Cyclooxygenase-2 (COX-2) inhibitors are essential drugs, which target the active site of COX-2. Celecoxib is one such drug, a Non-Steroidal Anti-Inflammatory Drug (NSAID) used to treat osteoarthritis, rheumatoid arthritis, acute pain, menstrual symptoms, and reduce polyps in familial adenomatous polyposis. (a) Locate within the Protein Data Bank (PDB) the 3-D structure of a complex between a COX-2 bound to celecoxib. State the PDB code of your chosen entry and download the coordinates for the structure to use with a molecular graphics program to investigate your chosen structure.
Inflammation is mediated in part by prostaglandins produced by the cyclooxygenase pathway. NSAIDs inhibit this pathway and serve as a combined anti-inflammatory, anti pyretic, and analgesic. Because NSAIDs are generally nonspecific and exert numerous side effects, there is great interest in more specific therapeutics such as selective COX-2 inhibitors and anti-cytokine agents.
Prostaglandins: Physiologic and Pathologic Functions
All cells in the body can synthesize prostaglandins. Arachidonic acid (AA) is separated from plasma phospholipids by phospholipase A2 in response to inflammatory stimuli. Cyclooxygenase metabolizes AA to the cycloendoperoxide prostaglandin H2 (PGH2), which is then converted to either PGD2, PGE2, PGF2α, PGI2 (prostacyclin) or TXA2 (thromboxane) by appropriate enzymes (i.e., thromboxane synthase in platelets, prostacyclin synthase in endothelial cells).
B (b) Using one of the programs RasMol, UCSF Chimera or Swiss-PDB Viewer, produce an image of the protein that you think clearly illustrates the major structural features within the enzyme and highlights the binding site of the inhibitor molecule. State the commands used within the selected program to obtain your image.
Anti-Inflammatory Drugs: NSAIDs, COX-2 Selective Inhibitors, Glucocorticoids and Anti-Cytokine Age
The prostaglandins exert numerous physiologic and pathophysiologic functions :
Physiologic: temperature homeostasis, bronchial tone, cytoprotection (gastric and renal mucosa), intestinal mobility, myometrial tone, semen viability (some prostaglandins like PGE1 have anti-inflammatory effects), renin secretion
Pathologic: fever (aberrant hypothalamic thermoregulation), asthma (airway responsiveness and immune hyperreactivity), ulcers (loss of cytoprotection), diarrhea (intestinal mobility), dysmenorrhea (myometrial tone), inflammation, bone erosion, pain (thought to be caused by PGD2)
Specific functions of prostaglandins in the context of inflammation include:
PGI2: inhibits platelet aggregation, vasodilatation, vascular permeability (edema)
PGE2: pain, hyperalgesia, heat, vasodilatation, bronchoconstriction, synergistically
act with other pro-inflammatory mediators (histamine, complement, LTB4)
TXA2: promotes platelet aggregation, vasoconstriction, bronchoconstriction
There are two forms of cyclooxygenase (COX) enzymes: COX-1 and COX-2. Though COX-1 and COX-2 catalyze the same reaction, their expression, functions, and properties are markedly different.
Constitutive (activated by physiologic stimuli)
“Housekeeping” and Maintenance
Inducible by pro-inflammatory stimuli (LPS, TNFα, IL-2, IFNγ, etc.)
Inflammatory and neoplastic sites (small amounts in kidney, uterus, ovary, CNS [neocortex, hippocampus])
Pro-inflammatory and mitogenic functions (? Neuronal plasticity)
COX-1 produces PGE2, PGI2, and TXA2 in platelets, GI mucosa, vascular endothelium, and the kidney. The housekeeping functions of these prostaglandins include maintaining renal and gastrointestinal blood flow (cytoprotection), regulation of vascular homeostasis, renal function, intestinal mucosal proliferation, and platelet function.
Pro-inflammatory functions of COX-2 produced prostaglandins include pain, fever, leukocyte proliferation, and inflammation. COX-2 produces prostaglandins at sites of inflammation (in macrophages, in synovial tissue of rheumatoid arthritis joint). Mitogenic functions of COX-2 produced prostaglandin include renal genesis and reproduction
C. (c) Using tools within the PDB discussed in Lecture 7, investigate the interactions between the inhibitor ligand and its binding site. Illustrate with images the different types of interactions and give specific details of these interactions in your discussion.
The goal of pharmacologic anti-inflammatory therapy has been to inhibit COX-2 produced prostaglandins. Nonspecific inhibition of COX-1 results in gastrointestinal and platelet side effects. Recent data on the toxicity of COX-2 selective NSAIDs illustrate that this is an overly simplistic view. The magnitude of the COX-2 problem is still unclear in this writing, but it will be considered at various points in this discussion.
(Note: There is an entire additional pathway of arachidonic acid metabolism by enzymes called lipoxygenases. 5-lipoxygenase is not present in all tissues but is limited to neutrophils, eosinophils, monocytes, and specific mast cell populations. Lipoxygenases produce leukotrienes (e.g., LTB4, LTD4), potent bronchoconstrictor and chemotactic agents. Leukotrienes have essential roles in asthma, glomerulonephritis and inflammatory bowel disease. (Refer to the Asthma case.)
NSAIDs (Non-steroidal anti-inflammatory drugs)
Most NSAIDs are polycyclic carboxylic acid derivatives with relatively low pKa values. NSAIDs are often classified based on their chemical structure
Salicylates: aspirin; diflunisal, 5-aminosalicylate, sodium salicylate, magnesium salicylate, sulfasalazine, olasalzine
Acetic acids: indomethacin, diclofenac, sulindac, etodolac, ketorolac, tolmetin
Propionic acids: ibuprofen, naproxen, fenoprofen, ketoprofen, flurbiprofen,
Fenamic acids: meclofenamate, mefenamate
Enolic acids (oxicam class): piroxicam
Ketones: nabumetone (converted to 6-naphthyl acetic acid in the liver)Qn2
(d) Discuss which types of bioinformatics tools and programs could be used to investigate protein-ligand interactions and to design new potentially improved inhibitors for COX-2.
NSAID General Pharmacodynamics
All NSAIDs (except aspirin) act as reversible, competitive cyclooxygenase inhibitors. They block the hydrophobic channel by which the substrate arachidonic acid accesses the enzyme active site. Aspirin covalently modifies and destroys the cyclooxygenase enzyme.
The ultimate function of the NSAID is to inhibit COX-2, preventing the generation of pro-inflammatory eicosanoids and thus limiting the extent of the inflammation and adverse signs and symptoms.
All NSAIDs have a ratio of inhibition of COX-2 / inhibition of COX-1. The higher the ratio, the more specific the therapeutic effect and fewer GI or platelet effects.
− NSAIDs with a high ratio (100:1 to 1000:1) are COX-2 Selective (Coxib). Despite the benefits of NSAIDs, they only provide symptomatic relief, as the
underlying pathophysiology of injury generally is unaffected.
NSAIDs have three immediate therapeutic effects:
Anti-pyrexia (decreasing hypothalamic PGE2) Anti-inflammatory
NSAIDs are also used as antithrombotics. Since they impair platelet aggregation, prolong bleeding time and function as anticoagulants. The COX-2 specific inhibitors do not exert antithrombotic effects.
Other functions of NSAIDs include inhibition of Superoxide generation
Lysosomal enzyme release
Indications for Specific NSAIDs
Cytokine release (IL-6)
There are four forms of creatine kinase expressed in humans. Two are expressed in the cytosol of cells, and two are mitochondrial. In this question, you will use protein-protein interaction databases to explore differences in the biochemical function of these two forms.
(a) Use UniProt to obtain the two files of cytosolic creatine kinase and the two files of mitochondrial creatine kinase. Carry out an alignment of the protein sequences using Kalign from the EBI portal. Comment on the similarities and
(b) For all forms of creatine kinase, use the STRING portal to identify possible protein partners. Use the Settings menu to display ten interacting partners in the shell in each case. Summarise your findings.
(c) Using the information obtained in parts (a) and (b) comment on the functional differences between the cytosolic and mitochondrial forms of creatine kinase.
Differences in the primary structure of the two forms. Please refer to Table 1 to find indications common to each structural class of NSAID. See below for COX-2 selective drugs.
Nonselective NSAIDs are used as analgesics for moderate pain of musculoskeletal and inflammatory origin (headaches, dysmenorrhea, osteoarthritis, rheumatoid arthritis, gout, surgical pain, tendonitis, and bursitis). NSAIDs also function as anti-inflammatory agents in many of these conditions and ulcerative colitis. Aspirin anti-platelet effects are used for MI and stroke prevention. Acetaminophen (technically not an NSAID) has no anti-inflammatory activity but is widely used as an analgesic and anti-pyretic.
As stated previously, NSAIDs are weak organic acids. They generally have Efficient GI absorption (nearly complete)
Low first-pass hepatic metabolism
Small volumes of distribution but extensive protein binding (>95%) slows the rate at which these drugs cross the capillary wall and penetrate tissue.
Accumulation in cells at sites of inflammation (acidic NSAIDs are preferentially sequestered in inflamed synovial tissues)
Efficient enterohepatic and renal excretion
Variable half-lives (the lower the pKa generally, the shorter the half-life)
Plasma Elimination Half Lives Another method to classify NSAIDs (besides the structure) Short Half-Life (< 6 hours): more immediate effect and clearance.
Aspirin (0.25-0.33 hrs), Diclofenac (1.1 ± 0.2 hrs), Ketoprofen (1.8 ± 0.4 hrs),
Ibuprofen (2.1 ± 0.3 hrs), Indomethacin (4.6 ± 0.7 hrs)
Long Half-Life (> 10 hours): slower onset of effect and slower clearance
Naproxen (14 ± 2 hrs), Sulindac (14 ± 8hrs), Namebutone (26 ± 5 hrs), Piroxicam (57 ± 22 hrs) (also COX-2 Selective Inhibitors)
Important Drug Interactions.
Using the H. sapiens sequence for the beta-adrenergic receptor kinase 2 (Uniprot reference code P35626), run homology modeling for this sequence using SWISS-MODEL to obtain a 3-dimensional structure.
DISCUSS, in detail, the modeling results that you obtain, including an in-depth discussion of all of the models obtained, the template used by the program for each model, and all of the critical features of the models produced and their quality as shown from the output generated.
Download the PDB coordinates of whichever model you consider the best model obtained, and create an image of your modeled structure using RasMol, UCSF Chimera or Swiss-PdbViewer, which clearly shows the main features of the model.
− Anticoagulants (warfarin): Bleeding risk significantly increased
− Phenytoin: (increased CNS toxicity, difficulty dosing)
− Oral Hypoglycemics: (increased hypoglycemic risk)
− Methotrexate: (increased toxicity)
Anti-Hypertensives (diuretics, beta-blockers, ACE inhibitors): NSAIDs may blunt the anti-hypertensive effects and cause renal decompensation or renal failure in patients receiving these drugs
Methotrexate, digoxin, aminoglycosides, lithium: NSAIDs inhibit clearance Probenecid: renal clearance of NSAIDs reduced by probenecid
Antacids: absorption of some NSAIDs inhibited by antacids
Aspirin: may lower levels of other NSAIDs, but side effects are additive
NSAIDs affect the gastrointestinal, CNS, hepatic, renal, hematologic, and skin systems. NSAIDs also cause allergic phenomena.
Gastrointestinal Toxicity of NSAIDs
Prostaglandins suppress gastric acid secretion and help maintain the gastric mucosal barrier, thus providing gastrointestinal protection. Because they suppress prostaglandin synthesis, NSAIDs cause gastric irritation, exacerbate peptic ulcer disease, cause mucosal lesions (superficial to penetrating ulcers), and induce bleeding.
NSAID-induced gastropathy typically includes gastritis, gastric bleeding, mucosal and subepithelial damage, and erosions, which may progress to ulcerations and perforations.
Occult blood loss may occur, and massive GI bleeding may also develop.
Symptoms including pain, dyspepsia, nausea, vomiting are frequent
Overall, there is a poor correlation of these symptoms with endoscopic findings. NSAID-induced gastric toxicity causes significant morbidity, requiring annual care
expenditures of $4 billion, and causes 7500 deaths per year.
FDA estimates that ulcers, bleeding, or perforation occurs in 1 to 2 % of patients using NSAIDs for three months and 2 to 5% of those using them for one year.
Specific risk factors for NSAID-induced GI toxicity include higher NSAID doses, older age, concurrent steroid use, history of peptic ulcer disease.
Treatment of NSAID Induced GI Toxicity
Discontinuation / Avoidance of NSAIDs / Use “Gastroprotective” NSAIDs Take medication with meal
− H2 Receptor Antagonists (high doses of ranitidine)
− Proton Pump Inhibitors (omeprazole)
− Misoprostol (PGE1 analog which restores cytoprotective effects) − Sucralfate
COX-2 Specific NSAIDs – use now called into question (see discussion below)
− Reduce risks of ulceration, bleeding, perforation vs. nonselective NSAIDs
CNS toxicity includes headache, confusion, tinnitus (aspirin), dizziness, mood alteration and depression, and aseptic meningitis (particularly in SLE patients). Aspirin is linked to Reye’s Syndrome (below).
NSAIDs may cause asymptomatic liver enzyme elevations or transaminitis (most common with diclofenac. Acute idiosyncratic hepatitis has also been reported. Reye’s Syndrome is an often fatal combination of microvesicular steatosis, and hepatic encephalopathy thought to be caused by aspirin administration to children post febrile viral infection (VZV, influenza B). For this reason, aspirin is generally not given to children.
PGE2 and PGI2 play no role in controlling renal function in healthy individuals with normal kidneys. Under certain conditions of localized circulatory stress often associated
with elevated levels of angiotensin II and catecholamines, locally produced vasodilating prostaglandins become essential to the maintenance of adequate renal function
Known post-translational modifications of human Spl and Sp3 and the amino acid residues modified.
(http://www.uniprot.org/uniprot/POB047 http://www.phosphosite.o g: http://www.uniprot.org/uniprot/Q02447; the Chu and Ferro 2005 Chuang et al. 2008. Wei et al., 2009)
http://www.ncbi.nlm.nih.gov/bioproject/PRINA392950/ ..https://www.ncbi.nlm.nih.gov/bioproject/PRINA378930/. The role of reticulation in the rapid diversification of organisms is attracting greater attention in evolutionary biology: Here, we report a population genomics approach to test the role of
Phylogenetic tree of representative orthologs of SR-BI from different species Amino acid sequences from the various SR. BI orthologs were analyzed using the multiple sequence alignment program Clustal Omega from EMBL-EB: http://www.ebi.ac.uk/Tools/msa/clustalo) Structural features of