Overview on the Discovery and Development of Anti-Inflammatory Drugs: Should the Focus Be on Synthesis or Degradation of PGE2?

Inflammation is a protective response that develops against tissue injury and infection. Chronic inflammation, on the other hand, is the key player in the pathogenesis of many inflammatory disorders including cancer. The cytokine storm, an inflammatory response flaring out of control, is mostly responsible for the mortality in COVID-19 patients. Anti-inflammatory drugs inhibit cyclooxygenases (COX), which are involved in the biosynthesis of prostaglandins that promote inflammation. The conventional non-steroidal anti-inflammatory drugs (NSAIDs) are associated with gastric and renal side-effects, as they inhibit both the constitutive COX-1 and the inducible COX-2. The majority of selective COX-2 inhibitors (COXIBs) are without gastric side-effects but are associated with cardiac side-effects on long-term use. The search for anti-inflammatory drugs without side-effects, therefore, has become a dream and ongoing effort of the Pharma companies. As PGE2 is the key mediator of inflammatory disorders, coming up with a strategy to reduce the levels of PGE2 alone without affecting other metabolites may form a better choice for the development of next generation anti-inflammatory drugs. In this direction the options being explored are on synthesis of PGE2-mPGES-1; PGE2 degradation through a specific PG dehydrogenase, 15-PGDH, and by blocking its activity mediated through a specific PGE receptor, EP4. As leukotrienes formed via the 5-lipoxygenase (5-LOX) pathway also play an important role in the mediation of inflammation, efforts are also being made to target both COX and LOX pathways. This review focuses on addressing the following three points: 1) How NSAIDs and COXIBs are associated with gastric, renal and cardiac side-effects; 2) Should the focus be on the targets upstream or downstream of PGE2; and 3) the status of alternative targets being explored for the discovery and development of anti-inflammatory drugs without side-effects.

Expression and Purification of Functionally Active Serotonin 5-HT2A Receptor in Insect Cells Using Low-titer Viral Stock.

The serotonin 5-HT2A receptor (5-HT2AR) is a member of the GPCR family that is important for various neurological functions and whose dysregulation causes many mental health disorders. Structural investigations of 5-HT2AR require the production of functionally active receptors expressed from eukaryotic cell cultures. In this protocol, we describe a step-by-step method to express and purify serotonin 5-HT2AR using a baculoviral expression vector system in Sf9 cell cultures, derived from our work with the rat (matching Uniprot ID P14842) and human (matching Uniprot ID P28223) 5-HT2ARs. A unique feature of this method is the utilization of cell culture additives to infect cells at low multiplicity of infection, thereby using several fold less quantity of viral titer compared to prior methods without the additive. This protocol can be tweaked to selectively over-express glycosylated or non-glycosylated forms of the receptor by varying the post-infection harvest times.

Cloning, Expression, Purification and Characterization of Oligomeric States of the Native 5HT2A G-Protein-Coupled Receptor.

BACKGROUND: The 5HT2A G-Protein Coupled Receptor (GPCR) is an important family of receptors involved in an array of neuromodulatory functions. Their dysregulation has been implicated in a number of psychiatric diseases. In spite of the importance of this GPCR, high resolution structure and mechanistic details of its function is unknown. Cholesterol plays an important role in the function of many receptors and reduced cholesterol levels can lead to disruption of serotonergic pathways. However, the role of cholesterol in the formation of GPCR oligomers has not been previously shown for this receptor. Given that receptor dimers have been shown to be the functional unit of this receptor, it is important to investigate the effect of cholesterol in the oligomeric state of 5HT2A receptor. OBJECTIVES: The main objective of this work is to clone, over-express and purify the 5HT2A receptor and investigate the effect of cholesterol in its oligomer formation. METHODS: The 5HT2A receptor (5HT2AR) DNA construct was subcloned into pFastBac-HT vector and the purified bacmid was used to transfect healthy Sf9 cells. After subsequent passages, a high titer baculovirus was used for over-expression in Sf9 cells. To verify whether the over-expressed receptor was localized in the membrane or cytosolic fraction, cells with and without baculoviral infection were analyzed by immunocytochemistry. Subsequently, the over-expression conditions required to obtain sufficient quantity of the receptor was optimized followed by the optimization of the purification conditions. Finally, the culture was scaled up and the receptor was purified by affinity chromatography. The over-expression of the receptor was checked by Western blotting and purity was analyzed by Coomassie stained SDS PAGE. Cryo-electron microscopy experiments were performed on the purified receptor in presence and absence of cholesterol and at multiple concentrations to rule out any concentration dependent effect on the oligomer formation. RESULTS: Immunocytochemistry experiments showed prominent nuclear staining; however, bright green staining along the cell membrane was observed only for the infected cells, suggesting appropriate trafficking of majority of the over-expressed receptors to the cell membrane. Results of cryoelectron microscopy show that the receptor with cholesterol had particles that were bigger in size (~11 - 12 nm) compared to the dimension of known GPCR homologs. In contrast, the receptor after removal of cholesterol revealed a uniform distribution of smaller particles (~5 - 6 nm) that is approximately half the size of 5HT2AR particles with cholesterol. Comparing the 2D average views of detergent-encapsulated 5HT2AR particles with the overall dimensions of other 5HT receptor analogs, we show that while a 5HT2AR dimer more closely matches the dimensions of particles with CHS, only a monomer can be fit to particles without CHS. Importantly, even at higher receptor concentration and particle density, the size for 5HT2AR particles without CHS remains the same, suggesting that dimerization is unlikely an effect of concentration. CONCLUSION: Our results indicated that 5HT2A receptor primarily forms a dimer in presence of cholesterol whereas it predominantly forms a monomer when cholesterol is removed.