A Phenotypic Screen Identifies Potent DPP9 Inhibitors Capable of Killing HIV-1 Infected Cells.

Although current antiretroviral therapy can control HIV-1 replication and prevent disease progression, it is not curative. Identifying mechanisms that can lead to eradication of persistent viral reservoirs in people living with HIV-1 (PLWH) remains an outstanding challenge to achieving cure. Utilizing a phenotypic screen, we identified a novel chemical class capable of killing HIV-1 infected peripheral blood mononuclear cells. Tool compounds ICeD-1 and ICeD-2 ("inducer of cell death-1 and 2"), optimized for potency and selectivity from screening hits, were used to deconvolute the mechanism of action using a combination of chemoproteomic, biochemical, pharmacological, and genetic approaches. We determined that these compounds function by modulating dipeptidyl peptidase 9 (DPP9) and activating the caspase recruitment domain family member 8 (CARD8) inflammasome. Efficacy of ICeD-1 and ICeD-2 was dependent on HIV-1 protease activity and synergistic with efavirenz, which promotes premature activation of HIV-1 protease at high concentrations in infected cells. This in vitro synergy lowers the efficacious cell kill concentration of efavirenz to a clinically relevant dose at concentrations of ICeD-1 or ICeD-2 that do not result in complete DPP9 inhibition. These results suggest engagement of the pyroptotic pathway as a potential approach to eliminate HIV-1 infected cells.

New-onset hypogammaglobulinaemia and infectious complications associated with rituximab use in childhood-onset rheumatic diseases.

OBJECTIVE: To investigate the incidence and risk factors for hypogammaglobulinaemia and infectious complications associated with rituximab treatment in childhood-onset rheumatic diseases. METHODS: We performed a single-centre retrospective study of patients (n = 85) treated at Boston Children's Hospital (BCH) from 2009 to 2019. Study subjects included patients (ages 6-24 years) who received rituximab for the treatment of a childhood-onset rheumatic disease. RESULTS: New-onset hypogammaglobulinaemia developed in 23 (27.1%) patients within 18 months of rituximab induction treatment. Twenty-two patients (25.9%) developed at least one infectious complication in the 18 months following the first rituximab infusion; of these, 11 (50%) had serious infections requiring inpatient treatment. After adjusting for potential confounders, exposure to pulse corticosteroid therapy in the month prior to rituximab use was a significant predictor of both new-onset hypogammaglobulinaemia (odds ratio [OR] 3.94; 95% CI: 1.07, 16.0; P = 0.044) and infectious complications (OR 15.3; 95% CI: 3.04, 126.8; P = 0.003). Post-rituximab hypogammaglobulinaemia was the strongest predictor of serious infectious complications (OR 7.89; 95% CI: 1.41, 65.6; P = 0.028). Younger age at rituximab use was also a significant predictor of new-onset hypogammaglobulinaemia (OR 0.83; 95% CI: 0.70, 0.97; P = 0.021). Compared with other rheumatic diseases, patients with vasculitis had a higher likelihood of developing infectious complications, including serious infections. CONCLUSION: Although rituximab was well tolerated in terms of infectious complications in the majority of patients with childhood-onset rheumatic diseases, a substantial proportion developed new-onset hypogammaglobulinaemia and infectious complications following treatment. Our study highlights a role for heightened vigilance of rituximab-associated hypogammaglobulinaemia and infections in paediatric patients with rheumatic conditions.

Phenotypic screen identifies calcineurin-sparing FK506 analogs as BMP potentiators for treatment of acute kidney injury.

Acute kidney injury (AKI) is a life-threatening disease with no known curative or preventive therapies. Data from multiple animal models and human studies have linked dysregulation of bone morphogenetic protein (BMP) signaling to AKI. Small molecules that potentiate endogenous BMP signaling should have a beneficial effect in AKI. We performed a high-throughput phenotypic screen and identified a series of FK506 analogs that act as potent BMP potentiators by sequestering FKBP12 from BMP type I receptors. We further showed that calcineurin inhibition was not required for this activity. We identified a calcineurin-sparing FK506 analog oxtFK through late-stage functionalization and structure-guided design. OxtFK demonstrated an improved safety profile in vivo relative to FK506. OxtFK stimulated BMP signaling in vitro and in vivo and protected the kidneys in an AKI mouse model, making it a promising candidate for future development as a first-in-class therapeutic for diseases with dysregulated BMP signaling.

Eosinophilic Granulomatosis With Polyangiitis: An Unusual Case of Pediatric Subglottic Stenosis.

Systemic disease is an uncommon cause of subglottic stenosis (SGS). We report a case of severe SGS due to underlying eosinophilic granulomatosis with polyangiitis (EGPA) in a child presenting with isolated stridor. EGPA is a rare systemic vasculitis with very limited cases reported in the pediatric population. While surgical intervention was required given the degree of stenosis in this case, medical management of the underlying systemic disease process is critical when there is clinical suspicion of SGS in the context of systemic vasculitis. Laryngoscope, 131:656-659, 2021.

Systematic Chemogenetic Library Assembly.

Chemogenetic libraries, collections of well-defined chemical probes, provide tremendous value to biomedical research but require substantial effort to ensure diversity as well as quality of the contents. We have assembled a chemogenetic library by data mining and crowdsourcing institutional expertise. We are sharing our approach, lessons learned, and disclosing our current collection of 4,185 compounds with their primary annotated gene targets (https://github.com/Novartis/MoaBox). This physical collection is regularly updated and used broadly both within Novartis and in collaboration with external partners.

Consensus treatment plans for periodic fever, aphthous stomatitis, pharyngitis and adenitis syndrome (PFAPA): a framework to evaluate treatment responses from the childhood arthritis and rheumatology research alliance (CARRA) PFAPA work group.

BACKGROUND: Periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis (PFAPA) syndrome is the most common periodic fever syndrome in children. There is considerable heterogeneity in management strategies and a lack of evidence-based treatment guidelines. Consensus treatment plans (CTPs) are standardized treatment regimens that are derived based upon best available evidence and current treatment practices that are a way to enable comparative effectiveness studies to identify optimal therapy and are less costly to execute than randomized, double blind placebo controlled trials. The purpose of this project was to develop CTPs and response criteria for PFAPA. METHODS: The CARRA PFAPA Working Group is composed of pediatric rheumatologists, infectious disease specialists, allergists/immunologists and otolaryngologists. An extensive literature review was conducted followed by a survey to assess physician practice patterns. This was followed by virtual and in-person meetings between 2014 and 2018. Nominal group technique (NGT) was employed to develop CTPs, as well as inclusion criteria for entry into future treatment studies, and response criteria. Consensus required 80% agreement. RESULTS: The PFAPA working group developed CTPs resulting in 4 different treatment arms: 1. Antipyretic, 2. Abortive (corticosteroids), 3. Prophylaxis (colchicine or cimetidine) and 4. Surgical (tonsillectomy). Consensus was obtained among CARRA members for those defining patient characteristics who qualify for participation in the CTP PFAPA study. CONCLUSION: The goal is for the CTPs developed by our group to lead to future comparative effectiveness studies that will generate evidence-driven therapeutic guidelines for this periodic inflammatory disease.

A Phenotypic Screen Identifies Calcium Overload as a Key Mechanism of ß-Cell Glucolipotoxicity.

Type 2 diabetes (T2D) is caused by loss of pancreatic ß-cell mass and failure of the remaining ß-cells to deliver sufficient insulin to meet demand. ß-Cell glucolipotoxicity (GLT), which refers to combined, deleterious effects of elevated glucose and fatty acid levels on ß-cell function and survival, contributes to T2D-associated ß-cell failure. Drugs and mechanisms that protect ß-cells from GLT stress could potentially improve metabolic control in patients with T2D. In a phenotypic screen seeking low-molecular-weight compounds that protected ß-cells from GLT, we identified compound A that selectively blocked GLT-induced apoptosis in rat insulinoma cells. Compound A and its optimized analogs also improved viability and function in primary rat and human islets under GLT. We discovered that compound A analogs decreased GLT-induced cytosolic calcium influx in islet cells, and all measured ß-cell-protective effects correlated with this activity. Further studies revealed that the active compound from this series largely reversed GLT-induced global transcriptional changes. Our results suggest that taming cytosolic calcium overload in pancreatic islets can improve ß-cell survival and function under GLT stress and thus could be an effective strategy for T2D treatment.

A small-molecule inhibitor of BamA impervious to efflux and the outer membrane permeability barrier.

The development of new antimicrobial drugs is a priority to combat the increasing spread of multidrug-resistant bacteria. This development is especially problematic in gram-negative bacteria due to the outer membrane (OM) permeability barrier and multidrug efflux pumps. Therefore, we screened for compounds that target essential, nonredundant, surface-exposed processes in gram-negative bacteria. We identified a compound, MRL-494, that inhibits assembly of OM proteins (OMPs) by the ß-barrel assembly machine (BAM complex). The BAM complex contains one essential surface-exposed protein, BamA. We constructed a bamA mutagenesis library, screened for resistance to MRL-494, and identified the mutation bamAE470K BamAE470K restores OMP biogenesis in the presence of MRL-494. The mutant protein has both altered conformation and activity, suggesting it could either inhibit MRL-494 binding or allow BamA to function in the presence of MRL-494. By cellular thermal shift assay (CETSA), we determined that MRL-494 stabilizes BamA and BamAE470K from thermally induced aggregation, indicating direct or proximal binding to both BamA and BamAE470K Thus, it is the altered activity of BamAE470K responsible for resistance to MRL-494. Strikingly, MRL-494 possesses a second mechanism of action that kills gram-positive organisms. In microbes lacking an OM, MRL-494 lethally disrupts the cytoplasmic membrane. We suggest that the compound cannot disrupt the cytoplasmic membrane of gram-negative bacteria because it cannot penetrate the OM. Instead, MRL-494 inhibits OMP biogenesis from outside the OM by targeting BamA. The identification of a small molecule that inhibits OMP biogenesis at the cell surface represents a distinct class of antibacterial agents.

Metabolic Enzyme Sulfotransferase 1A1 Is the Trigger for N-Benzyl Indole Carbinol Tumor Growth Suppression.

In an attempt to identify novel therapeutics and mechanisms to differentially kill tumor cells using phenotypic screening, we identified N-benzyl indole carbinols (N-BICs), synthetic analogs of the natural product indole-3-carbinol (I3C). To understand the mode of action for the molecules we employed Cancer Cell Line Encyclopedia viability profiling and correlative informatics analysis to identify and ultimately confirm the phase II metabolic enzyme sulfotransferase 1A1 (SULT1A1) as the essential factor for compound selectivity. Further studies demonstrate that SULT1A1 activates the N-BICs by rendering the compounds strong electrophiles which can alkylate cellular proteins and thereby induce cell death. This study demonstrates that the selectivity profile for N-BICs is through conversion by SULT1A1 from an inactive prodrug to an active species that induces cell death and tumor suppression.

Shrinking lung syndrome as a manifestation of pleuritis: a new model based on pulmonary physiological studies.

OBJECTIVE: The pathophysiology of shrinking lung syndrome (SLS) is poorly understood. We sought to define the structural basis for this condition through the study of pulmonary mechanics in affected patients. METHODS: Since 2007, most patients evaluated for SLS at our institutions have undergone standardized respiratory testing including esophageal manometry. We analyzed these studies to define the physiological abnormalities driving respiratory restriction. Chest computed tomography data were post-processed to quantify lung volume and parenchymal density. RESULTS: Six cases met criteria for SLS. All presented with dyspnea as well as pleurisy and/or transient pleural effusions. Chest imaging results were free of parenchymal disease and corrected diffusing capacities were normal. Total lung capacities were 39%-50% of predicted. Maximal inspiratory pressures were impaired at high lung volumes, but not low lung volumes, in 5 patients. Lung compliance was strikingly reduced in all patients, accompanied by increased parenchymal density. CONCLUSION: Patients with SLS exhibited symptomatic and/or radiographic pleuritis associated with 2 characteristic physiological abnormalities: (1) impaired respiratory force at high but not low lung volumes; and (2) markedly decreased pulmonary compliance in the absence of identifiable interstitial lung disease. These findings suggest a model in which pleural inflammation chronically impairs deep inspiration, for example through neural reflexes, leading to parenchymal reorganization that impairs lung compliance, a known complication of persistently low lung volumes. Together these processes could account for the association of SLS with pleuritis as well as the gradual symptomatic and functional progression that is a hallmark of this syndrome.

Consensus treatment plans for induction therapy of newly diagnosed proliferative lupus nephritis in juvenile systemic lupus erythematosus.

OBJECTIVE: To formulate consensus treatment plans (CTPs) for induction therapy of newly diagnosed proliferative lupus nephritis (LN) in juvenile systemic lupus erythematosus (SLE). METHODS: A structured consensus formation process was employed by the members of the Childhood Arthritis and Rheumatology Research Alliance after considering the existing medical evidence and current treatment approaches. RESULTS: After an initial Delphi survey (response rate = 70%), a 2-day consensus conference, and 2 followup Delphi surveys (response rates = 63-79%), consensus was achieved for a limited set of CTPs addressing the induction therapy of proliferative LN. These CTPs were developed for prototypical patients defined by eligibility characteristics, and included immunosuppressive therapy with either mycophenolic acid orally twice per day, or intravenous cyclophosphamide once per month at standardized dosages for 6 months. Additionally, the CTPs describe 3 options for standardized use of glucocorticoids, including a primarily oral, a mixed oral/intravenous, and a primarily intravenous regimen. There was consensus on measures of effectiveness and safety of the CTPs. The CTPs were well accepted by the pediatric rheumatology providers treating children with LN, and up to 300 children per year in North America are expected to be candidates for the treatment with the CTPs. CONCLUSION: CTPs for induction therapy of proliferative LN in juvenile SLE based on the available scientific evidence and pediatric rheumatology group experience have been developed. Consistent use of the CTPs may improve the prognosis of proliferative LN, and support the conduct of comparative effectiveness studies aimed at optimizing therapeutic strategies for proliferative LN in juvenile SLE.

Chemical approaches for investigating phosphorylation in signal transduction networks.

The power and scope of chemical synthesis offer considerable opportunities to broaden the lexicon of chemical tools that can be implemented for the study of complex biological systems. To investigate individual signaling proteins and pathways, chemical tools provide a powerful complement to existing genetic, chemical genetic and immunologic methods. In particular, understanding phosphorylation-mediated signaling in real time yields important information about the regulation of cellular function and insights into the origin of disease. Recent advances in the development of photolabile caged analogs of bioactive species and fluorescence-based sensors of protein kinase activities are useful for investigating protein phosphorylation and the roles of phosphoproteins. Photolabile caged analogs allow spatial and temporal control over the release of a compound, while fluorescence-based sensors allow the real-time visualization of kinase activity. Here, we discuss recent advances that have increased the specificity and availability of these tools.

Caged phosphoproteins.

We present the chemical and biological synthesis of caged phosphoproteins using the in vitro nonsense codon suppression methodology. Specifically, phosphoamino acid analogues of serine, threonine, and tyrosine with a single photocleavable o-nitrophenylethyl caging group were synthesized as the amino acyl tRNA adducts for insertion into full-length proteins. For this purpose, a novel phosphitylating agent was developed. The successful incorporation of these bulky and charged amino acids into the alpha-subunit of the nicotinic acetyl choline receptor (nAChR) and the vasodilator-stimulated phosphoprotein (VASP) using an in vitro translation system is reported.

Caged phosphopeptides reveal a temporal role for 14-3-3 in G1 arrest and S-phase checkpoint function.

Using classical genetics to study modular phosphopeptide-binding domains within a family of proteins that are functionally redundant is difficult when other members of the domain family compensate for the product of the knocked-out gene. Here we describe a chemical genetics approach that overcomes this limitation by using UV-activatable caged phosphopeptides. By incorporating a caged phosphoserine residue within a consensus motif, these reagents simultaneously and synchronously inactivate all phosphoserine/phosphothreonine-binding domain family members in a rapid and temporally regulated manner. We applied this approach to study the global function of 14-3-3 proteins in cell cycle control. Activation of the caged phosphopeptides by UV irradiation displaced endogenous proteins from 14-3-3-binding, causing premature cell cycle entry, release of G1 cells from interphase arrest and loss of the S-phase checkpoint after DNA damage, accompanied by high levels of cell death. This class of reagents will greatly facilitate molecular dissection of kinase-dependent signaling pathways when applied to other phosphopeptide-binding domains including SH2, Polo-box and tandem BRCT domains.

A new environment-sensitive fluorescent amino acid for Fmoc-based solid phase peptide synthesis.

A new 4-(N,N-dimethylamino) phthalimide-based environment-sensitive fluorescent building block for solid phase peptide synthesis, has been synthesized and incorporated into peptides. Peptides incorporating this residue show great potential for biological applications in sensing protein/protein interactions.

Caged phospho-amino acid building blocks for solid-phase peptide synthesis.

Three 1-(2-nitrophenyl)ethyl-caged phospho-amino acids have been synthesized for use in standard N(alpha)-fluorenylmethoxycarbonyl-based solid-phase peptide synthesis (SPPS). The most common naturally occurring phospho-amino acids, serine, threonine, and tyrosine, were prepared as protected caged building blocks by modification with a unique phosphitylating reagent. In previous work, caged phospho-peptides were made using an interassembly approach (Rothman, D. M.; Vazquez, M. E.; Vogel, E. M.; Imperiali, B. Org. Lett. 2002, 4, 2865-2868). However, this technique is limited to creating peptides without oxidation sensitive residues C-terminal to the amino acid to be modified and the methodology involves synthetic manipulations on the solid phase that may limit the utilization of the methodology. Herein we report the facile synthesis of N-alpha-Fmoc-phospho(1-nitrophenylethyl-2-cyanoethyl)-L-serine 1, N-alpha-Fmoc-phospho(1-nitrophenylethyl-2-cyanoethyl)-L-threonine 2, and N-alpha-Fmoc-phospho(1-nitrophenylethyl-2-cyanoethyl)-L-tyrosine 3. These building blocks allow the synthesis of any caged phospho-peptide sequence using standard Fmoc-based SPPS procedures.

General method for the synthesis of caged phosphopeptides: tools for the exploration of signal transduction pathways.

[reaction: see text] An interassembly approach for the synthesis of peptides containing 1-(2-nitrophenyl)ethyl-caged phosphoserine, -threonine, and -tyrosine has been developed. Photochemical uncaging of these peptides releases the 2-nitrophenylethyl protecting group to afford the corresponding phosphopeptide. The peptides described herein are based on phosphorylation sites of kinases involved in cell movement or cell cycle regulation and demonstrate the versatility of the method and compatibility with the synthesis of polypeptides, including a variety of encoded amino acids.