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Lipidic mediators
Esters or amides, like other eicosanoids are synthesized "on demand"
Oleoylethanolamide (regulates feeding and body weight) and palmitoylethanolamide (anti-inflammatory and analgesic) are closely related molecules but exert their actions through PPAR- .
Degradating enzymes
FAAH (Fatty acid amide hydrolase): Integral membrane protein present as homodimer, is a serine hydrolase that break down mainly amides. MAGL(Monoacylglycerol lipase): Esterase with a wide and hydropobic acces to the active site, homodimer, integral membrane protein. Partecipate to lipolysis in adipocytes.
Sn: Stereospecific Numbering. In order to designate the configuration of glycerol derivatives, the carbon atoms of glycerol are numbered stereospecifically. www.chem.qmul.ac.uk/iupac/lipid
FAAH,
a look to active site
Complex of FAAH protein and anandamide embedded in a 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE) lipid bilayer. FAAH is depicted in gray ribbons. The surface of residues belonging to the membrane access channel (MA), the acyl chain binding channel (AB), as well as the cytosolic port (CP) is shown in red, orange, and sky-blue, respectively. Phe432 and Trp531 at the intersection of the MA and AB channels are shown in green and violet.
Mechanism
The catalytic triad is not that characteristic of serine hydrolase Ser-His-Asp but Ser241, Lys142, Ser217. Ser241 was identified as the nucleophile in the active site of FAAH through mutational and affinity labeling studies. Lys142 acts as both a base to activate Ser241 and as an acid that helps protonate the substrate leaving group. Structural studies established that both of these activities of Lys142 are indirect and mediated through the hydroxyl side chain of Ser217.
J. Mol. Biol.(2010) 400, 743754
Determine structure
Analyze structure to determine possible inhibitor binding site
Dock and score compound from database against the selected site
Analyze ranked list of scored compound and optimize top pick for binding and selectivity
no
Can lead be ye optimized? s
Modify and optimize in silico
no
no
Is a nM inhibitor?
ye s
A) FAAH dimer is shown with the protein surface rendered in gray (hydrophilic) and green (hydrophobic). Electron density corresponding to the arachidonyl inhibitor is shown in violet. B) Aromatic and aliphatic residues in the substrate binding pocket surrounding the arachidonyl chain and their interactions are indicated
First structure determination of FAAH. Protein crystals by recombinantly expressing rat FAAH with 29 amino acids deleted at the NH2-terminus.
Science. 2002 Nov 29;298(5599):1793-6.
rFAAH
The availability of an X-ray structure of the PF-3845, complexed to FAAH allowed us to visualize the structural requirements needed to design a replacement for the core piperidinyl moiety, while maintaining other key interactions.
The position of the carbonyl made hydrogen bonds to several amide protons as part of the stabilization of transition state.
The 5-trifluoromethyl-pyridinyl moiety offered favorable potency and in vivo properties. (note the absence of H bond)
The HetAr-O-Ph moiety of PF-3845 formed both aromatic and van der Waals contacts with the protein. (note the absence of H bond)
PDB: 2wap
A computational approach was utilized to assist in the evaluation of various linkers, the docking result convinced us to explore the 3-carbon linker azetidine. kinact/Ki*
PF-3845 (green, X-ray), superimposed against model of compound 6 (red).
Established the linker, it was evaluated the effect of heteroaromatic substitution and conformational restraints .
*kinact: maximal rate of inactivation at saturation Ki: inhibitor concentration leading to 50% of kinact
The SAR revealed that the human FAAH was highly sensitive to tail pieces. The analogs with pyridazine urea head displayed the best human FAAH inhibition. Analogs with dimethyl oxazole showed a preference for the rat FAAH.
Modeling suggested that the saturated linker may occupy a transoid conformation. Superposition of conformationally optimized (covalently attached to Ser241) saturated (light green) 3carbon linker 6a, 5a trans (light blue), 13a trans (red), and 13a cis (orange). Results: The selectivity profile observed for PF-3845 was retained (ABPP proteome profiling*) . Compound 13 has good PK properties in vivo, low clearance and high bioavailability (73% and 93%); neither 13cis nor 13trans had comparable in vivo efficacy to PF-3845. Further optimization of azetidine would be required.
*ABPP: Activity based proteomics, probe consists of a reactive group and a tag. -ABPP-MudPIT: multidimensional protein identification technology, ABPP+MS/MS
Carbofuran
Fenobucarb
Carbaryl
These insecticides act inhibiting the enzyme acetylcholinesterase (serine hydrolase) in a time-dependent manner via carbamoylation. Carbaryl* was choosen for FAAH inhibitor design by progressive modification.
*This compound is sadly famous for the Bhopal disaster (India, 1984), the largest industrial accident in history with 11,000 deaths and
over 500,000 injuries.
Results:
Amongst carbaryl (19) analogue only 2-naftyl (21) show a weak activity. Passing from N-me (21) to N-cicloesyl (22) the IC50 decrease of 60 fold. Carbamate isosteric modification in 13, 16, 18 suggest that is a fondamental group. N-butyl derivatives (7, 23) has submicromolar potencies, the very low activity of 24 confirmed the essential role of the O substituent. Compound 7, 20-24 had no effect on AchE, CB1, CB2 and didnt affect AEA transport. Carbamic OR2 seems to act as leaving group. 9e, 25, 26 and 9a,d,f,g were synthetized for indagate the shape required for R2, the potency of the second series suggest a bent shape.
Superposition of the biphenyl moiety of 9i (orange carbons) to the lipophilic chain of arachidonic acid, cocrystallized with adipocyte lipid-binding protein (white carbons), COX-1 (yellow carbons), or COX-2 (green carbons).
Plot of the experimentally observed pIC50 data vs those calculated by the 3D-QSAR CoMSIA* model for the 14 compounds reported on the right.
3D-QSAR graphical depiction of the coefficients for the CoMSIA fields, calculated by PLS* analysis on the pIC50values of the 14 compounds reported in Table 2. All the compounds are represented as lines except7(white carbons) and 9g (orange carbons), which are represented as sticks. The colored volumes indicate the points where the influence of the steric potential on pIC50 is more significant. The color codes are the following: blue, very positive; green, positive; yellow, negative
*CoMSIA: Comparative molecular similarity indices analysis, computational tecniques that allow to build statistical and graphical models that relate the properties of molecules (including biological activity) to their structures. *PLS: Partial least squares regression
Chemistry
Phosgene
DMAP
Isocyanates synthesis:
Addition:
DOI: 10.1021/ol200695y
Amines addition:
Alcohols addition: