The nutlins were the first class of p53/MDM2 protein-protein interaction inhibitors to be synthesised. The nutlin library of compounds was initially investigated through high throughput screening by Pazgier and coworkers.78 They are based on a 4,5-dihydroimidazoline structure. The top hit compound, racemic nutlin-3, was found to have a Kdvalue of 263
nM using surface plasmon resonance.79
Further research showed that one enantiomer (nutlin-3a, figure1.19) was 150 times more active than its alternative stereochemistry enantiomer (nutlin-3b), with a Ki of 40 nM
as determined by fluorescence polarisation assays and an IC50 of 90 nM by competition
surface plasmon resonance alongside recombinant p53 ligand (compared to nutlin-3b, which was found to have an IC50 of 13.6 µM in the same assay).73 Nutlin-3a was more
potent as the stereochemistry permitted greater π-π-stacking interactions within the hydrophobic pocket, as the conformation allowed the compound to overlap better with the key wildtype-p53 interactions with MDM2.
N N O N NH O O O Cl Cl
Figure 1.19: Nutlin-3a, the original nutlin found to inhibit the p53/MDM2 interaction
Nutlin-3a was then tested in cell lines HCT116 (wt p53, key binding sequence SQETFS- DLWKLLP) and SJSA-1 (overexpressed MDM2), which were stained with in bromod- eoxyuridine (BrdU), to determine effects in the cell cycle. In both cases, 24 hours
following 4 µM nutlin-3a treatment, there were increased levels of G1 and G2/M activ-
ity in the cell cycle, with a significant reduction of S-phase activity, which is the point at which DNA replication occurs. Investigation of gene expression in the presence of nutlin-3a led to significant increases in the transcription of p53 and p21 (an associated tumour suppressor protein in the downstream pathway of p53).45
Additional cytotoxicity assays were undertaken to determine the effect of nutlin-3a on apoptosis through transferasemediated deoxyuridine triphosphate nick end labeling (TUNEL). As cells undergo damage and apoptosis, the labeling becomes more exten- sive and this can be quantified using flow cytometry and fluorescence microscopy.80 Using TUNEL, it was determined that 45% of SJSA-1 cells exposed to nutlin-3a be- came TUNEL-positive 48 h after incubation (at 24 h there were insignificant levels of TUNEL-positive cells).
In progressing from single cells to animal systems in preclinical studies of nutlin-3a, it was also determined that nude mice xenografts implanted with SJSA-1 cells had their tumour growth inhibited by 81% at 10 mg/kg inhibitor (the maximum tolerated dose). Solution-phase NMR-based studies combined with X-ray crystal structures showed that the disubstituted benzene ring occupied the Phe19 pocket, whilst the two chlorophenyl rings inhibit the Trp23 and Leu26 pockets.81 The piperidone-like moiety does not affect binding, however it works to improve aqueous solubility, as the compound is otherwise very lipophilic.
Although nutlin-3a showed great promise as an inhibitor, it was not a candidate for clinical trials due to its poor pharmacokinetic properties (its high lipophilicity made penetration into the desired tissues and drug formulation for drug delivery was diffi- cult) and therefore analogues of nutlin-3a were investigated, which retained or improved potency whilst also improving aqueous solubility.
1.4.2.1 Nutlins Optimisation: The Design of RG7112
In 2011, further SAR studies were undertaken on nutlin-3a to improve the potency of the nutlins and improve binding to MDMX, a secondary binding partner of both p53 and MDM2.82 This optimisation led to the development of RG7112 (figure1.18). RG7112 was the first p53/MDM2 interaction inhibitor to make it into Phase I clinical trials in advanced solid tumours, early-stage solid tumours, haematological cancers and liposar- comas; as well as acute myeloid leukaemia (AML) in combination with cytarabine.83 RG7112, the most potent nutlin generated to date (shown in figure 1.18), was devel- oped through the optimisation of nutlin-3a, in which the key binding modality was
the projection of the chlorophenyl groups into the Trp23 and Leu26 binding pockets of MDM2.83
During the development of RG7112, the methoxy group present on nutlin-3a was sus- ceptible to degradation and produced phenol as a breakdown product of metabolism. Substitution of the methoxy with a t -butyl functionality prevented the production of phenol as a toxic metabolite.84 Another key modification was the introduction of methyl functionalities to prevent the degradation of the imidazoline core to imidazole, which was completely inactive against MDM2. The isopropoxy functionality was replaced with an ethoxy to reduce the molecular weight and hence produce a more “druggable” compound (that is, a compound with desirable pharmacokinetics and potent pharmacodynamics permitting the formulation of the drug into a medicine). Finally, as the amide side chain was solvent-exposed, it was hypothesised that this could be used for solubilisation in aqueous media and therefore a variety of different polar substitutents were investi- gated. The most active compound, RG7112, contained a piperidine side chain linked to a methylsulfoxy by a propyl linker. The IC50 of RG7112 as determined by competition
SpR was 2.9 nM (compared to 90 nM for nutlin-3a as determined by competition SpR).
Despite the high potency of the nutlin class, resistance to RG7112 and other nutlins governed by mutations within the binding pocket and lid region of MDM2 has resulted in a need for further development of inhibitors.85 Interestingly, a recent paper has explored the cross-reactivity of nutlin-3a between both the anti-apoptotic Bcl family and the p52/MDM2 interaction,86 which could reduce the likelihood of resistance as well as increase its effectiveness in cancers. Using the X-ray crystal structure PDB-1YCR and nutlin-2 as a small molecule template, the stage was set for the design of novel small molecules that could inhibit this interaction.