Negative regulation of TLR signaling in myeloid cells--implications for autoimmune diseases.

Toll-like receptors (TLR) are transmembrane pattern recognition receptors that recognize microbial ligands and signal for production of inflammatory cytokines and type I interferon in macrophages and dendritic cells (DC). Whereas TLR-induced inflammatory mediators are required for pathogen clearance, many are toxic to the host and can cause pathological inflammation when over-produced. This is demonstrated by the role of TLR-induced cytokines in autoimmune diseases, such as rheumatoid arthritis, inflammatory bowel disease, and systemic lupus erythematosus. Because of the potent effects of TLR-induced cytokines, we have diverse mechanisms to dampen TLR signaling. Here, we highlight three pathways that participate in inhibition of TLR responses in macrophages and DC, and their implications in autoimmunity; A20, encoded by the TNFAIP3 gene, Lyp encoded by the PTPN22 gene, and the BCAP/PI3K pathway. We present new findings that Lyp promotes TLR responses in primary human monocytes and that the autoimmunity risk Lyp620W variant is more effective at promoting TLR-induced interleukin-6 than the non-risk Lyp620R protein. This suggests that Lyp serves to downregulate a TLR inhibitory pathway in monocytes, and we propose that Lyp inhibits the TREM2/DAP12 inhibitory pathway. Overall, these pathways demonstrate distinct mechanisms of negative regulation of TLR responses, and all impact autoimmune disease pathogenesis and treatment.

A potent and selective inhibitor for the UBLCP1 proteasome phosphatase.

The ubiquitin-like domain-containing C-terminal domain phosphatase 1 (UBLCP1) has been implicated as a negative regulator of the proteasome, a key mediator in the ubiquitin-dependent protein degradation. Small molecule inhibitors that block UBLCP1 activity would be valuable as research tools and potential therapeutics for human diseases caused by the cellular accumulation of misfold/damaged proteins. We report a salicylic acid fragment-based library approach aimed at targeting both the phosphatase active site and its adjacent binding pocket for enhanced affinity and selectivity. Screening of the focused libraries led to the identification of the first potent and selective UBLCP1 inhibitor 13. Compound 13 exhibits an IC50 of 1.0µM for UBLCP1 and greater than 5-fold selectivity against a large panel of protein phosphatases from several distinct families. Importantly, the inhibitor possesses efficacious cellular activity and is capable of inhibiting UBLCP1 function in cells, which in turn up-regulates nuclear proteasome activity. These studies set the groundwork for further developing compound 13 into chemical probes or potential therapeutic agents targeting the UBLCP1 phosphatase.

Overexpression of the autoimmunity-associated phosphatase PTPN22 promotes survival of antigen-stimulated CLL cells by selectively activating AKT.

A polymorphic variant of the phosphatase PTPN22 has been associated with increased risk for multiple autoimmune diseases. The risk allele is thought to function by diminishing antigen-receptor signals responsible for negative selection of autoreactive lymphocytes. We now show that PTPN22 is markedly overexpressed in chronic lymphocytic leukemia (CLL), a common malignancy of autoreactive B lymphocytes. We also show that overexpression of PTPN22 significantly inhibits antigen-induced apoptosis of primary CLL cells by blocking B-cell receptor (BCR) signaling pathways that negatively regulate lymphocyte survival. More importantly, we show that PTPN22 positively regulates the antiapoptotic AKT kinase, which provides a powerful survival signal to antigen-stimulated CLL cells. This selective uncoupling of AKT from other downstream BCR signaling pathways is a result of inhibition of a negative regulatory circuit involving LYN, CD22, and SHIP. Finally, we show that PTPN22 can be effectively down-regulated by the PKC inhibitors ruboxistaurin and sotrastaurin, resulting in enhanced killing of CLL cells exposed to proapoptotic BCR stimuli. Collectively, these data suggest that PTPN22 overexpression represents a protective mechanism that allows autoantigen-activated CLL cells to escape from negative selection and indicate that this mechanism could be exploited for therapeutic purposes by targeting PTPN22 with PKC inhibitors.

Role of SHP2 phosphatase in KIT-induced transformation: identification of SHP2 as a druggable target in diseases involving oncogenic KIT.

Intracellular mechanism(s) that contribute to promiscuous signaling via oncogenic KIT in systemic mastocytosis and acute myelogenous leukemia are poorly understood. We show that SHP2 phosphatase is essential for oncogenic KIT-induced growth and survival in vitro and myeloproliferative disease (MPD) in vivo. Genetic disruption of SHP2 or treatment of oncogene-bearing cells with a novel SHP2 inhibitor alone or in combination with the PI3K inhibitor corrects MPD by disrupting a protein complex involving p85α, SHP2, and Gab2. Importantly, a single tyrosine at position 719 in oncogenic KIT is sufficient to develop MPD by recruiting p85α, SHP2, and Gab2 complex to oncogenic KIT. Our results demonstrate that SHP2 phosphatase is a druggable target that cooperates with lipid kinases in inducing MPD.

Targeting mycobacterium protein tyrosine phosphatase B for antituberculosis agents.

Protein tyrosine phosphatases are often exploited and subverted by pathogenic bacteria to cause human diseases. The tyrosine phosphatase mPTPB from Mycobacterium tuberculosis is an essential virulence factor that is secreted by the bacterium into the cytoplasm of macrophages, where it mediates mycobacterial survival in the host. Consequently, there is considerable interest in understanding the mechanism by which mPTPB evades the host immune responses, and in developing potent and selective mPTPB inhibitors as unique antituberculosis (antiTB) agents. We uncovered that mPTPB subverts the innate immune responses by blocking the ERK1/2 and p38 mediated IL-6 production and promoting host cell survival by activating the Akt pathway. We identified a potent and selective mPTPB inhibitor I-A09 with highly efficacious cellular activity, from a combinatorial library of bidentate benzofuran salicylic acid derivatives assembled by click chemistry. We demonstrated that inhibition of mPTPB with I-A09 in macrophages reverses the altered host immune responses induced by the bacterial phosphatase and prevents TB growth in host cells. The results provide the necessary proof-of-principle data to support the notion that specific inhibitors of the mPTPB may serve as effective antiTB therapeutics.

Case report: Emergency presentation of Meckel's diverticulum in the 3rd trimester of pregnancy.

Background: Symptomatic Meckel's diverticulum (MD) is easily neglected in the acute abdomen during pregnancy. MD is the most common congenitally anomalous development of the intestines, with an incidence of 2% in the general population, although it is not easily diagnosed because of variable clinical features. Especially when complicated with pregnancy, doctors can easily overlook this disease, which directly threatens maternal and foetal life. Case Presentation: We report the case of a 25-year-old at 32 + 2 weeks of gestation complicated with MD volvulus who presented with progressive abdominal pain and finally peritonitis. She underwent exploratory laparotomy and small-bowel resection. The mother and the baby successfully recovered. Conclusions: MD-complicated pregnancy is not easily diagnosed. Once highly suspiciously diagnosed, especially with peritonitis, surgery should be arranged, which helps preserve maternal and foetal life.

Glucose-dependent insulinotropic polypeptide regulates body weight and food intake via GABAergic neurons in mice.

The development of single-molecule co-agonists for the glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) and glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) is considered a breakthrough in the treatment of obesity and type 2 diabetes. But although GIPR-GLP-1R co-agonism decreases body weight with superior efficacy relative to GLP-1R agonism alone in preclinical1-3 and clinical studies4,5, the role of GIP in regulating energy metabolism remains enigmatic. Increasing evidence suggests that long-acting GIPR agonists act in the brain to decrease body weight through the inhibition of food intake3,6-8; however, the mechanisms and neuronal populations through which GIP affects metabolism remain to be identified. Here, we report that long-acting GIPR agonists and GIPR-GLP-1R co-agonists decrease body weight and food intake via inhibitory GABAergic neurons. We show that acyl-GIP decreases body weight and food intake in male diet-induced obese wild-type mice, but not in mice with deletion of Gipr in Vgat(also known as Slc32a1)-expressing GABAergic neurons (Vgat-Gipr knockout). Whereas the GIPR-GLP-1R co-agonist MAR709 leads, in male diet-induced obese wild-type mice, to greater weight loss and further inhibition of food intake relative to a pharmacokinetically matched acyl-GLP-1 control, this superiority over GLP-1 vanishes in Vgat-Gipr knockout mice. Our data demonstrate that long-acting GIPR agonists crucially depend on GIPR signaling in inhibitory GABAergic neurons to decrease body weight and food intake.

Bicyclic benzofuran and indole-based salicylic acids as protein tyrosine phosphatase inhibitors.

Protein tyrosine phosphatases (PTPs) constitute a large and structurally diverse family of signaling enzymes that control the cellular levels of protein tyrosine phosphorylation. Malfunction of PTP activity has significant implications in many human diseases, and the PTP protein family provides an exciting array of validated diabetes/obesity (PTP1B), oncology (SHP2), autoimmunity (Lyp), and infectious disease (mPTPB) targets. However, despite the fact that PTPs have been garnering attention as novel therapeutic targets, they remain largely an untapped resource. The main challenges facing drug developers by the PTPs are inhibitor specificity and bioavailability. Work over the last ten years has demonstrated that it is feasible to develop potent and selective inhibitors for individual members of the PTP family by tethering together small ligands that can simultaneously occupy both the active site and unique nearby peripheral binding sites. Recent results with the bicyclic salicylic acid pharmacophores indicate that the new chemistry platform may provide a potential solution to overcome the bioavailability issue that has plagued the PTP drug discovery field for many years. Structural analysis of PTP-inhibitor complexes reveals molecular determinants important for the development of more potent and selective PTP inhibitors, thus offering hope in the medicinal chemistry of a largely unexploited protein class with a wealth of attractive drug targets.

Therapeutic Potential of Menstrual Blood-Derived Stem Cell Transplantation for Intrauterine Adhesions.

An increasing number of women experience intrauterine adhesion as a result of intrauterine operations, such as induced abortion, which can cause infertility, recurrent abortion and amenorrhea. Although some strategies have been applied clinically, such as hysteroscopy adhesiolysis of intrauterine adhesions, the results have not been promising. As regenerative medicine develops, research on menstrual blood-derived stem cell transplantation is increasing due to the properties of these cells, including self-renewal, differentiation, angiogenesis, anti-inflammation and immunomodulation. As a result, menstrual blood-derived stem cells may be an ideal cell source for the treatment of intrauterine adhesion. Excitingly, it has been reported that autologous menstrual blood stem cells could recovery injured endometrium and improve infertility in patients with refractory intrauterine adhesion. In this review, we discuss the possible potential of menstrual blood-derived stem cell transplantation for intrauterine adhesion, including the antifibrosis, angiogenesis, anti-inflammation and immunoregulation properties of the cells, which brings hopes for clinical therapy.

Germanium silicon oxide achieves multi-coloured ultra-long phosphorescence and delayed fluorescence at high temperature.

Colour-tuned phosphors are promising for advanced security applications such as multi-modal anti-counterfeiting and data encryption. The practical adoption of colour-tuned phosphors requires these materials to be responsive to multiple stimuli (e.g., excitation wavelength, excitation waveform, and temperature) and exhibit excellent materials stability simultaneously. Here we report germanium silicon oxide (GSO) - a heavy-metal-free inorganic phosphor - that exhibits colour-tuned ultra-long phosphorescence and delayed fluorescence across a broad temperature range (300 - 500 K) in air. We developed a sol-gel processing strategy to prepare amorphous oxides containing homogeneously dispersed Si and Ge atoms. The co-existence of Ge and Si luminescent centres (LC) leads to an excitation-dependent luminescence change across the UV-to-visible region. GSO exhibits Si LC-related ultra-long phosphorescence at room-temperature and thermally activated delayed fluorescence at temperatures as high as 573 K. This long-lived PL is sensitized via the energy transfer from Ge defects to Si LCs, which provides PL lifetime tunability for GSO phosphors. The oxide scaffold of GSO offers 500-day materials stability in air; and 1-week stability in strong acidic and basic solutions. Using GSO/polymer hybrids, we demonstrated colour-tuned security tags whose emission wavelength and lifetime can be controlled via the excitation wavelength, and temperature, indicating promise in security applications.

Next generation GLP-1/GIP/glucagon triple agonists normalize body weight in obese mice.

OBJECTIVE: Pharmacological strategies that engage multiple mechanisms-of-action have demonstrated synergistic benefits for metabolic disease in preclinical models. One approach, concurrent activation of the glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic peptide (GIP), and glucagon (Gcg) receptors (i.e. triagonism), combines the anorectic and insulinotropic activities of GLP-1 and GIP with the energy expenditure effect of glucagon. While the efficacy of triagonism in preclinical models is known, the relative contribution of GcgR activation remains unassessed. This work aims to addresses that central question. METHODS: Herein, we detail the design of unimolecular peptide triagonists with an empirically optimized receptor potency ratio. These optimized peptide triagonists employ a protraction strategy permitting once-weekly human dosing. Additionally, we assess the effects of these peptides on weight-reduction, food intake, glucose control, and energy expenditure in an established DIO mouse model compared to clinically relevant GLP-1R agonists (e.g. semaglutide) and dual GLP-1R/GIPR agonists (e.g. tirzepatide). RESULTS: Optimized triagonists normalize body weight in DIO mice and enhance energy expenditure in a manner superior to that of GLP-1R mono-agonists and GLP-1R/GIPR co-agonists. CONCLUSIONS: These pre-clinical data suggest unimolecular poly-pharmacology as an effective means to target multiple mechanisms contributing to obesity and further implicate GcgR activation as the differentiating factor between incretin receptor mono- or dual-agonists and triagonists.

A loss-of-function variant of PTPN22 is associated with reduced risk of systemic lupus erythematosus.

A gain-of-function R620W polymorphism in the PTPN22 gene, encoding the lymphoid tyrosine phosphatase LYP, has recently emerged as an important risk factor for human autoimmunity. Here we report that another missense substitution (R263Q) within the catalytic domain of LYP leads to reduced phosphatase activity. High-resolution structural analysis revealed the molecular basis for this loss of function. Furthermore, the Q263 variant conferred protection against human systemic lupus erythematosus, reinforcing the proposal that inhibition of LYP activity could be beneficial in human autoimmunity.

Cutting edge: the PTPN22 allelic variant associated with autoimmunity impairs B cell signaling.

PTPN22 is a gene encoding the protein tyrosine phosphatase Lyp. A missense mutation changing residue 1858 from cytosine to thymidine (1858C/T) is associated with multiple autoimmune disorders. Studies have demonstrated that Lyp has an inhibitory effect on TCR signaling; however, the presence of autoantibodies in all of the diseases associated with the 1858T variant and recent evidence that Ca(2+) flux is altered in B cells of 1858T carriers indicate a role for Lyp in B cell signaling. In this study we show that B cell signal transduction is impaired in individuals who express the variant. This defect in signaling is characterized by a deficit in proliferation, a decrease in phosphorylation of key signaling proteins, and is reversed by inhibition of Lyp. These findings suggest that the PTPN22 1858T variant alters BCR signaling and implicate B cells in the mechanism by which the PTPN22 1858T variant contributes to autoimmunity.

Structure, inhibitor, and regulatory mechanism of Lyp, a lymphoid-specific tyrosine phosphatase implicated in autoimmune diseases.

The lymphoid-specific tyrosine phosphatase (Lyp) has generated enormous interest because a single-nucleotide polymorphism in the gene (PTPN22) encoding Lyp produces a gain-of-function mutant phosphatase that is associated with several autoimmune diseases, including type I diabetes, rheumatoid arthritis, Graves disease, and systemic lupus erythematosus. Thus, Lyp represents a potential target for a broad spectrum of autoimmune disorders. Unfortunately, no Lyp inhibitor has been reported. In addition, little is known about the structure and biochemical mechanism that directly regulates Lyp function. Here, we report the identification of a bidentate salicylic acid-based Lyp inhibitor I-C11 with excellent cellular efficacy. Structural and mutational analyses indicate that the inhibitor binds both the active site and a nearby peripheral site unique to Lyp, thereby furnishing a solid foundation upon which inhibitors with therapeutic potency and selectivity can be developed. Moreover, a comparison of the apo- and inhibitor-bound Lyp structures reveals that the Lyp-specific region S(35)TKYKADK(42), which harbors a PKC phosphorylation site, could adopt either a loop or helical conformation. We show that Lyp is phosphorylated exclusively at Ser-35 by PKC both in vitro and in vivo. We provide evidence that the status of Ser-35 phosphorylation may dictate the conformational state of the insert region and thus Lyp substrate recognition. We demonstrate that Ser-35 phosphorylation impairs Lyp's ability to inactivate the Src family kinases and down-regulate T cell receptor signaling. Our data establish a mechanism by which PKC could attenuate the cellular function of Lyp, thereby augmenting T cell activation.

Aryl vinyl sulfonates and sulfones as active site-directed and mechanism-based probes for protein tyrosine phosphatases.

Protein tyrosine phosphatases (PTPs) play key roles in the regulation of normal and pathological processes ranging from cell proliferation, differentiation, metabolism, and survival to many human diseases including cancer and diabetes. Functional studies of PTP can be greatly facilitated by small molecule probes that covalently label the active site of a PTP through an activity-dependent chemical reaction. In this article, we characterize phenyl vinyl sulfonate (PVSN) and phenyl vinyl sulfone (PVS) as a new class of mechanism-based PTP probes. PVSN and PVS inactivate a broad range of PTPs in a time- and concentration-dependent fashion. The PVSN- and PVS-mediated PTP inactivation is active site-directed and irreversible, resulting from a Michael addition of the active-site Cys Sgamma onto the terminal carbon of the vinyl group. Structural and mechanistic analyses reveal the molecular basis for the preference of PVSN/PVS toward the PTPs, which lies in the ability of PVSN and PVS to engage the conserved structural and catalytic machinery of the PTP active site. In contrast to early alpha-bromobenzyl phosphonate-based probes, PVSN and PVS are resistant to solvolysis and are cell-permeable and thus hold promise for in vivo applications. Collectively, these properties bode well for the development of aryl vinyl sulfonate/sulfone-based PTP probes to interrogate PTP activity in complex proteomes.

The therapeutic potential of bone marrow mesenchymal stem cells in premature ovarian failure.

With the development of regenerative medicine, a variety of mesenchymal stem cells (MSCs) are increasingly considered for the treatment of premature ovarian failure (POF). Reportedly, bone marrow-derived MSCs (BMSCs) improve the ovarian reserve, which mainly depends on homing and paracrine activities. Furthermore, paracrine factors secreted by these stem cells play an important role in ovarian recovery. Relevant studies indicate that BMSC transplantation has some positive effects on the treatment of POF in animals, but BMSCs are not widely applied in clinical therapy. Clinical trials are ongoing despite the fact that several patients experiencing BMSC transplantation recover their normal menstrual cycles and even give birth to babies. In this review, we discuss the possible therapeutic mechanisms of BMSCs for POF, migration, antiapoptosis, antifibrosis, angiogenesis, anti-inflammation, immunoregulation, and oxidative stress, which provide the theoretical basis for further study and clinical therapy.

Mycobacterial Protein Tyrosine Phosphatases A and B Inhibitors Augment the Bactericidal Activity of the Standard Anti-tuberculosis Regimen.

Novel drugs are required to shorten the duration of treatment for tuberculosis (TB) and to combat the emergence of drug resistance. One approach has been to identify and target Mycobacterium tuberculosis (Mtb) virulence factors, which promote the establishment of TB infection and pathogenesis. Mtb produces a number of virulence factors, including two protein tyrosine phosphatases (PTPs), mPTPA and mPTPB, to evade the antimicrobial functions of host macrophages. To assess the therapeutic potential of targeting the virulent Mtb PTPs, we developed highly potent and selective inhibitors of mPTPA (L335-M34) and mPTPB (L01-Z08) with drug-like properties. We tested the bactericidal activity of L335-M34 and L01-Z08 alone or together in combination with the standard antitubercular regimen of isoniazid-rifampicin-pyrazinamide (HRZ) in the guinea pig model of chronic TB infection, which faithfully recapitulates some of the key histological features of human TB lesions. Following a single dose of L335-M34 50mg/kg and L01-Z08 20 mg/kg, plasma levels were maintained at levels 10-fold greater than the biochemical IC50 for 12-24 hours. Although neither PTP inhibitor alone significantly enhanced the antibacterial activity of HRZ, dual inhibition of mPTPA and mPTPB in combination with HRZ showed modest synergy, even after 2 weeks of treatment. After 6 weeks of treatment, the degree of lung inflammation correlated with the bactericidal activity of each drug regimen. This study highlights the potential utility of targeting Mtb virulence factors, and specifically the Mtb PTPs, as a strategy for enhancing the activity of standard anti-TB treatment.

Exploring the Existing Drug Space for Novel pTyr Mimetic and SHP2 Inhibitors.

Protein tyrosine phosphatases (PTPs) are potential therapeutic targets for many diseases. Unfortunately, despite considerable drug discovery efforts devoted to PTPs, obtaining selective and cell permeable PTP inhibitors remains highly challenging. We describe a strategy to explore the existing drug space for previously unknown PTP inhibitory activities. This led to the discovery of cefsulodin as an inhibitor of SHP2, an oncogenic phosphatase in the PTP family. Crystal structure analysis of SHP2 interaction with cefsulodin identified sulfophenyl acetic amide (SPAA) as a novel phosphotyrosine (pTyr) mimetic. A structure-guided and SPAA fragment-based focused library approach produced several potent and selective SHP2 inhibitors. Notably, these inhibitors blocked SHP2-mediated signaling events and proliferation in several cancer cell lines. Thus, SPAA may serve as a new platform for developing chemical probes for other PTPs.

Small molecule tools for functional interrogation of protein tyrosine phosphatases.

The importance of protein tyrosine phosphatases (PTPs) in the regulation of cellular signalling is well established. Malfunction of PTP activity is also known to be associated with cancer, metabolic syndromes and autoimmune disorders, as well as neurodegenerative and infectious diseases. However, a detailed understanding of the roles played by the PTPs in normal physiology and in pathogenic conditions has been hampered by the absence of PTP-specific small molecule agents. In addition, the therapeutic benefits of modulating this target class are underexplored as a result of a lack of suitable chemical probes. Potent and specific PTP inhibitors could significantly facilitate functional analysis of the PTPs in complex cellular signal transduction pathways and may constitute valuable therapeutics in the treatment of several human diseases. We highlight the current challenges to and opportunities for developing PTP-specific small molecule agents. We also review available selective small molecule inhibitors developed for a number of PTPs, including PTP1B, TC-PTP, SHP2, lymphoid-specific tyrosine phosphatase, haematopoietic protein tyrosine phosphatase, CD45, PTPß, PTPγ, PTPRO, Vaccinia H1-related phosphatase, mitogen-activated protein kinase phosphatase-1, mitogen-activated protein kinase phosphatase-3, Cdc25, YopH, mPTPA and mPTPB.

Organocatalytic multicomponent reaction for the acquisition of a selective inhibitor of mPTPB, a virulence factor of tuberculosis.

Mycobacterium protein tyrosine phosphatase B (mPTPB) is essential for the survival and persistence of Mycobacterium in the host. Thus small molecule inhibitors of mPTPB are potential anti-TB agents. We developed an efficient organocatalytic multicomponent reaction (MCR) between pyrrole, formaldehyde and aniline, affording a potent and selective mPTPB inhibitor with an IC(50) value of 1.5 µM and >50-fold specificity. Our studies provide a successful example of using organocatalysis as a discovery tool for the acquisition of PTP inhibitors.