Targeting next-generation PDE4 inhibitors in search of potential management of rheumatoid arthritis and psoriasis.

Immune-mediated inflammatory diseases (IMIDs) comprise a broad spectrum of conditions characterized by systemic inflammation affecting various organs and tissues, for which there is no known cure. The isoform-specific inhibition of phosphodiesterase-4B (PDE4B) over PDE4D constitutes an effective therapeutic strategy for the treatment of IMIDs that minimizes the adverse effects associated with non-selective PDE4 inhibitors. Thus, we report a new class of isoquinolone derivatives as next-generation PDE4 inhibitors for effective management of rheumatoid arthritis (RA) and psoriasis.

TGS1/PIMT knockdown reduces lipid accumulation in adipocytes, limits body weight gain and promotes insulin sensitivity in mice.

PRIP Interacting protein with Methyl Transferase domain (PIMT/TGS1) is an integral upstream coactivator in the peroxisome proliferator-activated receptor gamma (PPARγ) transcriptional apparatus. PPARγ activation alleviates insulin resistance but promotes weight gain. Herein, we show how PIMT regulates body weight while promoting insulin sensitivity in diet induced obese mice.

TGS1/PIMT regulates pro-inflammatory macrophage mediated paracrine insulin resistance: Crosstalk between macrophages and skeletal muscle cells.

Macrophage-driven chronic low-grade inflammatory response is intimately associated with pathogenesis of insulin resistance and type 2 diabetes (T2D). However, the molecular basis for skewing of pro-inflammatory macrophage is still elusive. Here, we describe the mechanism and significance of TGS1/PIMT (PRIP-Interacting protein with Methyl Transferase domain) in regulating macrophage activation and polarization and its impact on the development of insulin resistance in skeletal muscle cells.

PIMT Controls Insulin Synthesis and Secretion through PDX1.

Pancreatic beta cell function is an important component of glucose homeostasis. Here, we investigated the function of PIMT (PRIP-interacting protein with methyl transferase domain), a transcriptional co-activator binding protein, in the pancreatic beta cells. We observed that the protein levels of PIMT, along with key beta cell markers such as PDX1 (pancreatic and duodenal homeobox 1) and MafA (MAF bZIP transcription factor A), were reduced in the beta cells exposed to hyperglycemic and hyperlipidemic conditions.

PHLPP1 regulates PINK1-parkin signalling and life span.

Adaptability to intracellular or extracellular cues is essential for maintaining cellular homeostasis. Metabolic signals intricately control the morphology and functions of mitochondria by regulating bioenergetics and metabolism. Here, we describe the involvement of PHLPP1, a Ser/Thr phosphatase, in mitochondrial homeostasis.

PIMT regulates hepatic gluconeogenesis in mice.

The physiological and metabolic functions of PIMT/TGS1, a third-generation transcriptional apparatus protein, in glucose homeostasis sustenance are unclear. Here, we observed that the expression of PIMT was upregulated in the livers of short-term fasted and obese mice. Lentiviruses expressing Tgs1-specific shRNA or cDNA were injected into wild-type mice.

An efficient and cost-effective method for directed mutagenesis at multiple dispersed sites-a case study with Omicron Spike DNA.

Site-directed mutagenesis is an invaluable technique that enables the elucidation of the contribution of specific residues to protein structure and function. The simultaneous introduction of mutations at a large number of sites (>10), singly and in multiple combinations, is often necessary to fully understand the functional contributions. We report a simple, efficient, time and cost-effective method to achieve this using commonly available molecular biology reagents and protocols, as an alternative to gene synthesis.

Protocol to evaluate hyperlipidemia in zebrafish larvae.

In this report, we describe an approach to generate a zebrafish larval model of lipid accumulation that can be used as an system to study hyperlipidemic conditions such as atherosclerosis. Furthermore, we detail steps on staining techniques, lipid estimation assays, RNA isolation, and utilization of ImageJ to evaluate larval dimensions and to explore the model in the context of hyperlipidemia. Researchers should be aware of context specificity of the proposed protocols and interpret results accordingly.

PHLPPs: Emerging players in metabolic disorders.

That reversible protein phosphorylation by kinases and phosphatases occurs in metabolic disorders is well known. Various studies have revealed that a multi-faceted and tightly regulated phosphatase, pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP)-1/2 displays robust effects in cardioprotection, ischaemia/reperfusion (I/R), and vascular remodelling. PHLPP1 promotes foamy macrophage development through ChREBP/AMPK-dependent pathways.

Fe(III)-catalyzed regioselective and faster synthesis of isocoumarins with 3-oxoalkyl moiety at C-4: Identification of new inhibitors of PDE4.

In search of potent and new anti-inflammatory agents, we explored a new class of isocoumarin derivatives possessing the 3-oxoalkyl moiety at C-4 position. These compounds were synthesized via the FeCl catalyzed construction of isocoumarin ring. The methodology involved coupling of 2-alkynyl benzamides with alkyl vinyl ketone and proceeded via a regioselective cyclization to give the desired compound as a result of formation of CO and CC bonds.

PHLPP1 promotes neutral lipid accumulation through AMPK/ChREBP-dependent lipid uptake and fatty acid synthesis pathways.

Infiltration of arterial intima by foamy macrophages is a hallmark of early atherosclerotic lesions. Here, we investigated the potential role of Ser/Thr phosphatase PHLPP1 in foam cell development. PHLPP1 levels were elevated in OxLDL-exposed macrophages and high-fat diet (HFD)-fed zebrafish larvae.

Pd-catalysed general access to 7-membered N/O-heterocyclic compounds as potential agents against inflammation.

A Pd-catalysed regioselective synthesis of 4,5-disubstituted 7-membered N/O-heterocycles was achieved the 7- cyclization followed by C-C bond formation of 2-(1-alkynyl)phenylacetamide. The ligand/additive free cascade reaction proceeded in the presence of PdCl in aqueous MeCN when the separate and individual use of methyl vinyl ketone and allyl bromide generally afforded an O- and N-heterocycle, respectively. The pharmacological assay was performed to identify the first example of a 1-benzo[]azepin-2(3)-one based novel inhibitor of PDE4B.

CuCl-catalyzed inexpensive, faster and ligand/additive free synthesis of isoquinolin-1(2H)-one derivatives via the coupling-cyclization strategy: Evaluation of a new class of compounds as potential PDE4 inhibitors.

In spite of possessing a wide range of pharmacological properties the anti-inflammatory activities of isoquinolin-1(2H)-ones were rarely known or explored earlier. PDE4 inhibitors on the other hand in addition to their usefulness in treating inflammatory diseases have been suggested to attenuate the cytokine storm in COVID-19 especially TNF-α. In our effort, a new class of isoquinolin-1(2H)-ones derivatives containing an aminosulfonyl moiety were designed and explored as potential inhibitors of PDE4.

A variant in is associated with decreased disease severity in COVID-19.

Background: Mortality due to COVID-19 caused by SARS-CoV-2 infection varies among populations. Functional relevance of genetic variations in Angiotensin-converting enzyme 2 () and Transmembrane serine protease 2 (), two crucial host factors for viral entry, might explain some of this variation.

Methods: In this comparative study in Indian subjects, we recruited 510 COVID-19 patients and retrieved DNA from 520 controls from a repository.

PdCl-catalyzed synthesis of a new class of isocoumarin derivatives containing aminosulfonyl / aminocarboxamide moiety: First identification of a isocoumarin based PDE4 inhibitor.

While anti-inflammatory properties of isocoumarins are known their PDE4 inhibitory potential was not explored previously. In our effort the non-PDE4 inhibitor isocoumarins were transformed into the promising inhibitors via introducing an aminosulfonyl/aminocarboxamide moiety to the C-3 benzene ring attached to the isocoumarin framework. This new class of isocoumarins were synthesized via a PdCl-catalyzed construction of the 4-allyl substituted 3-aryl isocoumarin ring starting from the appropriate 2-alkynyl benzamide derivative.

Synthesis of 11,12-dihydro benzo[c]phenanthridines via a Pd-catalyzed unusual construction of isocoumarin ring/FeCl-mediated intramolecular arene-allyl cyclization: First identification of a benzo[c]phenanthridine based PDE4 inhibitor.

In spite of their various pharmacological properties the anti-inflammatory potential of benzo[c]phenanthridines remained underexplored. Thus, for the first time PDE4 inhibitory potential of 11,12-dihydro benzo[c]phenanthridine/benzo[c]phenanthridine was assessed in vitro. Elegant synthesis of these compounds was performed via a multi-step sequence consisting of a Pd-catalyzed unusual construction of 4-allyl isocoumarin ring and FeCl-mediated intramolecular regio- as well as site-selective arene-allyl cyclization as key steps.

InCl mediated heteroarylation of indoles and their derivatization via CH activation strategy: Discovery of 2-(1H-indol-3-yl)-quinoxaline derivatives as a new class of PDE4B selective inhibitors for arthritis and/or multiple sclerosis.

A new class of PDE4 inhibitors were designed and synthesized via the InCl mediated heteroarylation of indoles and their further derivatization through the Pd(II)-catalyzed CH activation strategy. This effort allowed us to discover a series of 2-(1H-indol-3-yl)-quinoxaline based inhibitors possessing PDE4B selectivity over PDE4D and PDE4C. One of these compounds i.

2-[2-(4-(trifluoromethyl)phenylamino)thiazol-4-yl]acetic acid (Activator-3) is a potent activator of AMPK.

AMPK is considered as a potential high value target for metabolic disorders. Here, we present the molecular modeling, in vitro and in vivo characterization of Activator-3, 2-[2-(4-(trifluoromethyl)phenylamino)thiazol-4-yl]acetic acid, an AMP mimetic and a potent pan-AMPK activator. Activator-3 and AMP likely share common activation mode for AMPK activation.

ERK1/2 activated PHLPP1 induces skeletal muscle ER stress through the inhibition of a novel substrate AMPK.

Nutritional abundance associated with chronic inflammation and dyslipidemia impairs the functioning of endoplasmic reticulum (ER) thereby hampering cellular responses to insulin. PHLPP1 was identified as a phosphatase which inactivates Akt, the master regulator of insulin mediated glucose homeostasis. Given the suggestive role of PHLPP1 phosphatase in terminating insulin signalling pathways, deeper insights into its functional role in inducing insulin resistance are warranted.

In-house made nucleofection buffer for efficient and cost effective transfection of RAW 264.7 macrophages.

Electroporation is the most widely employed method of gene transfer into macrophages which are hard to transfect. RAW 264.7 is a widely used cell line for studying macrophage responses.

LPS depletes PHLPP levels in macrophages through the inhibition of SP1 dependent transcriptional regulation.

We have previously reported that bacterial endotoxin LPS attenuates expression of PHLPP, a ser/thr phosphatase, at both transcript and protein levels in different immune cells, however the underlying molecular mechanism is unknown and is of significant interest. Here, in line with the decreased transcript levels upon LPS treatment, we observed that LPS caused significant reduction in PHLPP promoter activity. We observed that SP1, a transcription factor frequently associated with inflammation, was recruited to the PHLPP promoter region.

Deubiquitinase USP12 promotes LPS induced macrophage responses through inhibition of IκBα.

Post translational modifications, ubiquitination and its reversal by deubiquitination play an important role in regulating innate immune system. USP12 is a poorly studied deubiquitinase reported to regulate T-cell receptor signalling however the functional role of USP12 in macrophages, the principal architects of inflammation, is unknown. Thus, in this study we probed the involvement of USP12 in macrophage mediated inflammatory responses using bacterial endotoxin, LPS, as the model system.

MicroRNA-712 restrains macrophage pro-inflammatory responses by targeting LRRK2 leading to restoration of insulin stimulated glucose uptake by myoblasts.

Chronic inflammatory diseases such as insulin resistance, Type 2 diabetes, neurodegenerative diseases etc., are shown to be caused due to imbalanced activation states of macrophages. MicroRNAs which are transcriptional/post-transcriptional regulators of gene expression drive several pathophysiological processes including macrophage polarization.

Co-activator binding protein PIMT mediates TNF-α induced insulin resistance in skeletal muscle via the transcriptional down-regulation of MEF2A and GLUT4.

The mechanisms underlying inflammation induced insulin resistance are poorly understood. Here, we report that the expression of PIMT, a transcriptional co-activator binding protein, was up-regulated in the soleus muscle of high sucrose diet (HSD) induced insulin resistant rats and TNF-α exposed cultured myoblasts. Moreover, TNF-α induced phosphorylation of PIMT at the ERK1/2 target site Ser(298).