Drug design: FAAH inhibitorsDifferent approaches at work

Diego Maria CannasJ Mol Biol. 1989 Jul 20;208(2):307-25.

Endocannabinoid systemAn overview

Complex system involved in a variety of physiological functions such: memory, neurogenesis, mood, appetite, analgesia, sleep, immune system, reproduction,

thermoregulation.

Nature Reviews Urology 8, 553-561 (October 2011)

Endocannabinoid system in prostate cancer

NATURE MEDICINE • VOLUME 8 • NUMBER 6 • JUNE 2002

Possible future developments of the use of endocannabinoid-based drugs in cancer therapy

Gi/0 protein coupled receptor

CB1 receptors are amongst the most numerous in the brain (hippocampus, hypothalamus, cerebellum) are expressed also in peripheral tissues. Scarcely present in the trunk.

CB2 receptor has 45% of homology with CB1, mainly located in lymphoid tissues. The transduction mechanism don’t affect the Ca2+ channels.

Redraw by Devane et al. Copyright © Elsevier All rights reserved.

Anandamide e N-arachidonoyl dopamine acts also as TRPV1* agonist, a ion channel implied in nociception.

*transient receptor potential cation channel subfamily V member 1

Lipidic mediators

Pharmacol Rev58:389–462, 2006

Oleoylethanolamide (regulates feeding and body weight) and palmitoylethanolamide (anti-inflammatory and analgesic) are closely related molecules but exert their actions through PPAR-α .

Esters or amides, like other eicosanoids are synthesized "on demand"

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

J.Chem.TheoryComput. 2013, 9, 1202−1213

Complex of FAAH protein and anandamide embedded in a 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (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 greenand 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, 743–754

Enzyme bind to PF-3845

Ligand basedVs

Structure based

1) cloning, purification and structure determination of target protein.

2) In silico screening, compounds are scored and ranked by steric and electrostatic interactions. Best compounds are tested with biochemical assays.

3) Structure determination of target-lead complex and lead optimization.

Structure based drug design

Choose drug target

Analyze ranked list of scored compound and optimize top pick for binding and selectivity

Analyze structure to determine possible inhibitor binding site

Dock and score compound from database against the selected site

Purchase or synthetize lead and test for binding

Modify and optimize in silico

Homology modeling, use known structure and modify sequence for desidered target.

Pick next lead.Analyze and optimize

Can lead be optimized?

Is lead a micromolar inhibitor?

Clinical trials

Analyze structure for interaction

Determine structure of target and lead

Commercial drug

Obtain pure preparation of target

Determine structure

Is a nM inhibitor?

Make lead bioavailable and test for potency

no

no

no

yes

yes

yes

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.

Structure based design of novel irreversible FAAH inhibitorsBioorg. Med. Chem. Lett. 19 (2009) 5970–5974 doi:10.1016/j.bmcl.2009.07.101

Overlap PF-3845 (gray) and PF-750 (green) complexes with h/rFAAH

Overlap PF-3845-h/rFAAH and MAP-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 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

The 5-trifluoromethyl-pyridinyl moiety offered favorable potency and in vivo properties.(note the absence of H bond)

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 toSer241) saturated (light green) 3-carbon linker 6a, 5a trans (light blue), 13a trans (red), and 13a cis (orange).

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.

Results:

*ABPP: Activity based proteomics, probe consists of a reactive group and a tag.-ABPP-MudPIT: multidimensional protein identification technology, ABPP+MS/MS

Discovery of FAAH inhibitor

Carbofuran CarbarylFenobucarb

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.

Inhibitor Potencies (IC50) of Test Compounds on FAAH Activity

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 didn’t 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

CarbonyldiimidazolePhosgene DMAP

DMAP catalisys mechanism

Phosgene acyl chloride formation mechanism

Two main mechanisms:

1) SNac substitution

2) Addition to isocyanatesIsocyanates synthesis:

Amines addition to phosgene:

Addition: DOI: 10.1021/ol200695y

Isonitriles oxidation (Org. Let. 2011):

Amines addition: Alcohols addition:

Lead Compound: URB 524IC50: 63 nM

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