• The large contact surfaces between proteins (~1,500–3,000 Å2).
• The flat and relatively featureless nature of these surfaces as compared to the active-sites on more traditional targets.
• The lack of a natural small molecule ligand to use as a lead molecule.

However, as the authors go on to discuss, it is often found that a small number of amino acid residues contribute disproportionately to the binding interaction between proteins. Targeting these so-called hot-spots represents an approach to tackling these targets. The review provides a nice analysis of six examples of the successful discovery of inhibitors of protein-protein interactions.

Recently, scientists at the Universities of Newcastle and Oxford have published the optimisation of a series of isoindolinone inhibitors of the interaction between Murine Double Minute 2 (MDM2) and p53.  The tumor suppressor protein p53 acts as a signalling node and is involved in the response of cells to a range of stresses. Inactivation of p53 is often involved in the development and subsequent progression of cancer. Inhibitors of the MDM2-p53 interaction have been proposed as anti cancer agents.  The same team has previously reported isoindolinone 49 that has an IC50 for the MDM2-p53 interaction of 15.9 μM. This paper outlines the optimisation of this compound to generate 74a with an IC50 of 0.17 μM. Ideally the optimisation would have been supported by X-ray crystallography, however the team were unable to obtain co-crystals of the series bound to their target, the MDM2 protein. Instead a combination of NMR studies and docking were used to predict binding modes for the inhibitors.

Through the preparation and testing of analogues of 49 it was established that increases in potency were achievable through the introduction of a para nitro substituent on the N-benzyl group and by steric restriction of the 3-carbon chain through introduction of a cyclopropyl group. Despite considerable efforts to find a suitable replacement for the nitro-group the team were unable to find a similarly active replacement. Separation and testing of the enantiomers of 74 showed that the (R) form, 74a, is primarily responsible for the MDM2-p53 inhibition.

The authors go on to demonstrate cellular activity for 74a in a range of assays. This paper provides nice evidence of the inroads that medicinal chemists are making into the challenging protein-protein interaction target class.

The authors postulate the following on the basis of these findings:

• Inactive structures of GPCRs are sufficiently good templates for activated state models (also see recent structures of β1 and β2 with agonists bound in the inactive state).
• The molecular switching between inactive and active receptor forms appears to be controlled by significant residue movements (and disruption of a hydrophobic network) one turn beyond the so-called “toggle switch” (W286 in β2).  This information could enable better modeling of ligand-induced activation.
• The high similarity of binding sites in inactive and active receptors may make in silico discrimination between agonists and antagonists extremely difficult, if not impossible.

However, they emphasize the importance of obtaining additional structural data for activated GPCR complexes in order to establish the generality of these early observations and to elucidate the best methods for incorporating these findings into computational approaches to predicting ligand activity.  It’ll be interesting to see how this area develops as additional structures become available.

1. An in vitro cell viability screen in THLE cells (+/- P450 transfection) (See a key reference describing a related screen from Pfizer).
2. A HepG2 ‘Crabtree effect’ toxicity assay – to detect mitochondrial poisons (in galactose medium cultured cells – see presentation from AstraZeneca).
3. Biliary transport inhibition via a membrane vesicle assay in insect cells overexpressing specific transporters. Quantification of inhibition of ATP-dependent biliary transport (e.g. inhibition of bile salt transporter [BSEP]) correlates with DILI – see Greer (2009)).

The latest  Nature Reviews|Drug Discovery contains an extensive review article which summarises much of this material as well as additional background information, in particular reviewing the pros & cons of reactive metabolite screening using GSH/CN trapping assays (and highlighting an interesting new hard & soft nucleophilic trapping agent containing both Cys & Lys residues).  A number of decision trees and methodologies are reviewed, including data arising from Merck’s extensive use of early covalent binding studies (which require commitment to the preparation of radio-labelled drug substance and are generally viewed as definitive go/no go experiments) and which, with no correlation between the incidence of liver toxicity and the observed levels of covalent binding, actually questioned the whole approach.  The review finishes somewhat philosophically by challenging a number of existing opinions/dogma and emphasises the need to continue informatics approaches to help to identify potentially harmful protein adducts (e.g. use of the Target Protein Database which is a repository for all publicly available information relating to known covalent adducts of mammalian proteins and chemically-reactive metabolites of xenobiotic agents, including drugs).

For this analysis, the authors calculated an ADMET score (i.e. the deviation from oral drug space as defined by AlogP and molecular mass) to characterize the data set.  The plot below shows 1791 oral drugs colour-coded according to their ADMET score (on the left), and then (on the right) the same drugs compared with the scores for the total content of the ChEMBL database (approximately 200k compounds).  A greater deviation from oral drug space is observed in the latter case (14% of drugs have ADMET scores >2 compared with 39% of ChEMBL molecules).

Further detailed graphics and analyses are also presented to arrive at the following conclusions:

1. Average oral drug potency is approx 50 nM (Another clear observation was that 8% of oral drugs have both mol mass > 400 and AlogP > 4, compared to 30% of all ChEMBL molecules and 41% of ChEMBL molecules with nanomolar potency).
2. The majority of oral drugs have off-target pharmacological activities (This analysis was clearly complicated by any intentional poly-pharmacology).   The graphic below shows, for 392 oral drugs, N = number of off-target hits with reported potencies ≤ 1 µM (i.e. only 29% in the N=0 segment are totally selective) :                                                                                    
3. There was no clear relationship between in vitro potency and therapeutic dose (which is perhaps unsurprising given the numerous additional factors involved).

Whilst the points made in the paper mainly serve to reinforce the conclusions of earlier publications, they nonetheless highlight the importance, and usefulness, of open-source information sources and the prospect of their more widespread utility in future.

• Design strategies for optimizing unbound brain concentration in the CNS.  Recent publications (e.g. Wager, et.al., Defining Desirable Central Nervous System Drug Space…;  and the earlier Hitchcock, et.al., Structure−Brain Exposure Relationships) have highlighted the key physicochemical property space within which CNS drugs are located and to which drug discovery efforts ought to ostensibly be aligned in order to achieve the required balance of efficacy and safety. However, the authors stress that optimizing unbound brain and plasma concentration ratios (or brain availability) by adopting a more holistic, multi-parameter approach is crucial to success (and they refer back to another of their own recent publications in this vein).  Structural elements associated with blood-brain barrier (BBB) penetration and computational approaches to predicting BBB permeability are also discussed briefly.

Physicochemical property space - CNS drugs and candidates (click to enlarge)

• Screening strategies to optimise brain availability.  This section looks at the role of transporters (e.g. P-gp) in controlling molecular access to the CNS and at in-vitro and in-vivo screening approaches to addressing this issue in drug discovery.  The recent publication of the mouse P-gp x-ray crystal structure (PDB3G60) and it’s implications are also discussed.
• Brain Delivery Approaches. Molecules with properties that fall outside the property space of known CNS drugs (e.g. antibodies, siRNA, peptides) offer novel approaches to the treatment of CNS disease and numerous innovative approaches to the delivery of such agents are under investigation.  The final section summarizes progress in this area.

• Isosteres of Hydrogen:
  • Deuterium as an isostere of hydrogen.
  • Deuterium substitution to modulate metabolism.
  • Deuterium substitution to modulate metabolism and toxicity.
  • Deuterium to slow epimerization.
  • Fluorine as an isostere of hydrogen.
  • Fluorine for hydrogen exchange to modulate metabolism.
  • Deploying fluorine to modulate basicity in KSP inhibitors.
  • Substitution of hydrogen by fluorine as a strategy for influencing conformation.
  • Fluorine/hydrogen exchange to influence membrane permeability.
• Isosteres of Carbon and Alkyl Moieties:
  • CH2, O, and S isosterism.
  • CH2/O isosterism in PGI2 mimetics.
  • CH2/O isosterism in non-prostanoid PGI2 mimetics.
  • Silicon as an isostere of carbon.
  • Silicon in p38α MAP kinase inhibitors.
  • Silicon as a carbon isostere in biogenic amine reuptake inhibitors.
  • Silicon as a carbon isostere in haloperidol.
  • Silicon/carbon isosterism in retinoids.
  • Silicon/carbon exchange in protease inhibitors.
  • Silanediols as HIV-1 protease inhibitors.
  • Silanediols in ACE inhibitors.
  • Silicon in thermolysin inhibitors.
  • Cyclopropyl rings as alkyl isosteres.
  • Cyclopropyl in T-Type calcium channel antagonists.
  • Cyclopropyl in CRF-1 antagonists.
  • Oxetanes as mimetics of alkyl moieties.
  • CF3 as a substitute for methyl in a tert-butyl moiety.
  • CF2 as an isostere of C(CH3)2.
• N Substitution for CH in Benzene Rings:
  • N for CH in phenyl rings in CRF-1 antagonists.
  • N for CH in phenyl rings in calcium sensing receptor antagonists.
  • N for C substitution in dihydropyridine derivatives.
• Biphenyl and Phenyl Mimetics:
  • Biphenyl mimetics in factor Xa inhibitors.
  • Phenyl mimetics in glutamate antagonists.
  • Phenyl mimetics in oxytocin antagonists.
• Phenol, Alcohol, and Thiol Isosteres:
  • Phenol and catechol isosteres.
  • Phenol isosteres in dopamine D1/D5 antagonists.
  • Alcohol and thiol mimetics:
    • Sulfoximine moiety as an alcohol isostere.
    • RCHF2 as an isostere of ROH.
    • RCHF2 as a thiol mimetic in HCV NS3 protease inhibitors.
    • Application of RCHF2 as an alcohol isostere in lysophosphatidic acid.
• Hydroxamic Acid Isosteres:
  • Application of RCHF2 to hydroxamic acid isosteres.
  • Hydroxamic acids in carbonic anhydrase II inhibitors.
  • Hydroxamic acid mimetics in TNFα-converting enzyme (TACE) inhibitors.
• Carboxylic Acid Isosteres:
  • Carboxylic acid isosteres in angiotensin II receptor antagonists.
  • Acylsulfonamide in HCV NS3 protease inhibitors.
  • Acylsulfonamide in EP3 antagonists.
  • Acylsulfonamides in Bcl-2 inhibitors.
  • 2,6-Difluorophenol as a CO2H mimetic.
  • Squaric acid derivatives as CO2H mimetics.
  • Aminosquarate derivatives as amino acid mimetics.
  • Heterocycles as amino acid mimetics.
• Isosteres of Heterocycles:
  • Avoiding quinonediimine formation in bradykinin B1 antagonists.
  • Isosterism between heterocycles in drug design.
  • Heterocycles as hydrogen bond acceptors.
  • H-bonding capacity of common functional groups.
  • Heterocycles and H-bonds: Filling the gaps.
  • H-bonding and Brønsted basicity differences.
  • Pyridazine H-bonding in p38α MAP kinase inhibitors.
  • Heterocycle substituents and H-bonding.
  • Heterocycle isosteres in p38α MAP kinase inhibitors.
  • Heteroatoms as H-bond acceptors.
  • Oxygen atoms and H-bonding.
  • Isosterism between heterocycles in non-prostanoid PGI2 mimetics.
  • Isosterism between heterocycles in estradiol 17β-dehydrogenase inhibitors.
  • Role of pyrimidine nitrogen atoms in cathepsin S inhibitors.
  • Heterocycles and metal coordination.
  • Heterocycles and C-H bonding.
  • Heterocycle C-H bond donors in GSK3 inhibitors.
  • Evolution of GSK3 inhibitors.
  • Janus kinase 2 (JAK2) inhibitors.
  • Electron demand of heterocycles and applications.
  • Heterocycles and activation of a C=O in serine protease inhibitors.
• Isosteres of the Nitro Group:
  • Nitro isosteres in α1α adrenoreceptor antagonists.
• Isosteres of Carbonyl Moieties:
  • Ketone isosteres.
  • Oxetane as a C=O isostere.
  • Fluorine as a C=O isostere in factor VIIa and thrombin inhibitors.
  • Fluorine as a C=O isostere in FKBP12 mimetics.
  • Amide and ester isosteres.
  • Trifluoroethylamines as amide isosteres.
  • Trifluoroethylamines as amide isosteres in cathepsin K inhibitors.
  • Trifluoroethylamine as an amide replacement in the HCV NS3 inhibitor Telaprevir.
  • Amide isosteres in adenosine 2B antagonists.
• Ether/sulfone and glyoxamide/sulfone isosterism:
  • Ether/sulfone isosterism in HIV-1 protease inhibitors.
  • Sulfone/glyoxamide isosterism in HIV attachment inhibitors.
• Urea isosteres:
  • Urea isosteres in histamine antagonists.
  • Urea-type isosteres in KATP openers.
  • Urea isosteres in CXCR2 antagonists.
  • Additional squaric acid urea isosteres.
  • Urea/thiourea isosteres in factor Xa inhibitors.
• Guanidine and Amidine Isosteres:
  • Arginine isosteres in factor Xa inhibitors.
• Phosphate and Pyrophosphate Isosteres:
  • Phosphate and pyrophosphate mimetics.
  • Phosphate isosteres in PTP-1B.
  • Squarates as phosphate isosteres.
  • HIV-1 integrase inhibitors and phosphate isosterism.
  • Phosphate isosteres in HIV-1 RNase H and HCV NS5B inhibitors.
  • Pyrophosphate isosteres in Ras inhibitors.
  • Phosphate isosteres in HIV-1 reverse transcriptase substrates.
  • Phosphate isosterism in balanol.
  • Phosphate shape mimetics.
• Isosteres of Ligand-Water Complexes:
  • Displacing water from the binding site of HIV-1 protease.
  • Displacing water in poly(ADP-ribose) polymerase-1 (PARP) inhibitors.
  • Displacing water in scytalone dehydratase.
  • Displacing water in p38α MAP kinase inhibitors.
  • Displacing water in epidermal growth factor receptor kinase inhibitors.

A recent paper from Pfizer describes some of their work in this area.  Following high-throughput screening, an early lead (2) with good pI3Kα potency and high ligand efficiency (0.48) was identified.  Compound 2 had moderate activity at mTOR but otherwise was selective across a panel of other protein kinases.  An x-ray co-crystal structure (PDB3ML8) of 2 bound into the active site of PI3Kγ  was used to guide subsequent optimisation efforts (key binding motifs are highlighted below).  Compound 2 had poor pharmacokinetics (high in vitro clearance) and the group’s design strategy focused on replacing any metabolic hot spots whilst maintaining drug-like physical properties and, at the same time, improving the mTOR activity.

The benzyl alcohol was the first group identified as conferring a metabolic liability and N-dealkylation of the N-methylaminopyrimidine was also anticipated to be a likely metabolic pathway.  Isosteric replacements for the benzyl alcohol were introduced and the desmethyl aminopyrimidine analogues were prepared whilst ensuring that logP and MW were not increased significantly.  2-Methoxypyridines were identified as the preferred benzyl alcohol replacements and, in combination with the desmethyl amino modification, were found to lower clearance and improve mTOR potency (the N-Me substituent was postulated to make an unfavourable interaction with the mTOR protein).  The cyclopentyl group in 2 was then modified to optimise potency and improve solubility through the addition of polar substituents.  Hydroxyethyl-substituted cyclohexanol in this position gave the optimal balance of properties and led to the identification of their clinical candidate, PF-04691502 (Ki=0.57 and 16 nM at PI3Kα and mTOR respectively), which was subsequently advanced into a phase I clinical trial.  The progression from 2 to PF-04691502 nicely illustrates the use of structure-based design to optimise multiple physicochemical properties and the authors illustrate this succinctly (see below) using a plot of the mPI3Kα potency vs cLogP overlaid with contours of equivalent lipophilic efficiency (LipE).  In moving from compound 2 to the eventual candidate 1 (PF-04691502) the LipE was increased from under 6 to about 8.  The x-ray co-crystal structure (PDB3ML9) of PF-04691502 revealed a number of interesting features (see above), including a water-mediated H-bond and an internal H-bond.

The current front line therapy for HCV infection (which can lead to liver cirrhosis) involves treatment with PEGylated interferon α (PEGasys or PEG-Intron, by injection) and ribavirin (orally).  Unfortunately this approach has limited efficacy due, in part, to the low completion rates observed for the lengthy (~1 year) course of therapy.   The clear need for improved therapies and the associated commercial opportunity has led to many companies progressing molecules into the clinic (see this overview of the area), a number of which have targeted NS3/4A protease.   The NS3/4A protease represents an attractive target as it is essential for viral polyprotein maturation.  Boehringer Ingelheim (BI) initially led the field in this area and achieved proof of concept with BILN-2061, although this molecule was subsequently terminated due to cardiac toxicity in pre-clinical species.  More recently, both Vertex (Telaprevir, VX-950) and Schering-Plough (Boceprevir, SCH 503034) have advanced molecules to Phase III, with several other companies close behind.

A previous report from Merck had described their entry into this field using a molecular modelling approach to design analogues of BILN-2061.  Interestingly, this approach led to the surprisingly straightforward switch from a P1-P3 tether (highlighted above) to a P2-P4 tether (see below) and subsequently to the identification of a clinical candidate, MK-7009, following painstaking optimisation of the P2 and P3 substituents.

This highly potent candidate intriguingly contains 2 carbamate groups within its macrocyle.  Good plasma exposures were observed in dogs and chimpanzees and whilst high liver exposures (which is clearly key for the target indication) were obtained in rats and rhesus monkeys, disappointingly low plasma exposures (which are required to access the non-hepatic HCV replication sites) were observed in these species.

In an effort to address these potential shortcomings, the letter describes the optimisation of the isoquinoline template, originally reported in the J.Am.Chem.Soc. paper referenced above, rather than the isoindoline carbamate present in the first clinical candidate (MK-7009).  The authors describe how they sought to improve the potency and rat plasma/liver concentrations, relative to their previous lead molecule, mainly by changes to the P3 group and to the isoquinoline substituents.  Unfortunately, and the authors are to be admired for their refreshing honesty in this respect, the PK of this series was unpredictable and there was no alternative but to screen a large number of compounds to identify those which combined the highest rat oral plasma and liver exposures with the requisite potency.  A seemingly clear link was observed between increased lipophilicity at P3 and both an increase in plasma concentrations and a reduction in potency in the serum protein-containing assay system that was used.   However, this understandable link to lipophilicity did not hold for all changes or indeed for all of the isoquinoline substituents that were explored.  In the end, it appears that the clinical candidate, MK-1220, was chosen following the detailed characterisation of a relatively large set of similar analogues.

Nonetheless, these data do provide the opportunity to reflect on the fact that some of these macrocyclic templates are able to achieve good levels of cell permeability and display favourable pharmacokinetic profiles whilst clearly lying in an increasingly taboo physicochemical and molecular weight territory (a recent paper from Ensemble Therapeutics Corp. gives some further insight into this area of drug discovery in which the rule of five is clearly violated).

The latter approach is the subject of a J.Med.Chem. perspective by authors from the University of Tokyo.  The paper describes some of their own work in the area and also reviews relevant examples from the literature.  The various examples are grouped according to the specific structural modification:

• Removal of aromaticity
• Increased dihedral angle/disruption of molecular symmetry  (see example below)
• Introduction of substituents into benzylic position
• Twisting of fused rings

The examples presented in this paper clearly demonstrate the utility of this approach and the importance of considering these types of molecular modifications alongside the more classical introduction of polar, solubilizing groups.