 = 2 µg/mL) and other Gram-positive pathogens.

1,3-Dioxane-Linked Bacterial Topoisomerase Inhibitors with Enhanced Antibacterial Activity and Reduced hERG Inhibition.

The development of new therapies to treat methicillin-resistant Staphylococcus aureus (MRSA) is needed to counteract the significant threat that MRSA presents to human health. Novel inhibitors of DNA gyrase and topoisomerase IV (TopoIV) constitute one highly promising approach, but continued optimization is required to realize the full potential of this class of antibiotics. Herein, we report further studies on a series of dioxane-linked derivatives, demonstrating improved antistaphylococcal activity and reduced hERG inhibition. A subseries of analogues also possesses enhanced inhibition of the secondary target, TopoIV.

Identification of halogenated polycyclic aromatic hydrocarbons in biological samples from Alberta Oil-Sands Region.

Halogenated polycyclic aromatic hydrocarbons (HPAHs) were identified in biological samples from the Alberta Oil-Sands Region (AOSR) using gas chromatography coupled with high-resolution time-of-flight mass spectrometry (GC-HRTOF-MS) at a resolving power of 25,000. Knowledge of the electron ionization (EI) fragmentation behavior of individual HPAH isomers, achieved by injecting authentic standards in full-scan MS mode, was paramount in identifying a suite of HPAHs in samples from the AOSR. Confirmation of compounds in biological samples was based on the measured mass accuracy (±3 ppm) of 2 characteristic ions prominent in the EI mass spectra of each compound. Numerous compounds were detected in the high resolution total ion chromatogram in liver extracts of 4 biological species from the AOSR: river otter (Lontra Canadensis), northern pike (Esox lucius), lake whitefish (Coregonus clupeaformis) and snails (Gastropod sp.) many of which remain unidentified. Careful examination of the high-resolution accurate mass data suggests that dichloro-anthracene/phenanthrene, bromo-anthracene/phenanthrene and dibromo-fluorene were present in the biological samples. Lipid corrected concentrations of dichloro-PAHs were estimated to be 16.3 ±â€¯11.4 (n = 4) and 5.5 (n = 1) ng/g in lake whitefish and river otter, respectively. Concentrations of mono-bromo-PAHs were an order of magnitude greater in snails (170.5 ng/g) than in northern pike (12.5 ng/g) while concentrations of dibromo-PAHs were 4 times greater in snails than in northern pike. The detection of these compounds in biota implies that these compounds are bioaccumulative. The liver-based biomagnification factor of the dichloro-PAH congener in the river otter/lake whitefish feeding relationship is much smaller than 1 implying that this compound does not biomagnify.

Ionic mechanisms underlying tonic and burst firing behavior in subfornical organ neurons: a combined experimental and modeling study.

Subfornical organ (SFO) neurons exhibit heterogeneity in current expression and spiking behavior, where the two major spiking phenotypes appear as tonic and burst firing. Insight into the mechanisms behind this heterogeneity is critical for understanding how the SFO, a sensory circumventricular organ, integrates and selectively influences physiological function. To integrate efficient methods for studying this heterogeneity, we built a single-compartment, Hodgkin-Huxley-type model of an SFO neuron that is parameterized by SFO-specific in vitro patch-clamp data. The model accounts for the membrane potential distribution and spike train variability of both tonic and burst firing SFO neurons. Analysis of model dynamics confirms that a persistent Na+ and Ca2+ currents are required for burst initiation and maintenance and suggests that a slow-activating K+ current may be responsible for burst termination in SFO neurons. Additionally, the model suggests that heterogeneity in current expression and subsequent influence on spike afterpotential underlie the behavioral differences between tonic and burst firing SFO neurons. Future use of this model in coordination with single neuron patch-clamp electrophysiology provides a platform for explaining and predicting the response of SFO neurons to various combinations of circulating signals, thus elucidating the mechanisms underlying physiological signal integration within the SFO. NEW & NOTEWORTHY Our understanding of how the subfornical organ (SFO) selectively influences autonomic nervous system function remains incomplete but theoretically results from the electrical responses of SFO neurons to physiologically important signals. We have built a computational model of SFO neurons, derived from and supported by experimental data, which explains how SFO neurons produce different electrical patterns. The model provides an efficient system to theoretically and experimentally explore how changes in the essential features of SFO neurons affect their electrical activity.

Synthesis and anti-staphylococcal activity of novel bacterial topoisomerase inhibitors with a 5-amino-1,3-dioxane linker moiety.

Novel bacterial type II topoisomerase inhibitors (NBTIs) constitute a promising new class of antibacterial agents. We report a series of NBTIs with potent anti-staphylococcal activity and diminished hERG inhibition. Dioxane-linked compound 9 demonstrated MICs ≤1 µg/mL against both methicillin-susceptible (MSSA) and -resistant Staphylococcus aureus (MRSA), accompanied by reduced hERG inhibition as compared to cyclohexane- or piperidine-linked analogs.

Identification of N-{cis-3-[Methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]cyclobutyl}propane-1-sulfonamide (PF-04965842): A Selective JAK1 Clinical Candidate for the Treatment of Autoimmune Diseases.

Janus kinases (JAKs) are intracellular tyrosine kinases that mediate the signaling of numerous cytokines and growth factors involved in the regulation of immunity, inflammation, and hematopoiesis. As JAK1 pairs with JAK2, JAK3, and TYK2, a JAK1-selective inhibitor would be expected to inhibit many cytokines involved in inflammation and immune function while avoiding inhibition of the JAK2 homodimer regulating erythropoietin and thrombopoietin signaling. Our efforts began with tofacitinib, an oral JAK inhibitor approved for the treatment of rheumatoid arthritis. Through modification of the 3-aminopiperidine linker in tofacitinib, we discovered highly selective JAK1 inhibitors with nanomolar potency in a human whole blood assay. Improvements in JAK1 potency and selectivity were achieved via structural modifications suggested by X-ray crystallographic analysis. After demonstrating efficacy in a rat adjuvant-induced arthritis (rAIA) model, PF-04965842 (25) was nominated as a clinical candidate for the treatment of JAK1-mediated autoimmune diseases.