Discovery of a Tunable Heterocyclic Electrophile 4-Chloro-pyrazolopyridine That Defines a Unique Subset of Ligandable Cysteines.

Electrophilic small molecules with novel reactivity are powerful tools that enable activity-based protein profiling and covalent inhibitor discovery. Here, we report a reactive heterocyclic scaffold, 4-chloro-pyrazolopyridine (CPzP) for selective modification of proteins via a nucleophilic aromatic substitution (SNAr) mechanism. Chemoproteomic profiling reveals that CPzPs engage cysteines within functionally diverse protein sites including ribosomal protein S5 (RPS5), inosine monophosphate dehydrogenase 2 (IMPDH2), and heat shock protein 60 (HSP60). Through the optimization of appended recognition elements, we demonstrate the utility of CPzP for covalent inhibition of prolyl endopeptidase (PREP) by targeting a noncatalytic active-site cysteine. This study suggests that the proteome reactivity of CPzPs can be modulated by both electronic and steric features of the ring system, providing a new tunable electrophile for applications in chemoproteomics and covalent inhibitor design.

Covalent Inhibition by a Natural Product-Inspired Latent Electrophile.

Strategies to target specific protein cysteines are critical to covalent probe and drug discovery. 3-Bromo-4,5-dihydroisoxazole (BDHI) is a natural product-inspired, synthetically accessible electrophilic moiety that has previously been shown to react with nucleophilic cysteines in the active site of purified enzymes. Here, we define the global cysteine reactivity and selectivity of a set of BDHI-functionalized chemical fragments using competitive chemoproteomic profiling methods. Our study demonstrates that BDHIs capably engage reactive cysteine residues in the human proteome and the selectivity landscape of cysteines liganded by BDHI is distinct from that of haloacetamide electrophiles. Given its tempered reactivity, BDHIs showed restricted, selective engagement with proteins driven by interactions between a tunable binding element and the complementary protein sites. We validate that BDHI forms covalent conjugates with glutathione S-transferase Pi (GSTP1) and peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1), emerging anticancer targets. BDHI electrophile was further exploited in Bruton's tyrosine kinase (BTK) inhibitor design using a single-step late-stage installation of the warhead onto acrylamide-containing compounds. Together, this study expands the spectrum of optimizable chemical tools for covalent ligand discovery and highlights the utility of 3-bromo-4,5-dihydroisoxazole as a cysteine-reactive electrophile.

Role of Serine Proteases at the Tumor-Stroma Interface.

During tumor development, invasion and metastasis, the intimate interaction between tumor and stroma shapes the tumor microenvironment and dictates the fate of tumor cells. Stromal cells can also influence anti-tumor immunity and response to immunotherapy. Understanding the molecular mechanisms that govern this complex and dynamic interplay, thus is important for cancer diagnosis and therapy. Proteolytic enzymes that are expressed and secreted by both cancer and stromal cells play important roles in modulating tumor-stromal interaction. Among, several serine proteases such as fibroblast activation protein, urokinase-type plasminogen activator, kallikrein-related peptidases, and granzymes have attracted great attention owing to their elevated expression and dysregulated activity in the tumor microenvironment. This review highlights the role of serine proteases that are mainly derived from stromal cells in tumor progression and associated theranostic applications.

Covalent Small Molecule Immunomodulators Targeting the Protease Active Site.

Cells of the immune system utilize multiple proteases to regulate cell functions and orchestrate innate and adaptive immune responses. Dysregulated protease activities are implicated in many immune-related disorders; thus, protease inhibitors have been actively investigated for pharmaceutical development. Although historically considered challenging with concerns about toxicity, compounds that covalently modify the protease active site represent an important class of agents, emerging not only as chemical probes but also as approved drugs. Here, we provide an overview of technologies useful for the study of proteases with the focus on recent advances in chemoproteomic methods and screening platforms. By highlighting covalent inhibitors that have been designed to target immunomodulatory proteases, we identify opportunities for the development of small molecule immunomodulators.

Increased local expression of P-glycoprotein on CD4+ T-cells in vitreous of patients with non-infectious uveitis: a pilot study.

Nearly a third of uveitis patients are unable to achieve adequate inflammation control with conventional anti-inflammatory therapy. Several factors are known to influence responsiveness to anti-inflammatory therapy. In this pilot study, we have investigated the local expression of P-glycoprotein, an efflux-transport protein with role in multi-drug resistance, in vitreous CD4+ T-cells of patients with non-infectious uveitis (NIU). CD4+ T-cells were isolated from vitreous and peripheral venous blood samples of NIU patients undergoing therapeutic vitrectomy. Rhodamine-123, a substrate of P-glycoprotein, whose retention inside cells is inversely proportional to P-glycoprotein function, was used to assay this transporter protein. In addition, cells were stained with IFN-γ, IL-17, GM-CSF and FoxP3, and analysed by flow cytometry. T-cell mitochondria were imaged by Mitotracker Red and confocal microscopy. Vitreous CD4+ T-cells expressed significantly higher P-gp and pro-inflammatory (IL-17+, IFNγ+IL17+) cytokine expression than matching blood samples. Mitochondrial fission was noted in vitreous T-cells and fusion in blood. We concluded that NIU is associated with higher P-glycoprotein expression and pro-inflammatory state in vitreous than in blood. This supports P-glycoprotein inhibition and adjunctive local anti-inflammatory treatment in management of NIU.

Role of Multidrug Resistance Proteins in Nonresponders to Immunomodulatory Therapy for Noninfectious Uveitis.

Purpose: Nearly a third to half of patients with noninfectious uveitis (NIU) fail to achieve control with immunomodulatory therapy (IMT). Multidrug resistance (MDR) proteins are transmembrane proteins that allow efflux of intracellular drugs, leading to drug resistance. The aim of our study was to compare MDR protein function in blood CD4+ cells between responders and nonresponders to IMT. Methods: We included NIU patients on IMT for ≥6 months and corticosteroid dose ≤10 mg/d. Nonresponders to treatment were those with worsening (two or more steps) of inflammation in the past 3 months on full-dose immunosuppressive therapy. MDR function was assessed by Rhodamine-123 dye retention in blood CD4+ cells. Three nonresponders were treated with adjunctive oral cyclosporine A (CSA, MDR inhibitor) therapy for 2 months and reevaluated. Results: Fourteen NIU patients were recruited. Most (n = 8) had Vogt-Koyanagi-Harada disease. These included nine nonresponders and five responders to IMT. Nonresponders produced significantly higher MDR function and proinflammatory cytokines (interferon γ, tumor necrosis factor α, interleukin 17, and Granulocyte Macrophage Colony Stimulating Factor (GM-CSF)) than responders. In vitro CSA treatment of CD4+ cells inhibited MDR expression and proinflammatory cytokine production while increasing Foxp3. Finally, adjunctive oral CSA therapy led to improvement in clinical inflammatory scores with a concurrent decrease in MDR function and proinflammatory cytokine secretion. Conclusions: MDR function is significantly higher in CD4+ T cells of nonresponders to IMT. Adjunctive CSA therapy may decrease MDR function and allow improvement in treatment response to IMT. Translational Relevance: Our study highlights the need for MDR inhibition strategies in NIU patients not responding to IMT for improving the efficacy of anti-inflammatory therapy.

Identification and expression analysis of miRNAs and elucidation of their role in salt tolerance in rice varieties susceptible and tolerant to salinity.

Soil salinization is a serious problem for cultivation of rice, as among cereals rice is the most salt sensitive crop, and more than 40% of the total agricultural land amounting to approximately 80 million ha the world over is salt affected. Salinity affects a plant in a varieties of ways, including ion toxicity, osmotic stress and oxidative damage. Since miRNAs occupy the top place in biochemical events determining a trait, understanding their role in salt tolerance is highly desirable, which may allow introduction of the trait in the rice cultivars of choice through biotechnological interventions. High throughput sequencing of sRNAs in the root and shoot tissues of the seedlings of the control and NaCl treated Pokkali, a salt-tolerant rice variety, identified 75 conserved miRNAs and mapped 200 sRNAs to the rice genome as novel miRNAs. Expression of nine novel miRNAs and two conserved miRNAs were confirmed by Northern blotting. Several of both conserved and novel miRNAs that expressed differentially in root and/or shoot tissues targeted transcription factors like AP2/EREBP domain protein, ARF, NAC, MYB, NF-YA, HD-Zip III, TCP and SBP reported to be involved in salt tolerance or in abiotic stress tolerance in general. Most of the novel miRNAs expressed in the salt tolerant wild rice Oryza coarctata, suggesting conservation of miRNAs in taxonomically related species. One of the novel miRNAs, osa-miR12477, also targeted L-ascorbate oxidase (LAO), indicating build-up of oxidative stress in the plant upon salt treatment, which was confirmed by DAB staining. Thus, salt tolerance might involve miRNA-mediated regulation of 1) cellular abundance of the hormone signaling components like EREBP and ARF, 2) synthesis of abiotic stress related transcription factors, and 3) antioxidative component like LAO for mitigation of oxidative damage. The study clearly indicated importance of osa-miR12477 regulated expression of LAO in salt tolerance in the plant.

Autoreactive T Cells in Immunopathogenesis of TB-Associated Uveitis.

Purpose: Intraocular inflammation in tuberculosis-associated uveitis (TBU) is usually widespread, and responds unpredictably to treatment. Herein, we analyze the intraocular T-cell response in TBU for its surface phenotype, antigenic specificity, and functional characteristics to explain the above observations. Methods: We isolated T cells from vitreous humor samples of patients with TBU and non-TB uveitis (controls). These were directly stained for surface markers CD4, CD8, CD45RO, CD45RA, CCR7, as well as intracellular cytokines IFN-γ, TNF-α, and IL-17 and analyzed on flow cytometry. Antigenic specificity was determined by activating with Mycobacterium tuberculosis-specific antigen Early Secreted Antigenic Target-6 (ESAT-6) or retinal crude extract (RCE). Activation-induced cell death (AICD) characteristics of each T-cell population were analyzed by staining for PI-Annexin V, Fas-FasL, phospho-Akt, and phospho-Erk1/2. Results: Immunophenotyping of vitreous humor samples demonstrated polyfunctional effector and central memory CD4+ T helper cells coexpressing IFN-γ, TNF-α, and IL-17. Both ESAT-6 and RCE (autoreactive) specificity was found in T cells extracted from TBU samples; however, the mycobacterial and autoreactive T-cell populations differed in their sensitivity to AICD. Autoreactive T cells appeared to resist AICD through decreased expression of apoptotic markers, FasL and caspase-3, sustained phosphorylation of Akt, and lowered Erk1/2 activity. Conclusions: Autoreactive T cells are present in TBU eyes and are relatively resistant to AICD. An understanding of this epiphenomenon could be crucial in planning treatment of TBU patients, and interpreting response to anti-TB therapy.

Inherent low Erk and p38 activity reduce Fas Ligand expression and degranulation in T helper 17 cells leading to activation induced cell death resistance.

Activation Induced Cell Death of T helper cells is central to maintaining immune homeostasis and a perturbation often manifests in aberrant T helper cells that is associated with immunopathologies. Significant presence of T cells positive for IL-17A (Th17) and dual positive for IFN-γ/IL-17A (Th1/Th17) in both effector (CD45RA+RO+) and memory (CD45RA-RO+) compartments with differential FasL protein in RA peripheral blood suggested their differential TCR AICD sensitivity. Lowered active caspase-3 in Th17 and Th1/Th17 over Th1 cells confirmed their capability to resist AICD and pointed to early upstream events. Differential MAPK activities, FasL protein and downstream caspase-3 activities in murine Th1 and Th17 cells established distinct TCR mediated signaling pathways and suggested low Erk and p38 activity as pivotal for AICD sensitivity. We extrapolated our mouse and human data and report that Fas-FasL is the preferred death pathway for both Th1 and Th17 and that inherently low Erk2 activity protected Th17 cells from TCR AICD. The presence of significantly higher numbers of aberrant T helper cells in RA also suggest an inflammatory cytokine milieu and AICD insensitive T cell link to sustained inflammation. Re sensitization to apoptosis by targeting MAPK activity especially Erk2 in RA might be of therapeutic value.

Synergy of iron, high sensitivity C-reactive protein and ceruloplasmin with oxidative stress in non-diabetic normo-tensive South Indian obese men.

BACKGROUND: Oxidative stress and inflammation are implicated in the pathogenesis of obesity and its related complications. Previous studies have suggested a potential link between obesity and altered iron metabolism. The present study was designed to evaluate iron, C-reactive protein, ceruloplasmin and oxidative stress and their association, if any, in non-diabetic normo-tensive South Indian obese men. METHODS: 30 obese men and 30 age-matched males with normal body weight were recruited in the study. Serum iron, copper, ceruloplasmin, high sensitivity C-reactive protein (hs-CRP), malondialdehyde, protein carbonyl, total oxidant status and total antioxidant status were estimated in all the subjects. RESULTS: Serum iron, ceruloplasmin, high sensitivity C-reactive protein (hs-CRP), malondialdehyde (MDA), protein carbonyl and total oxidant status were significantly increased and total antioxidant status was significantly reduced in obese men, compared to controls. Linear regression analysis shows highly significant positive association of iron with hs-CRP. CONCLUSION: The data from the present study concludes that oxidative stress parameters, hs-CRP, iron and ceruloplasmin were significantly elevated in obese Indian men, suggesting they are more prone to develop cardiovascular disease, than age-matched men with normal body weight.