A Massively Parallel CRISPR-Based Screening Platform for Modifiers of Neuronal Activity.

Understanding the complex interplay between gene expression and neuronal activity is crucial for unraveling the molecular mechanisms underlying cognitive function and neurological disorders. Here, we developed pooled screens for neuronal activity, using CRISPR interference (CRISPRi) and the fluorescent calcium integrator CaMPARI2. Using this screening method, we evaluated 1343 genes for their effect on excitability in human iPSC-derived neurons, revealing potential links to neurodegenerative and neurodevelopmental disorders.

Systematic perturbation screens identify regulators of inflammatory macrophage states and a role for TNF mRNA m6A modification.

Macrophages exhibit remarkable functional plasticity, a requirement for their central role in tissue homeostasis. During chronic inflammation, macrophages acquire sustained inflammatory 'states' that contribute to disease, but there is limited understanding of the regulatory mechanisms that drive their generation. Here we describe a systematic functional genomics approach that combines genome-wide phenotypic screening in primary murine macrophages with transcriptional and cytokine profiling of genetic perturbations in primary human macrophages to uncover regulatory circuits of inflammatory states.

Overloading And unpacKing (OAK) - droplet-based combinatorial indexing for ultra-high throughput single-cell multiomic profiling.

Multiomic profiling of single cells by sequencing is a powerful technique for investigating cellular diversity. Existing droplet-based microfluidic methods produce many cell-free droplets, underutilizing bead barcodes and reagents. Combinatorial indexing on microplates is more efficient for barcoding but labor-intensive.

Loss of the intracellular enzyme QPCTL limits chemokine function and reshapes myeloid infiltration to augment tumor immunity.

Tumor-associated macrophages are composed of distinct populations arising from monocytes or tissue macrophages, with a poorly understood link to disease pathogenesis. Here, we demonstrate that mouse monocyte migration was supported by glutaminyl-peptide cyclotransferase-like (QPCTL), an intracellular enzyme that mediates N-terminal modification of several substrates, including the monocyte chemoattractants CCL2 and CCL7, protecting them from proteolytic inactivation. Knockout of Qpctl disrupted monocyte homeostasis, attenuated tumor growth and reshaped myeloid cell infiltration, with loss of monocyte-derived populations with immunosuppressive and pro-angiogenic profiles.

Ru(II)-diimine complexes and cytochrome P450 working hand-in-hand.

With a growing interest in utilizing visible light to drive biocatalytic processes, several light-harvesting units and approaches have been employed to harness the synthetic potential of heme monooxygenases and carry out selective oxyfunctionalization of a wide range of substrates. While the fields of cytochrome P450 and Ru(II) photochemistry have separately been prolific, it is not until the turn of the 21st century that they converged. Non-covalent and subsequently covalently attached Ru(II) complexes were used to promote rapid intramolecular electron transfer in bacterial P450 enzymes.

Design and Evaluation of Highly Selective Human Immunoproteasome Inhibitors Reveal a Compensatory Process That Preserves Immune Cell Viability.

The pan-proteasome inhibitor bortezomib demonstrated clinical efficacy in off-label trials of Systemic Lupus Erythematosus. One potential mechanism of this clinical benefit is from the depletion of pathogenic immune cells (plasmablasts and plasmacytoid dendritic cells). However, bortezomib is cytotoxic against nonimmune cells, which limits its use for autoimmune diseases.

c-Maf-dependent T cell control of intestinal T17 cells and IgA establishes host-microbiota homeostasis.

Foxp3 regulatory T cells (T cells) are crucial for the maintenance of immune homeostasis both in lymphoid tissues and in non-lymphoid tissues. Here we demonstrate that the ability of intestinal T cells to constrain microbiota-dependent interleukin (IL)-17-producing helper T cell (T17 cell) and immunoglobulin A responses critically required expression of the transcription factor c-Maf. The terminal differentiation and function of several intestinal T cell populations, including RORγt T cells and follicular regulatory T cells, were c-Maf dependent.

Tumor suppressor BAP1 is essential for thymic development and proliferative responses of T lymphocytes.

Loss of function of the nuclear deubiquitinating enzyme BRCA1-associated protein-1 (BAP1) is associated with a wide spectrum of cancers. We report that tamoxifen-induced BAP1 deletion in adult mice resulted in severe thymic atrophy. BAP1 was critical for T cell development at several stages.

Disruption of XIAP-RIP2 Association Blocks NOD2-Mediated Inflammatory Signaling.

Inflammatory responses mediated by NOD2 rely on RIP2 kinase and ubiquitin ligase XIAP for the activation of nuclear factor κB (NF-κB), mitogen-activated protein kinases (MAPKs), and cytokine production. Herein, we demonstrate that selective XIAP antagonism blocks NOD2-mediated inflammatory signaling and cytokine production by interfering with XIAP-RIP2 binding, which removes XIAP from its ubiquitination substrate RIP2. We also establish that the kinase activity of RIP2 is dispensable for NOD2 signaling.

Inherited GINS1 deficiency underlies growth retardation along with neutropenia and NK cell deficiency.

Inborn errors of DNA repair or replication underlie a variety of clinical phenotypes. We studied 5 patients from 4 kindreds, all of whom displayed intrauterine growth retardation, chronic neutropenia, and NK cell deficiency. Four of the 5 patients also had postnatal growth retardation.

Discovery of oxa-sultams as RORc inverse agonists showing reduced lipophilicity, improved selectivity and favorable ADME properties.

Modification of the δ-sultam ring of RORc inverse agonist 2 led to the discovery of more polar oxa-sultam 65. The less lipophilic inverse agonist (65) displayed high potency in a biochemical assay, which translated into inhibition of IL-17 production in human peripheral blood mononuclear cells. The successful reduction of lipophilicity of this new analog gave rise to additional improvements in ROR selectivity and aqueous kinetic solubility, as well as reduction in plasma protein binding, while maintaining high cellular permeability.

Post-translational regulation of RORγt-A therapeutic target for the modulation of interleukin-17-mediated responses in autoimmune diseases.

Retinoic acid-related orphan receptor gamma t (RORγt) is a nuclear receptor, which is selectively expressed by various lymphocytes. RORγt is critical for the development of secondary and tertiary lymphoid organs, and for the thymic development of the T cell lineage. RORγt has been extensively studied as the master transcription factor of IL-17 expression and Th17 cells, which are strongly associated with various inflammatory and autoimmune conditions.

Deciphering the crosstalk among IL-1 and IL-10 family cytokines in intestinal immunity.

The IL-1 and IL-10 family cytokines are important regulators of intestinal immunity. Whereas these cytokines have protective roles in response to mucosal damage or infection, they also contribute to pathology in certain settings. How these cytokines function to maintain intestinal homoeostasis, and under what circumstances they contribute to disease is poorly understood.

Discovery of 1-{4-[3-fluoro-4-((3s,6r)-3-methyl-1,1-dioxo-6-phenyl-[1,2]thiazinan-2-ylmethyl)-phenyl]-piperazin-1-yl}-ethanone (GNE-3500): a potent, selective, and orally bioavailable retinoic acid receptor-related orphan receptor C (RORc or RORγ) inverse agonist.

Retinoic acid receptor-related orphan receptor C (RORc, RORγ, or NR1F3) is a nuclear receptor that plays a major role in the production of interleukin (IL)-17. Considerable efforts have been directed toward the discovery of selective RORc inverse agonists as potential treatments of inflammatory diseases such as psoriasis and rheumatoid arthritis. Using the previously reported tertiary sulfonamide 1 as a starting point, we engineered structural modifications that significantly improved human and rat metabolic stabilities while maintaining a potent and highly selective RORc inverse agonist profile.

Discovery of imidazo[1,5-a]pyridines and -pyrimidines as potent and selective RORc inverse agonists.

The nuclear receptor (NR) retinoic acid receptor-related orphan receptor gamma (RORγ, RORc, or NR1F3) is a promising target for the treatment of autoimmune diseases. RORc is a critical regulator in the production of the pro-inflammatory cytokine interleukin-17. We discovered a series of potent and selective imidazo[1,5-a]pyridine and -pyrimidine RORc inverse agonists.

Minor Structural Change to Tertiary Sulfonamide RORc Ligands Led to Opposite Mechanisms of Action.

A minor structural change to tertiary sulfonamide RORc ligands led to distinct mechanisms of action. Co-crystal structures of two compounds revealed mechanistically consistent protein conformational changes. Optimized phenylsulfonamides were identified as RORc agonists while benzylsulfonamides exhibited potent inverse agonist activity.

Interleukin-22 induces interleukin-18 expression from epithelial cells during intestinal infection.

T helper 1 (Th1) cell-associated immunity exacerbates ileitis induced by oral Toxoplasma gondii infection. We show here that attenuated ileitis observed in interleukin-22 (IL-22)-deficient mice was associated with reduced production of Th1-cell-promoting IL-18. IL-22 not only augmented the expression of Il18 mRNA and inactive precursor protein (proIL-18) in intestinal epithelial cells after T.

Deubiquitinase DUBA is a post-translational brake on interleukin-17 production in T cells.

T-helper type 17 (TH17) cells that produce the cytokines interleukin-17A (IL-17A) and IL-17F are implicated in the pathogenesis of several autoimmune diseases. The differentiation of TH17 cells is regulated by transcription factors such as RORγt, but post-translational mechanisms preventing the rampant production of pro-inflammatory IL-17A have received less attention. Here we show that the deubiquitylating enzyme DUBA is a negative regulator of IL-17A production in T cells.

A reversed sulfonamide series of selective RORc inverse agonists.

The identification of a new series of RORc inverse agonists is described. Comprehensive structure-activity relationship studies of this reversed sulfonamide series identified potent RORc inverse agonists in biochemical and cellular assays which were also selective against a panel of nuclear receptors. Our work has contributed a compound that may serve as a useful in vitro tool to delineate the complex biological pathways involved in signalling through RORc.

Interleukin-22 alleviates metabolic disorders and restores mucosal immunity in diabetes.

The connection between an altered gut microbiota and metabolic disorders such as obesity, diabetes, and cardiovascular disease is well established. Defects in preserving the integrity of the mucosal barriers can result in systemic endotoxaemia that contributes to chronic low-grade inflammation, which further promotes the development of metabolic syndrome. Interleukin (IL)-22 exerts essential roles in eliciting antimicrobial immunity and maintaining mucosal barrier integrity within the intestine.

Reduction in lipophilicity improved the solubility, plasma-protein binding, and permeability of tertiary sulfonamide RORc inverse agonists.

Using structure-based drug design principles, we identified opportunities to reduce the lipophilicity of our tertiary sulfonamide RORc inverse agonists. The new analogs possessed improved RORc cellular potencies with >77-fold selectivity for RORc over other nuclear receptors in our cell assay suite. The reduction in lipophilicity also led to an increased plasma-protein unbound fraction and improvements in cellular permeability and aqueous solubility.

Role of IL-22 in microbial host defense.

Interleukin (IL)-22 is a member of the IL-10 family of cytokines, which, besides IL-10, contains seven additional cytokines. Although the founding member IL-10 is an important immunoregulatory cytokine that represses both innate and adaptive immunity, the other family members preferentially target epithelial cells and enhance innate host defense mechanisms against various pathogens such as bacteria, yeast, and viruses. Based on their functions, the IL-10 family can be further divided into three subgroups, IL-10 itself, the IL-20 subfamily, and the IFNλ subfamily.