Stephen P.
Andrews
* and
Benjamin
Tehan
Heptares Therapeutics Limited, BioPark, Broadwater Road, Welwyn Garden City, Hertfordshire AL7 3AX, UK. E-mail: steve.andrews@heptares.com
First published on 11th September 2012
Significant progress has been made with stabilising G protein-coupled receptors (GPCRs) in recent years, and this has enabled the structures of several members of this important target class to be solved by X-ray crystallography. High resolution structural data is improving our understanding of GPCR activation and function, and is beginning to impact the drug discovery community. StaR® proteins are GPCRs which have been minimally engineered to impart thermostability. StaRs® are stable in detergent micelles and are suitable reagents for use with X-ray crystallography, biophysical screening techniques and fragment screening. This article reviews the role that StaRs® can play in the identification and optimisation of novel ligands for GPCRs by examining a specific case in which a preclinical candidate for the treatment of Parkinson's disease was developed. Compound 13 was identified following the virtual screening of experimentally enabled homology models of COMPOUND LINKS
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Download mol file of compoundadenosine A2A receptor (A2AR) and was subsequently optimised using the structural insight provided by X-ray crystallography and Biophysical Mapping of closely related molecules. Compound 19 is an exemplar from this chemical series which displays low molecular weight and high oral bioavailability; it has a good pharmacokinetic profile and is highly efficacious in preclinical models of Parkinson's disease.
Initially, these high resolution structures were solved with the receptors in inactive states; however, several structures have now been solved with GPCRs in active-state conformations,17–19 and as co-complexes with agonist, inverse agonist and neutral antagonistligands, as well as with nanobodies,20 antibodies21 and G proteins.22 These recent advances in generating high-quality GPCR structural information have sparked much interest in the field and have greatly enhanced our understanding of receptor activation and function.16,23–30 Furthermore, this structural insight may now be used to drive the rational design of drug-like molecules with high degrees of potency and selectivity for their intended GPCR target by expediting the analysis of shape- and electrostatic-complementarity between the ligands and their binding sites. Such structure-based drug design (SBDD) approaches have been highly successful with soluble targets such as kinases and proteases, but the inherent challenges with stabilising, isolating and purifying GPCRs have precluded their use with these techniques until very recently.31–33
StaR® proteins are GPCRs which have been thermostabilised by the introduction of a small number of point mutations.34 These modified proteins are stable in detergent micelles and can be removed from their native cell membranes, purified, crystallised and used with a number of biophysical screening techniques.
This article reviews the applications of StaR® proteins in the discovery and development of new ligands for the adenosine A2A receptor (A2AR). These reagents have allowed, for the first time, the application of Biophysical Mapping techniques and the generation of X-ray structures of small molecules in co-complex with GPCRs during an on-going medicinal chemistry campaign. Furthermore, these techniques have enabled SBDD of a chemical series identified by virtual screening of A2AR which has led to the nomination of a preclinical candidate for the treatment of Parkinson's disease (PD).
The four COMPOUND LINKS
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Download mol file of compoundadenosine receptor sub-types (A1, A2A, A2B and A3) all belong to GPCR family A and are activated by extracellular COMPOUND LINKS
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Download mol file of compoundadenosine. Selective agonist and antagonistligands are known for all of these receptors and have been reviewed elsewhere;36 a representative selection of A2AR ligands is shown in Fig. 1. The known A2AR agonists are almost exclusively derived from purine nucleosides and have been examined therapeutically as anti-inflammatory agents. Regadenoson is the only clinically approved A2AR agonist and is used as a coronary vasodilator in myocardial perfusion imaging.37
Fig. 1 A representative selection of A2AR ligands. A2AR agonists are typically closely related to COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundadenosine, such as NECA and the coronary vasodilator, regadenoson.37 A2AR antagonists are generally derived from purine bases such as COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundxanthine (e.g.COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundcaffeine, XAC and istradefylline95), COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundadenine (e.g. ZM241385) or COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundguanine (e.g. vipadenant97); some antagonists which are not derived from purines have recently shown efficacy in preclinical models of PD, such as Lu AA47070,98,99 SYN-115 (ref. 100) and 2-amino-8-(4-methylpiperazine-1-carbonyl)-4-phenyl-5H-indeno[1,2-d]pyrimidin-5-one.101 Preladenant is phase III clinical trials for the treatment of PD.45 |
A2AR antagonists have generally been derived directly from COMPOUND LINKS
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Download mol file of compoundxanthine, or contain heterocyclic scaffolds which are closely related to other purines such as COMPOUND LINKS
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Download mol file of compoundadenine.36,38 Typical examples include the purine-like derivative ZM241385, a potent and selective A2AR inverse agonist which has been used widely as a research tool,39,40 and the xanthine derivative, COMPOUND LINKS
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Download mol file of compoundcaffeine, a weak, non-selective adenosine receptor antagonist.
Parkinson's disease (PD) is a neurological disease in which motor function is progressively impaired by the gradual loss of dopaminergic neurons in the striatum.41 Current treatments for PD rely upon restoring COMPOUND LINKS
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Download mol file of compounddopamine D2 receptor (D2R) signalling, most commonly by administering Levadopa (L-DOPA, a dopamine precursor).42 This can initially be effective at restoring motor function; however, efficacy often reduces as the disease progresses and the benefits of the treatment are generally accompanied by undesirable side effects such as dyskinesias.
A2AR antagonists have been evaluated as alternative therapeutic agents for the treatment of PD; they have shown efficacy in several preclinical models of PD and advantages in reducing the dyskinesias experienced by patients undergoing standard COMPOUND LINKS
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Download mol file of compounddopamine replacement therapies.38,43 A2AR is co-expressed with D2R in the striatum where the functional effects of the two receptors are in opposition: i.e. A2AR antagonism and D2R agonism invoke complementary mechanisms for the treatment of PD. A1R is widely distributed throughout the CNS and periphery; however, the low density of A2BR and A3R in the brain, as well as the relatively discrete localisation of A2AR in the striatum with respect to COMPOUND LINKS
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Download mol file of compounddopamine receptors, make A2AR an attractive CNS target for the treatment of PD.44
Preladenant (SCH420814; Merck) has shown efficacy in multiple preclinical models of PD, including the haloperidol-induced catalepsy and 6-hydroxydopamine-lesion turning models in rodents, as well as the MPTP-lesion model in cynomolgus monkeys.45 Preladenant is currently being evaluated in phase III clinical trials for the treatment of PD.
Fig. 2 The topology of a GPCR, as exemplified by the crystal structure of A2A StaR2 in complex with ZM241385 (PDB code 3PWH; simulated lipid bilayer). |
These membrane-spanning proteins are inherently unstable when isolated from cells, whereupon they rapidly unfold. However, this stability can be modulated with point mutations introduced viaprotein engineering, or by preparing co-complexes of GPCRs with antibodies, both of which can facilitate crystallisation.46 Alternatively, chimeric fusion proteins have been prepared with T4 lysozyme47 (which has a high propensity to crystallise), and other fusion partners48 which have allowed the structures of several GPCRs to be solved.29,49
A2AR is soluble in detergents such as dodecylmaltoside (DDM), and the binding activity of the inverse agonist ZM241385 is maintained in this system at 4 °C.50 For wild-type (WT) A2AR solubilised in 2% DDM, the temperature at which 50% of binding is retained after 30 minutes of incubation (melting temperature, Tm) is 23 °C. However, the stability of the receptor was significantly increased following SDM experiments which identified point mutations to improve its apparent Tm. In some cases it is possible to predict these mutations based on the large in-house database of known stabilising mutations or from those in the public domain.46 Stabilising mutations are then combined to give a StaR®.
A2A StaR1 contained four thermostabilising mutations (A54L, T88A, K122A and V239A), and its Tm was found to be 12 °C higher than that of WT A2AR in decylmaltoside (DM; Table 1).34 A2A StaR1 was sufficiently stable for use with some biophysical screening technologies. A2A StaR2 contained a further four mutations (R107A, L202A, L235A and S277A) and was found to have a Tm that was 25 °C higher than that of WT A2AR in DM; it was stable enough for crystallisation.51
StaR® proteins can be prepared in different pharmacological conformations (i.e. active or inactive states of the receptor), and it is possible to control this by judicious choice of the ligand used in the thermostabilisation process. This has particular relevance when generating StaRs® for use as reagents in drug discovery as, for example, StaRs® in agonist (active) conformations generally bind to agonistligands with similar or increased affinities to the corresponding WT receptor, whereas they typically have lower affinity towards inverse agonistligands.18
StaR1 and StaR2 were both stabilised in inverse agonist (inactive) conformations (Table 1). StaR1 was found to bind to a number of neutral antagonists and inverse agonists with similar affinities to the native receptor (Fig. 3); however, the agonists CGS21680 and NECA showed a loss of affinity for StaR1 of approximately 100-fold. StaR2 was found to bind to antagonists and inverse agonists with higher affinity than the WT receptor whilst showing a lowered affinity towards agonists (Fig. 3).
Fig. 3 Binding affinities of A2AR ligands measured against StaR1 and StaR2, each plotted against affinities measured at WT A2AR. Filled circles denote neutral antagonist and inverse agonistligands; open circles denote agonistligands. |
Several A2A StaRs® have been generated in agonist (active) conformations, including GL26 and GL31.18,52 GL26 contained four thermostabilising mutations (L48A, Q89A, T65A and A54L) and was found to have a Tm 20 °C higher than that of WT A2AR in DM (Table 1). GL31 contained the same four thermostabilising mutations as well as a mutation to remove a potential glycosylation site (N154A). GL31 was sufficiently stable to be co-crystallised with the agonistligandsCOMPOUND LINKS
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Download mol file of compoundadenosine and NECA.52 As might be expected from the discussion above, GL26 and GL31 bind to agonistligands with similar affinity to the native receptor, whereas inverse agonistligands show a marked decrease in their affinity for these receptors in active-state conformations (e.g. ZM241385 shows ∼350-fold drop in affinity for GL26 vs. WT A2AR).18 However, neutral antagonistligands, such as istradefylline, bind to WT A2AR and GL26 with similar affinities.
The ability to evaluate the pharmacological profile of a ligand in binding assays with active and inactive StaRs® is a potentially powerful tool, particularly when used in combination with molecular modelling. This ‘reverse pharmacology’ approach contrasts with the conventional requirement to determine a ligand's pharmacology in a cellular or phenotypic assay, and may herald a complementary new approach to GPCR drug discovery. This technique enables the identification of compounds which have an ability to select either an active or inactive receptor conformation, independently of the cellular system. This topic will be the subject of future publications.
Fig. 4 The residues which were found to reduce the binding of [3H]-ZM241385 during the StaR® stabilisation process and are within 5 Å of residues known to affect the binding of triazolotriazineantagonists are shown in green (mapped onto the crystal structure 3PWH with ZM241385 shown in pink).62,63 |
Experimentally enabled homology models of A2AR have been created using both MODELLER58 and MOE,59 with manual readjustment of the clustalW60 alignment onto the β1 adrenergic GPCR crystal where necessary.61 Special consideration was given to residues which were shown to affect ligand binding in both published SDM studies62,63 and during the thermostabilisation process, and the majority of these residues were found to line the anticipated ligand binding site. Further validation was obtained by docking a small set of known A2Aligands (including ZM241385) into each of the structures and comparing these models' enrichment rates against a decoy set of similarly sized molecules. Thus, two models were used for parallel virtual screening experiments with no added constraints (virtual screening results are discussed in Part II).
In some cases, hit rates greater than 35% have been achieved, highlighting the significant improvements that can be achieved with insights from crystal structures.68,69 However, significant user bias is typically used in the preparation of these models; for example, in one case, the dipole moment of a residue shown to be essential for all previously identified ligands was modified,68 and in another case, side chains were tuned and “structural waters” were retained to ensure high hit rates in test sets prior to engaging in final virtual screens.69
It is common practise to prepare models in this way to ensure that significant hit rates are obtained during virtual screening, and post processing of pharmacophores is often carried out in order to maximise the chance of success. However, as others have pointed out, care must be taken when preparing a model and post-processing the data generated so that ligands of interest are not excluded.70 These authors review a virtual screen based on the β2 receptor crystal structure which gave a hit rate of ∼30%, as well as numerous other virtual screens using homology models of GPCRs that achieved hit rates ranging from <1% to ∼45%. This variation is not surprising; as an earlier docking and scoring assessment study has highlighted, significant variation is often observed in virtual screening hit rates across a wide range of targets.71 This study showed that docking programs may be successful in generating poses similar to known binding modes; however, associated scoring functions were less successful at correctly identifying them. It was also noted that, while the application of knowledge about a target can improve hit rates, it is often to the detriment of the diversity of the leads identified.
During our own virtual screening against the experimentally enabled homology models of A2AR described above, no residue-specific constraints, modified weightings or structural waters were used, so as to maximise the novelty and diversity of the ligands identified (vide infra).
Once validated, this SPR screening protocol was used to assess the binding of a library of fragment-sized molecules.75 Single-point screening was initially performed at 200 μM with injections of XAC as a positive control at regular intervals. XAC was found to perform consistently and the receptor remained active throughout the study. Fragment hits were re-analysed in a concentration–response format to determine their binding affinities, which ranged from 10–5000 μM.
A2A StaR2 has been immobilised on sepharose resin and screened by the TINS method using one third of the Zobio fragment collection (molecules selected at random). Thus 531 fragment-sized molecules were screened at 500 μM using OmpA (a stable membrane protein) as a reference protein.77 Using a target/reference ratio of ≤0.7 for the well-resolved 1H NMR signals, 94 fragments were found to bind specifically to StaR2 (conversely, only 6 fragments were found to bind specifically to OmpA which is known to exhibit minimal specific binding to small molecules).
Having established which molecules bound to A2AR via the TINS technique, the 94 hit molecules were screened against WT A2AR in radioligand binding assays with [3H]-ZM241385 and [3H]-NECA.77 Competitive binding and dissociation rates of the radioligands were examined with the fragments in order to determine whether they were acting as orthosteric or allosteric ligands.
Seven competitive orthosteric ligands with diverse chemical structures were identified, with IC50 values measuring between 70 and 1000 μM in concentration–response studies with [3H]-ZM241385. Several non-competitive ligands were also identified. Measurement of their effects on the dissociation rates of radioligands at both A2AR and A1R suggested that the TINS methodology had identified positive and negative allosteric modulators which were selective for A2AR.77
Fig. 5 Electropherograms showing the relative migration time of the A2AR antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), under different conditions (corrected for differences in electro-osmotic flow).80,102 (A) DPCPX is injected into the capillary and gives a single peak. (B) DPCPX is injected with the competitive ligand, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundcaffeine, present in both the injection buffer and running buffer. In this control run, COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundcaffeine does not produce an additional peak or cause a change in the DPCPX peak profile.102 (C) DPCPX is injected into the capillary containing A2A StaR1. The broadening and shift of the peak are indicative of an interaction between A2A StaR1 and DPCPX. (D) DPCPX and COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundcaffeine are injected into the capillary containing A2A StaR1 with COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundcaffeine in the running buffer. The DPCPX peak partly returned to its unbound peak profile, indicating that competitive binding had occurred between the two ligands and the receptor. Negative control ligands such as COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundaspirin were found not to affect the profile of DPCPX in this way. |
Using the chosen StaR® as a template, it is possible to make an additional point mutation in the binding site of interest and compare the effect of this mutation on the binding of various ligands. By making a panel of such StaRs®, each with a different binding site mutation, it is possible to ‘map’ and rank the residues required for ligand binding. This can assist with prioritisation where a number of predicted binding modes exists, and can allow the comparison of binding interactions across different chemical series. The choice of mutants to be prepared can be influenced by SDM data and by residues thought to be of importance from molecular modelling.
In the case of COMPOUND LINKS
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Download mol file of compoundadenosine A2AR, a set of eight StaRs® was prepared, each with a different point mutation in the orthosteric binding site of the StaR1 backbone (Table 2). The receptors were then individually captured onto SPR chips to allow cross-screening against an array of ligands from different chemotypes and with a wide range of binding affinities.81
The study provided key structural information which facilitated lead optimisation of compounds identified by virtual screening before X-ray crystallography data was available for this receptor. The BPM process was also used to build knowledge and gain an understanding of the modes of binding of non-proprietary A2Aligands, such as ZM241385 and the xanthineligands XAC and COMPOUND LINKS
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Download mol file of compoundcaffeine (Fig. 6 and 7).
Fig. 6 BPM heat maps of COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundcaffeine (left) and ZM241385 (right) to show the most significant ligand-binding site interactions. Heat map colouring is explained in Table 2. |
Fig. 7 (A) The best-scoring binding mode of XAC observed during molecular modelling studies; (B) the refined binding mode of XAC which takes into account BPM data. This refined binding mode was later found to be broadly in agreement with the binding mode observed by X-ray crystallography (see Fig. 9). Heat map colouring is explained in Table 2. |
For example, docking XAC into the homology model of A2AR initially resulted in several plausible binding modes but these could be more accurately ranked by taking into account the BPM data generated for this ligand (Fig. 7). SPR screening of XAC against the panel of binding-site mutants revealed that, in particular, mutation to COMPOUND LINKS
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Download mol file of compoundalanine of Ile66, Leu85, Tyr271 or Asn181 resulted in reduced binding, while the mutant S277A showed a higher affinity towards XAC than the parent receptor, StaR1. These data are in agreement with the binding mode shown in Fig. 7B, in which one of the hydrophobic propyl chains of XAC (N3-propyl) sits in a lipophilic pocket lined by Ile66 and the other (N1-propyl) sits in a pocket containing Ser277. Thus the mutant I66A (which has a reduced lipophilic contact with N3-propyl compared to StaR1) shows a reduction in binding, whereas S277A (which, after removal of the polar hydroxyl group, has a more favourable interaction with N1-propyl) shows an increase in binding relative to StaR1. The binding mode predicted during this process was later found to be broadly in agreement with the X-ray crystal structure of XAC in complex with A2AR (vide infra).
The first A2AR structure to be reported was of an inactive receptor conformation, solved to 2.6 Å resolution with ZM241385 bound, using the T4 lysosyme technology (PDB code 3EML).5 Structures of A2AR have since been solved in both active and inactive conformations, including by using fusion protein (T4 lysosyme) techniques in combination with LCP crystallization,5,19 as well as with thermostabilisation51,52,85 and antibody co-complexation21 techniques.
Fig. 8 The X-ray crystal structure of ZM241385 in co-complex with A2A StaR2 (ligand shown in green; PDB code 3PWH). Overlaid in pink is the binding mode of ZM241385 observed in the T4 lysosyme structure (PDB code 3EML). |
In the StaR® structure, the phenolic ring of ZM241385 binds in a cleft between helices TM1, 2 and 7 (defined by Glu13, Ala63, Ile66, Ser67, Leu267, Met270, Ile274, His278, and Tyr271), with the hydroxyl group H-bonding to the backbone carbonyl oxygen of Ala63.51 This additional H-bond may, in some part, explain the relatively high affinity of ZM241385 when compared to its non-hydroxylated counterpart, as well as its high selectivity for A2AR vs. A1R.57 In the T4 lysosyme and antibody structures (which show close overall agreement in their ligand orientations), the phenolic tail of ZM241385 has been placed in solvent, suggesting that there may be multiple binding modes for this ligand.
A2A StaR2 has also been crystallised with two xanthine derivatives: the high affinity ligand, XAC (PDB code 3REY), and the low affinity fragment-sized ligand, COMPOUND LINKS
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Download mol file of compoundcaffeine (PDB code 3RFM).51 Both xanthine derivatives show similarities to ZM241385 in that their core heterocycles H-bond to the side chain carbonyl oxygen of Asn253, π-stack with Phe168 and make hydrophobic contacts with Ile274 (Fig. 9). Interestingly, however, the side chain amide of Asn253 in the XAC structure undergoes a rotation to optimise the H-bond formed. Furthermore, XAC extends much further out of the core of the binding site, where the propyl side chains can be seen to interact with Ile66 to one side, as well as Asn181 and Leu85 to the other. These interactions are consistent with BPM data (vide supra), as are the interactions between the phenyl group and polar tail with Tyr271. The polar tail of XAC binds in the cleft between TMs 1, 2 and 7 (in a similar way to ZM241385), although the residues in question are in different rotameric states (particularly Tyr9 and Tyr271), effectively creating a deeper and narrower cleft than is seen in the 3PWH structure. Interestingly, the cleft between TMs 1, 2 and 7 seen in the XAC structure is identical to that seen in the structure with COMPOUND LINKS
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Download mol file of compoundcaffeine, even though COMPOUND LINKS
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Download mol file of compoundcaffeine does not bind in this region, suggesting that this is a low energy orientation of these residues.
Fig. 9 The crystal structure of A2A StaR2 in co-complex with COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundcaffeine (ligand shown in green; PDB code 3RFM). Overlaid in pink is the X-ray structure of XAC from its co-complex with A2A StaR2 (PDB code 3REY) to show the similarities in the placement of the xanthine cores. Note that in the 3REY structure, the amide head group of Asn253 had rotated 180°. |
These inactive A2A StaR2 co-complexes with the inverse agonists ZM241385 and XAC, and the neutral antagonist, COMPOUND LINKS
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Download mol file of compoundcaffeine, differ significantly from the active-state A2AR structures which have been produced either with T4 lysozyme technology (bound to UK-432097 (ref. 19)) or the A2A StaR®, GL31 (bound to NECA and COMPOUND LINKS
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Download mol file of compoundadenosine52). These differences shed some light on the activation process of A2AR, as the binding of agonistligands results in a significant contraction of the binding site when compared to that seen with inverse agonist or neutral antagonistligands. This contraction results from an inward shift of TM7 toward the core of the receptor, an extracellular upward movement of TM3 by 2 Å, an inward movement of the proline bulge in TM5, and the rigid body rotation of TM6 around Phe242. At a molecular level, changes in the binding site from the ground state A2A StaR2 structure to the active state GL31 are driven by the agonists forming H-bonds from the adenine core to Asn253, and from the ribose group to Ser277 and His278.
By examining the binding of agonists, inverse agonists and neutral antagonists to both active and inactive StaRs® in this way, it is possible to understand the subtleties of the different interactions that these ligand classes make with the receptor. Indeed, by solving crystal structures of these different receptor conformations, it is possible to visualise the movements of the receptor and understand the driving forces of activation, which, in turn, can expedite the discovery and development of pharmaceutical agents.86
The remainder of this article will show how the application of these techniques facilitated the rapid and efficient optimisation of a hit A2AR antagonist which was identified by virtual screening of experimentally enabled homology models. The techniques described in Part I were used in the development of a preclinical candidate for the treatment of PD.
Following 3D visualisation and post-processing analyses, 230 compounds were screened against WT A2AR in a radioligand binding assay with [3H]-ZM241385; 20 compounds were active at concentrations below 55 μM, which corresponded to a 9% hit rate.61 Of particular note, the most potent compound showed pKi = 8.5, ligand efficiency (LE)89,90 = 0.52 and lipophilic ligand efficiency (LLE)91 = 5.4 (MW = 310.4, clogP = 3.1). The 10 most potent hits, which showed pKis of 5.53–8.46, LEs of 0.27–0.52 and LLEs between 2.1 and 5.4, are shown in Table 3 and Fig. 10.
Compound | pKi | LE | LLE | clogP | PSA | MW |
---|---|---|---|---|---|---|
1 | 8.46 | 0.52 | 5.4 | 3.1 | 84.9 | 310.4 |
2 | 5.15 | 0.47 | 4.5 | 0.7 | 72.2 | 222.3 |
3 | 5.75 | 0.44 | 3.9 | 1.9 | 61.7 | 264.3 |
4 | 6.15 | 0.36 | 3.2 | 3.0 | 66.6 | 327.4 |
5 | 5.65 | 0.33 | 3.7 | 1.9 | 85.7 | 331.3 |
6 | 5.62 | 0.31 | 2.6 | 3.0 | 76.7 | 367.9 |
7 | 5.91 | 0.30 | 3.2 | 2.7 | 78.4 | 367.4 |
8 | 5.33 | 0.29 | 3.4 | 1.9 | 79.8 | 340.4 |
9 | 5.70 | 0.29 | 3.9 | 1.8 | 80.1 | 363.4 |
10 | 5.53 | 0.27 | 2.1 | 3.4 | 95.9 | 382.4 |
Fig. 10 The chemical structures of the top 10 hits identified by virtual screening with experimentally enabled homology models of A2AR. |
These molecules represented a diverse range of chemotypes and showed very little resemblance to A2AR antagonists that were known at the time. Indeed, the structural similarity scores of these top 10 hits ranged from 0.31–0.19, when compared to the most similar A2AR ligands in the literature (Tanimoto similarities on FCFP4 fingerprints).61
Fig. 11 Selected SAR within the 2-amino-1,3,5-triazine series. |
BPM analysis of several members of this 1,3,5-triazine series highlighted that the molecules bound deeply within the binding site and revealed a general trend with key interactions between these ligands and binding site residues Leu85, Tyr271, Ile66, Ser277 and Asn181 (see compound series 2 in ref. 81).
Molecular modelling and BPM were used in synergy to further increase the potency and selectivity of this series. For example, it was found that one of the substituents of compound 12 occupies the same part of the binding site as the ribose ring of the natural ligandCOMPOUND LINKS
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13 | 14 | 15 | 16 | 17 | 18 | 19 | |
---|---|---|---|---|---|---|---|
A2AR pKi | 6.93 | 7.29 | 8.40 | 7.67 | 8.11 | 8.85 | 8.46 |
LE | 0.50 | 0.50 | 0.55 | 0.50 | 0.53 | 0.57 | 0.48 |
A1R pKi | 6.56 | 7.25 | 7.36 | 6.71 | 7.07 | 9.79 | 7.50 |
rPPB (%) | ND | 98 | 99 | 98 | 82 | 98 | 92 |
Consideration was given to designing molecules in this series which bind more favourably to A2AR than A1R and, to this end, GRID maps were calculated from homology models for each receptor.92,93 This systematic energetic analysis allowed the comparison of the overall shape, size and electrostatics of the binding sites of each of the receptor sub-types.23 In particular, there are two residues in the A2AR binding site which are correspondingly larger in the A1R binding site (A2AR: Ser277 and Ala59 vs. A1R: COMPOUND LINKS
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Download mol file of compoundThr and Val, respectively), giving an overall larger pocket for A2AR.
Furthermore, analysis of BPM data and GRID maps generated with H-bond acceptor and donor probes, as well as with COMPOUND LINKS
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Download mol file of compoundwater and lipophilic probes, facilitated the rational introduction of both lipophilic and polar substituents onto the template of compound 13 which could increase potency and selectivity. It was found that small substituents at position 5 of the phenyl group on triazine-C6 were well-placed to occupy a lipophilic hot spot within the A2AR/A1R binding sites and gave an overall increase in affinity, whereas substituents at position 3 of the same ring would project out of the tighter A1R pocket, giving selectivity against this off-target receptor (Fig. 12). This hypothesis was supported by SAR for a number of 1,2,4-triazine derivatives in which A2AR potency and selectivity could be increased, for example, as exemplified with chloride substituents in 13, 14 and 15 (Table 4).
Fig. 12 Calculated GRID maps to show the surfaces of A2AR (grey) and A1R (blue mesh) binding sites, as well as lipophilic hot spots within A2AR (yellow). 3,5-Disubstituted aryl groups at triazine-C6 were found to increase potency and selectivity for A2AR; small substituents at position 3 could break the surface of A1R binding site, imparting selectivity (blue circle), while lipophilic groups at position 5 were found to sit favourably within a lipophilic hot spot and increase affinity (red circle). |
Furthermore, it was hypothesised that the introduction of a hydrogen-bonding group at the 4-position of the same ring may introduce an additional interaction with His278 or binding-site COMPOUND LINKS
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Download mol file of compoundwater molecule. Indeed, pyridyl analogues such as 17 were found to be more potent than their phenyl counterparts and the introduction of this additional heteroatom favourably modified the physicochemical properties of the compounds which showed good solubility, long half-lives in rat liver microsomes and relatively low levels of binding to plasma proteins (Tables 4 and 5).85
In vitro ADME profile | In vivo rat pharmacokinetic profile | ||||
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1 mg kg−1 (IV) | 2 mg kg−1 (PO) | ||||
RLM T½ | 86 min | Plasma clearance | 42 mL min−1 kg−1 | T max | 0.4 h |
rPPB | 92% | V d (ss) | 4.6 L kg−1 | C max | 244 ng mL−1 |
Kinetic solubility | 35 μM | Terminal T½ | 1.2 h | Terminal T½ | 1.1 h |
AUCinf | 397 ng h mL−1 | AUCinf | 846 ng h mL−1 | ||
Brain:plasma (0.5 h) | 3.2 | F po | 100% | ||
CSF:brain (0.5 h) | 0.036 |
The corresponding phenol derivatives were also found to have very high affinities for A2AR. Compound 18 was found to be a potent ligand in which the hydroxyl substituent was placed directly onto a GRID hotspot identified with the water and H-bond donor/acceptor probes (hotspots not shown; see WaterMap discussion below). This compound also displayed very slow receptor off-rates, as determined by SPR analysis against StaR1 (kd = 0.001 and >1.0 s−1 for compounds 18 and 13, respectively).81 The ability to examine receptor kinetics as part of the screening cascade during lead optimisation can be advantageous and, during this programme of work, facilitated the identification of compounds which later showed potent in vivo efficacy.85
Fig. 13 The X-ray crystal structures of (A) compound 17 in complex with A2A StaR2 (PDB code 3UZA) and (B) compound 18 in complex with A2A StaR2 (PDB code 3UZC). In both structures, the aminotriazine can be seen H-bonding to Asn253; in (B), the hydroxyl group of 18 can be seen to H-bond to His278. Helices 3 and 4 have been removed from the GPCRs for clarity. |
An analysis of the binding site of the 3PWH structure (after removal of the ligand), using WaterMap software from Schrödinger, showed that these highly ligand efficient molecules completely displace the two clusters of COMPOUND LINKS
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Download mol file of compoundwater molecules that are high in energy (relative to bulk solvent), located in the ribose binding pocket and in the hydrophobic pocket occupied by the phenyl substituent on triazine-C5.23,85 The WaterMap software was used to calculate the enthalpic and entropic energies of waters relative to bulk solvent using a molecular dynamics simulation on a full, explicit COMPOUND LINKS
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Download mol file of compoundwater network. It has also been calculated using SZMAP that pyridyl analogues such as 17 are able to stabilise high energy COMPOUND LINKS
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Download mol file of compoundwater molecules within this network via interactions with their pyridylnitrogen atom, whereas phenol derivatives such as 18 are able to completely displace these COMPOUND LINKS
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Download mol file of compoundwater molecules with their additional hydroxyl substituent (Fig. 14).23,94
Fig. 14 Relative energies of COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundwater molecules within the binding site networks for compound 13 (A), compound 17 (B) and compound 18 (C); calculations performed with SZMAP. In all cases, several destabilised and highly destabilised COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundwater molecules are displaced by the ligands. For waters indicated by arrows: (A) COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundwater is highly destabilised relative to bulk solvent; (B) COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundwater is stabilised by pyridyl N and is comparable in energy to bulk solvent; (C) COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundwater is displaced by phenolic OH. |
On the basis of their in vitro ADME and in vivo pharmacokinetic profiles, compounds such as 19 were progressed into a haloperidol-induced catalepsy model of PD in rats. In this pharmacodynamic model a loss of striatal COMPOUND LINKS
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Download mol file of compounddopamine receptor function is induced by blockade of the receptors with the antagonist haloperidol, causing a Parkinsonian like-state which can be reversed by A2AR antagonism. Istradefylline was used as a control during these studies as it has previously shown efficacy in various models of PD as well in clinical trials with human patients suffering from PD.95,96 An example dataset for the effect of compound 19 measured in this model is shown in Fig. 15. Compound 19 was found to potently reverse the cataleptic state induced in the rats, with measured ED50 values of 0.2 mg kg−1 at both 1 and 2 hour time points after administration. Indeed, 0.3 mg kg−1 doses of compound 19 were found to have comparable efficacy to 1.0 mg kg−1 doses of istradefylline.
Fig. 15 The effects of compound 19 in the haloperidol-induced catalepsy model. A dose-dependent reversal of catalepsy was observed with compound 19 in rats; istradefylline was used as a positive control. |
Biophysical Mapping of hits identified through virtual screening against homology models of A2AR allowed their rapid progression into lead series. Molecular modelling, BPM and X-ray crystallography were then used in synergy to optimise the potency, selectivity and receptor kinetics of a series of 1,2,4-triazine derivatives, culminating in the nomination of a low molecular weight and orally bioavailable candidate for the treatment of CNS disease. The characterisation of this agent will be the subject of future publications.
PD | Parkinson's disease |
GPCR | G protein-coupled receptor |
SBDD | Structure-based drug design |
BPM | Biophysical Mapping |
PO | Oral route of administration |
IV | Intravenous route of administration |
T ½ | Half-life |
RLM | Rat liver microsomes |
rPPB | Rat plasma protein binding |
V d (ss) | Volume of distribution at steady state |
AUCinf | Total drug exposure (area under the curve to infinite time) |
CSF | Cerebral spinal fluid |
C max | Maximum drug concentration |
T max | Time at which maximum drug concentration is reached |
F po | Oral bioavailability |
XAC | Xanthine amine congener |
NECA | 5′-N-Ethylcarboxamidoadenosine |
DPCPX | 8-Cyclopentyl-1,3-dipropylxanthine |
WT | Wild-type. |
This journal is © The Royal Society of Chemistry 2013 |