A Novel C-C Chemoattractant Cytokine (Chemokine) Receptor 6 (CCR6) Antagonist (PF-07054894) Distinguishes between Homologous Chemokine Receptors, Increases Basal Circulating CCR6+ T Cells, and Ameliorates Interleukin-23-Induced Skin Inflammation.

Blocking chemokine receptor C-C chemoattractant cytokine (chemokine) receptor (CCR) 6-dependent T cell migration has therapeutic promise in inflammatory diseases. PF-07054894 is a novel CCR6 antagonist that blocked only CCR6, CCR7, and C-X-C chemoattractant cytokine (chemokine) receptor (CXCR) 2 in a ß-arrestin assay panel of 168 G protein-coupled receptors. Inhibition of CCR6-mediated human T cell chemotaxis by (R)-4-((2-(((1,4-Dimethyl-1H-pyrazol-3-yl)(1-methylcyclopentyl)methyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)-3-hydroxy-N,N-dimethylpicolinamide (PF-07054894) was insurmountable by CCR6 ligand, C-C motif ligand (CCL) 20. In contrast, blockade of CCR7-dependent chemotaxis in human T cells and CXCR2-dependent chemotaxis in human neutrophils by PF-07054894 were surmountable by CCL19 and C-X-C motif ligand 1, respectively. [3H]-PF-07054894 showed a slower dissociation rate for CCR6 than for CCR7 and CXCR2 suggesting that differences in chemotaxis patterns of inhibition could be attributable to offset kinetics. Consistent with this notion, an analog of PF-07054894 with fast dissociation rate showed surmountable inhibition of CCL20/CCR6 chemotaxis. Furthermore, pre-equilibration of T cells with PF-07054894 increased its inhibitory potency in CCL20/CCR6 chemotaxis by 10-fold. The functional selectivity of PF-07054894 for inhibition of CCR6 relative to CCR7 and CXCR2 is estimated to be at least 50- and 150-fold, respectively. When administered orally to naïve cynomolgus monkeys, PF-07054894 increased the frequency of CCR6+ peripheral blood T cells, suggesting that blockade of CCR6 inhibited homeostatic migration of T cells from blood to tissues. PF-07054894 inhibited interleukin-23-induced mouse skin ear swelling to a similar extent as genetic ablation of CCR6. PF-07054894 caused an increase in cell surface CCR6 in mouse and monkey B cells, which was recapitulated in mouse splenocytes in vitro. In conclusion, PF-07054894 is a potent and functionally selective CCR6 antagonist that blocks CCR6-mediated chemotaxis in vitro and in vivo. SIGNIFICANCE STATEMENT: The chemokine receptor, C-C chemoattractant cytokine (chemokine) receptor 6 (CCR6) plays a key role in the migration of pathogenic lymphocytes and dendritic cells into sites of inflammation. (R)-4-((2-(((1,4-Dimethyl-1H-pyrazol-3-yl)(1-methylcyclopentyl)methyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)-3-hydroxy-N,N-dimethylpicolinamide (PF-07054894) is a novel CCR6 small molecule antagonist that illustrates the importance of binding kinetics in achieving pharmacological potency and selectivity. Orally administered PF-07054894 blocks homeostatic and pathogenic functions of CCR6, suggesting that it is a promising therapeutic agent for the treatment of a variety of autoimmune and inflammatory diseases.

Patterns and prevalence of cognitive dysfunction in systemic lupus erythematosus.

OBJECTIVE: Systemic lupus erythematosus (SLE) is a chronic autoimmune disease, in which cognitive dysfunction is common, but poorly understood. This study aims to characterize the prevalence and patterns of cognitive dysfunction in SLE. METHOD: SLE patients (n = 95) and demographically matched healthy controls (n = 48) underwent cross-sectional cognitive testing using the 1-hr conventional neuropsychological test battery recommended by the American College of Rheumatology for use in SLE. We used standard deviations (SD) from the healthy control group to define impairment. For each cognitive test we compared SLE and control groups using independent samples t-tests (or alternatives when needed). We performed cluster analysis using a machine learning algorithm to look for patterns of cognitive dysfunction. RESULTS: The SLE group performed significantly worse than healthy controls on every cognitive test. The largest differences were in the domains of verbal fluency, working memory and attention, while fine motor and psychomotor speed were the least affected domains. As expected, the prevalence of cognitive dysfunction varied depending on the SD cut-off used, with 49% of participants being >1.5 SD below the healthy control mean in at least two cognitive domains. Heat mapping showed variability in the pattern of dysfunction between individual patients and cluster analysis confirmed the presence of two clusters of patients, which were those significantly impaired versus those having preserved cognition. CONCLUSIONS: Cognitive dysfunction is common in SLE but markedly heterogeneous across both cognitive domains and across the SLE group. Cluster analysis supports the use of a binary definition of cognitive dysfunction in SLE.

Primary aldosteronism: an unsuspected culprit of hypertension in systemic lupus erythematosus?

Primary aldosteronism (PA) is the most common endocrine cause of secondary hypertension and is associated with a high risk of cardiovascular disease in the general population. Patients suffering from systemic lupus erythematosus (SLE), a multisystem and multifactorial autoimmune disease, experience a high burden of hypertension and cardiovascular disease. Importantly, cardiovascular disease is one of the leading causes of death in SLE. Very limited evidence suggests an increased proportion of autoimmune diseases such as SLE in patients with PA. However, studies evaluating the prevalence of PA in the SLE population are lacking. Despite the potential for curative or targeted treatments, guidelines for the management of hypertension in SLE do not currently recommend testing for PA. This review highlights PA as a potentially over-looked secondary cause of hypertension in SLE, and offers future directions in research to improve the detection of this highly modifiable cardiovascular risk factor in the SLE population.

The TACI receptor regulates T-cell-independent marginal zone B cell responses through innate activation-induced cell death.

Activation-induced cell death (AICD) plays a critical role in immune homeostasis and tolerance. In T-cell-dependent humoral responses, AICD of B cells is initiated by Fas ligand (FasL) on T cells, stimulating the Fas receptor on B cells. In contrast, T-cell-independent B cell responses involve innate-type B lymphocytes, such as marginal zone (MZ) B cells, and little is known about the mechanisms that control AICD during innate B cell responses to Toll-like receptor (TLR) activation. Here, we show that MZ B cells undergo AICD in response to TLR4 activation in vivo. The transmembrane activator, calcium modulator, and cyclophilin ligand interactor (TACI) receptor and TLR4 cooperate to upregulate expression of both FasL and Fas on MZ B cells and also to repress inhibitors of Fas-induced apoptosis signaling. These findings demonstrate an unappreciated role for TACI and its ligands in the regulation of AICD during T-cell-independent B cell responses.

The advantages of describing covalent inhibitor in vitro potencies by IC50 at a fixed time point. IC50 determination of covalent inhibitors provides meaningful data to medicinal chemistry for SAR optimization.

Recent years have seen a resurgence in drug discovery efforts aimed at the identification of covalent inhibitors which has led to an explosion of literature reports in this area and most importantly new approved therapies. These reports and breakthroughs highlight the significant investments made across the industry in SAR campaigns to optimize inhibitors. The potency of covalent inhibitors is generally considered to be more accurately described by the time-independent kinetic parameter kinact/Ki rather than a by a simple IC50 since the latter is a time-dependent parameter. Enzyme substrate concentrations are an additional important factor to consider when attempting to translate parameters derived from enzymology experiments to phenotypic behavior in a physiologically relevant cell-based system. Theoretical and experimental investigations into the relationship between IC50, time, substrate concentration and Kinact/Ki provided us with an effective approach to provide meaningful data for SAR optimization. The data we generated for our JAK3 irreversible covalent inhibitor program using IC50 values provided by enzyme assays with long incubations (>1h) coupled with physiological substrate concentration provided the medicinal chemist with optimal information in a rapid and efficient manner. We further document the wide applicability of this method by applying it to other enzymes systems where we have run covalent inhibitor programs.

Design and optimization of a series of 4-(3-azabicyclo[3.1.0]hexan-3-yl)pyrimidin-2-amines: Dual inhibitors of TYK2 and JAK1.

Herein, we disclose a new series of TYK2/ JAK1 inhibitors based upon a 3.1.0 azabicyclic substituted pyrimidine scaffold. We illustrate the use of structure-based drug design for the initial design and subsequent optimization of this series of compounds. One advanced example 19 met program objectives for potency, selectivity and ADME, and demonstrated oral activity in the adjuvant-induced arthritis rat model.

Effect of storage duration on cytokine stability in human serum and plasma.

Quantification of analytes such as cytokines in serum samples is intrinsic to translational research in immune diseases. Optimising pre-analytical conditions is critical for ensuring study quality, including evaluation of cytokine stability. We aimed to evaluate the effect on cytokine stability of storage duration prior to freezing of serum, and compare to plasma samples obtained from patients with systemic lupus erythematosus (SLE). Protein stability was analysed by simultaneously quantifying 18 analytes using a custom multi-analyte profile in SLE patient serum and plasma samples that had been prospectively stored at 4 °C for pre-determined periods between 0 and 30 days, prior to freezing. Six analytes were excluded from analysis, because most tested samples were above or below the limit of detection. Amongst the 12 analysed proteins, 11 did not show significant signal degradation. Significant signal degradation was observed from the fourth day of storage for a single analyte, CCL19. Proteins levels were more stable in unseparated serum compared to plasma for most analytes, with the exception of IL-37 which appears slightly more stable in plasma. Based on this, a maximum 3 days of storage at 4 °C for unseparated serum samples is recommended for biobanked samples intended for cytokine analysis in studies of human immune disease.

Roles of ligands from the TNF superfamily in B cell development, function, and regulation.

Most ligands from the tumour necrosis factor (TNF) superfamily play very important roles in the immune system, and particularly so in B lymphocyte biology. TNF ligands are essential to many aspects of normal B cell biology from development in the bone marrow to maturation in the periphery as well as for activation and differentiation into germinal centre, memory or plasma cells. TNF ligands also influence other aspects of B cell biology such as their ability to present antigens or regulate immune responses. Importantly, inadequate regulation of many TNF ligands is associated with B cell disorders including autoimmunity and cancers. As a result, inhibitors of a number of TNF ligands have been tested in the clinic, with some becoming very successful approved treatments alleviating B cell-mediated pathologies.

Small molecule screening in human induced pluripotent stem cell-derived terminal cell types.

A need for better clinical outcomes has heightened interest in the use of physiologically relevant human cells in the drug discovery process. Patient-specific human induced pluripotent stem cells may offer a relevant, robust, scalable, and cost-effective model of human disease physiology. Small molecule high throughput screening in human induced pluripotent stem cell-derived cells with the intent of identifying novel therapeutic compounds is starting to influence the drug discovery process; however, the use of these cells presents many high throughput screening development challenges. This technology has the potential to transform the way drug discovery is performed.

BAFF-driven autoimmunity requires CD19 expression.

B cell activating factor of the tumor necrosis factor family (BAFF or BLyS) is a critical factor for B cell survival and maturation. BAFF-transgenic (BAFF-Tg) mice develop autoimmunity that resembles systemic lupus erythematosus (SLE) in a T cell-independent but MyD88-dependent manner, implicating toll-like receptor (TLR) signaling. The specific B cell subtypes that make pro-inflammatory autoantibodies in BAFF-Tg mice are TLR-activated innate B cells known as marginal zone (MZ) and B1 B cells. These cells infiltrate the salivary glands and kidneys of diseased BAFF-Tg mice. However, loss of B1a or MZ B cells does not protect BAFF-Tg mice against disease, suggesting that B1b B cells might be the important pathogenic B cell subset. To test this hypothesis, we have generated BAFF-Tg mice that retained follicular B cells, but are deficient in B1a, B1b and MZ B cells, by crossing BAFF-Tg mice to CD19-deficient mice (BTg-CD19(-/-)). The BTg-CD19(-/-) mice did not produce autoantibodies and were protected from splenomegaly, kidney pathology and all signs of autoimmunity. This work suggests that B1b B cells, rather than MZ or B1a B cells, are sufficient and possibly required for the development of autoimmunity. Loss of the majority of innate-like B cells was able to protect BAFF-Tg mice from developing disease, so we can now conclude that autoimmunity induced by excessive BAFF production requires B1b B cells and CD19 signaling.

Identification of orally-bioavailable antagonists of the TRPV4 ion-channel.

Antagonists of the TRPV4 receptor were identified using a focused screen, followed by a limited optimization program. The leading compounds obtained from this exercise, RN-1665 23 and RN-9893 26, showed moderate oral bioavailability when dosed to rats. The lead molecule, RN-9893 26, inhibited human, rat and murine variants of TRPV4, and showed excellent selectivity over related TRP receptors, such as TRPV1, TRPV3 and TRPM8. The overall profile for RN-9893 may permit its use as a proof-of-concept probe for in vivo applications.

Deregulated Cdk5 triggers aberrant activation of cell cycle kinases and phosphatases inducing neuronal death.

Aberrant activation of cell cycle proteins is believed to play a critical role in Alzheimer's disease (AD) pathogenesis; although, the molecular mechanisms leading to their activation in diseased neurons remain elusive. The goal of this study was to investigate the mechanistic link between Cdk5 deregulation and cell cycle re-activation in ß-amyloid(1-42) (Aß(1-42))-induced neurotoxicity. Using a chemical genetic approach, we identified Cdc25A, Cdc25B and Cdc25C as direct Cdk5 substrates in mouse brain lysates. We show that deregulated Cdk5 directly phosphorylates Cdc25A, Cdc25B and Cdc25C at multiple sites, which not only increases their phosphatase activities but also facilitates their release from 14-3-3 inhibitory binding. Cdc25A, Cdc25B and Cdc25C in turn activate Cdk1, Cdk2 and Cdk4 kinases causing neuronal death. Selective inhibition of Cdk5 abrogates Cdc25 and Cdk activations in Aß(1-42)-treated neurons. Similarly, phosphorylation-resistant mutants of Cdc25 isoforms at Cdk5 sites are defective in activating Cdk1, Cdk2 and Cdk4 in Aß(1-42)-treated primary cortical neurons, emphasizing a major role of Cdk5 in the activation of Cdc25 isoforms and Cdks in AD pathogenesis. These results were further confirmed in human AD clinical samples, which had higher Cdc25A, Cdc25B and Cdc25C activities that were coincident with increased Cdk5 activity, as compared to age-matched controls. Inhibition of Cdk5 confers the highest neuroprotection against Aß(1-42) toxicity, whereas inhibition of Cdc25 isoforms was partially neuroprotective, further emphasizing a decisive role of Cdk5 deregulation in cell-cycle-driven AD neuronal death.

Autoimmune rheumatic disease in Australian Aboriginal and Torres Strait Islander Peoples: What do we know?

Autoimmune rheumatic disease (AIRD) is a collective term, which comprises a group of multisystem inflammatory autoimmune diseases, including connective tissue disease, chronic inflammatory arthritis, sarcoidosis and systemic vasculitis. Some AIRD are prevalent in the general population, and all can cause significant morbidity and reduced quality of life, with some increasing the risk of premature mortality, such as systemic lupus erythematosus (SLE), a connective tissue disease that is more prevalent and severe in Australian Aboriginal and Torres Strait Islander Peoples with high mortality rates. To ensure that management of AIRD can be optimised for all Australians, it is important that we understand the prevalence and potential phenotypic variations of AIRD across the Australian population. However, to date there have been few described cases of AIRD other than SLE in Aboriginal and Torres Strait Islander Peoples. In this review, we summarise what is known about AIRD other than SLE in Aboriginal and Torres Strait Islander Peoples, particularly with regards to prevalence, phenotype and disease outcomes, and highlight the current gaps in knowledge.

Systemic lupus erythematosus in Aboriginal and Torres Strait Islander peoples in Australia: addressing disparities and barriers to optimising patient care.

The first inhabitants of Australia and the traditional owners of Australian lands are the Aboriginal and Torres Strait Islander peoples. Aboriginal and Torres Strait Islander peoples are two to four times more likely to have systemic lupus erythematosus (SLE) than the general Australian population. Phenotypically, SLE appears distinctive in Aboriginal and Torres Strait Islander peoples and its severity is substantially increased, with mortality rates up to six times higher than in the general Australian population with SLE. In particular, Aboriginal and Torres Strait Islander peoples with SLE have increased prevalence of lupus nephritis and increased rates of progression to end-stage kidney disease. The reasons for the increased prevalence and severity of SLE in this population are unclear, but socioeconomic, environmental, and biological factors are all likely to be implicated, although there are no published studies investigating these factors in Aboriginal and Torres Strait Islander peoples with SLE specifically, indicating an important knowledge gap. In this Review, we summarise the data on the incidence, prevalence, and clinical and biological findings relating to SLE in Aboriginal and Torres Strait Islander peoples and explore potential factors contributing to its increased prevalence and severity in this population. Importantly, we identify health disparities and deficiencies in health-care provision that limit optimal care and outcomes for many Aboriginal and Torres Strait Islander peoples with SLE and highlight potentially addressable goals to improve outcomes.

Discovery, Characterization, and Structure of a Cell Active PAD2 Inhibitor Acting through a Novel Allosteric Mechanism.

Peptidyl arginine deiminases (PADs) are important enzymes in many diseases, especially those involving inflammation and autoimmunity. Despite many years of effort, developing isoform-specific inhibitors has been a challenge. We describe herein the discovery of a potent, noncovalent PAD2 inhibitor, with selectivity over PAD3 and PAD4, from a DNA-encoded library. The biochemical and biophysical characterization of this inhibitor and two noninhibitory binders indicated a novel, Ca2+ competitive mechanism of inhibition. This was confirmed via X-ray crystallographic analysis. Finally, we demonstrate that this inhibitor selectively inhibits PAD2 in a cellular context.

Structural basis for CCR6 modulation by allosteric antagonists.

The CC chemokine receptor 6 (CCR6) is a potential target for chronic inflammatory diseases. Previously, we reported an active CCR6 structure in complex with its cognate chemokine CCL20, revealing the molecular basis of CCR6 activation. Here, we present two inactive CCR6 structures in ternary complexes with different allosteric antagonists, CCR6/SQA1/OXM1 and CCR6/SQA1/OXM2. The oxomorpholine analogues, OXM1 and OXM2 are highly selective CCR6 antagonists which bind to an extracellular pocket and disrupt the receptor activation network. An energetically favoured U-shaped conformation in solution that resembles the bound form is observed for the active analogues. SQA1 is a squaramide derivative with close-in analogues reported as antagonists of chemokine receptors including CCR6. SQA1 binds to an intracellular pocket which overlaps with the G protein site, stabilizing a closed pocket that is a hallmark of inactive GPCRs. Minimal communication between the two allosteric pockets is observed, in contrast to the prevalent allosteric cooperativity model of GPCRs. This work highlights the versatility of GPCR antagonism by small molecules, complementing previous knowledge of CCR6 activation, and sheds light on drug discovery targeting CCR6.

Antidrug antibodies (ADAb) to tumour necrosis factor (TNF)-specific neutralising agents in chronic inflammatory diseases: a real issue, a clinical perspective.

The introduction of biologics, especially tumour necrosis factor (TNF) inhibitors, has revolutionized the management of chronic inflammatory diseases. However, at least one third of patients with these diseases, receiving TNF inhibitors either do not respond to treatment, or lose initial responsiveness. For a significant proportion, improvement of clinical response is achieved after switching to another anti-TNF drug, suggesting a basis for failure unrelated to the therapeutic target itself. A likely explanation for this is immunogenicity, as all biologics are potentially immunogenic, and the resulting anti-drug antibodies (ADAb) can theoretically decrease the efficacy of biologics and/or induce adverse events. Indeed, in these chronic inflammatory diseases, many studies have now established correlations between ADAb formation, low serum drug levels, and the failure or loss of response to anti-TNF antibodies. This article will review key findings related to ADAb, and propose a model wherein monitoring of drug levels and ADAb may be a predictive tool leading to a better choice of biologics. Such an approach could improve chronic inflammatory disease management toward a personalized and more cost-effective approach.

BAFF and innate immunity: new therapeutic targets for systemic lupus erythematosus.

Recently, the B cell has emerged as a cornerstone of systemic lupus erythematosus (SLE) pathogenesis. This has been highlighted by studies of the cytokine B-cell-activating factor of the tumour necrosis factor (TNF) family (BAFF), a crucial factor regulating B-cell maturation, survival and function. Overexpression of BAFF in mice leads to the development of an SLE-like disease, independent of T cells but instead relying on innate immunity mechanisms. Moreover, BAFF has been shown to be elevated in the serum of patients suffering from autoimmune conditions, especially SLE, and may correlate with disease activity. These findings challenge the previous notion that T:B-cell collaboration is the sole driver of SLE. In recent years, controlled trials have for the first time tested targeted therapeutics for SLE. However, agents designed to target B cells failed to meet primary endpoints in clinical trials in SLE, suggesting that a more complex role for B cells in SLE awaited elucidation. By contrast, on 9 March 2011, the US Food and Drug Administration approved belimumab, a fully human anti-BAFF monoclonal antibody, as a new B-cell-specific treatment for SLE. This article will review over 10 years of research on the BAFF system, key findings that led to this recent positive clinical outcome and propose a model potentially explaining why this B-cell-specific therapy has yielded positive results in clinical trials. We will also review promising therapies presently in clinical trials targeting innate immunity, which are likely to revolutionize SLE management towards a personalized and targeted therapy approach.

Phenotypic drug discovery: recent successes, lessons learned and new directions.

Many drugs, or their antecedents, were discovered through observation of their effects on normal or disease physiology. For the past generation, this phenotypic drug discovery approach has been largely supplanted by the powerful but reductionist approach of modulating specific molecular targets of interest. Nevertheless, modern phenotypic drug discovery, which combines the original concept with modern tools and strategies, has re-emerged over the past decade to systematically pursue drug discovery based on therapeutic effects in realistic disease models. Here, we discuss recent successes with this approach, as well as consider ongoing challenges and approaches to address them. We also explore how innovation in this area may fuel the next generation of successful projects.