Lesley Ann
Howell
*a and
Andrew Michael
Beekman
*b
aSchool of Biological and Chemical Sciences, Queen Mary University of London, Mile End Road, London E1 4NS, UK. E-mail: L.Howell@qmul.ac.uk
bSchool of Pharmacy, University of East Anglia, Norwich Research Park, Norwich, Norfolk, NR47TJ, UK. E-mail: A.Beekman@uea.ac.uk
First published on 19th November 2020
Using the protein–protein interaction of Mcl-1/Noxa, two methods for efficient modulator discovery are directly compared. In silico peptide-directed ligand design is evaluated against experimental peptide-directed binding, allowing for the discovery of two new inhibitors of Mcl-1/Noxa with cellular activity. In silico peptide-directed ligand design demonstrates an in vitro hit rate of 80% (IC50 < 100 μM). The two rapid and efficient methods demonstrate complementary features for protein–protein interaction modulator discovery.
In an attempt to further improve the economy of drug discovery for PPIs we recently reported peptide-directed binding24 as a method to identify selective small molecule modulators. This technique extends on the REPLACE technique,25–27 utilising high affinity peptides as scaffolds for small molecule fragment identification. Using the PPI of apoptosis influencing Mcl-1/Noxa as a paradigm, we took the NoxaB-(75–93)-C75A peptide (AAQLRRIGDKVNLRQKLLN, IC50 650 nM) and divided it into two half peptides, amino acids 75–84 (AAQLRRIGD) and 85–93 (KVNLRQKLLN, Fig. 1: step 1), each containing two key binding residues (L78, I81, V85, and Q89). Reactive terminals were added to the termini of the half peptides, an alkyne on the C terminus of AAQLRRIGD and an azide on the N terminus of KVNLRQKLLN. These half peptides, with and without reactive termini, possessed no discernible binding affinity for Mcl-1. The reactive termini where used to “click” small molecule fragments to the peptides, with copper catalysed alkyne azide cycloaddition. The binding affinity of the peptide-small molecule hybrids was examined. The fragments which were shown to restore binding affinity in some way emulate the peptide segment they have replaced (Fig. 1: step 2). The small molecule fragments that appeared to emulate amino acids 75–84 where then clicked to the small molecule fragments which emulated amino acids 85–93, providing small molecules with a high probability of emulating the entire NoxaB peptide (Fig. 1: step 3). The choice of small molecule fragments to be clicked to peptides was guided by in silico modelling of the covalent reaction, allowing for a high efficiency.
To further improve the efficiency of this method we applied a fully computational peptide-directed ligand design to the PPIs of hDM2/hDMX and p53.28 This process mirrored that of peptide-directed binding, but performed steps 1–3 (Fig. 1) computationally, identifying a number of small molecules triazoles for synthesis. Analogous to our Mcl-1/Noxa example, the crystal structures of a modified p53 peptide bound to both hDM2 and hDMX were modified to generate two half peptides with reactive azide and alkyne termini. In silico covalent docking was performed to identify fragments which are likely to restore binding of the semi-peptide. Here the two methods diverge. Instead of preparing the identified peptide-small molecule hybrids, the top 10 azide fragments and top 10 alkyne fragments were combined virtually to identify 100 small molecule triazoles. This library was redocked to the protein target and the top 10 results were chosen for synthesis.
Our report of in silico peptide-directed ligand design28 demonstrated a higher efficiency at discovering small molecule inhibitors of PPIs when compared to the analogous experimental peptide-directed binding.24 The in silico method required the preparation of only 20 compounds to obtain a 50% hit rate of compounds which demonstrated an IC50 < 100 μM in in vitro protein fluorescence anisotropy assays. The experimental method revealed 54% of the small molecule compounds prepared demonstrated an IC50 < 100 μM, slightly higher than the in silico only route, but also required the preparation of 60 peptide-small molecule hybrids, adding time and cost. The 60 hybrids prepared identified 23 hits (reducing the hit rate to 44% for total compounds synthesised), which suggested 104 possible small molecule triazoles, of which we chose to prepare 35, based on availability and cost of components. It may be by chance that our hit rate was as high as observed, but seems unlikely given the number of compounds observed. The question remained, is one method clearly superior to the other, or are they complementary?
Structure | FA IC50 (μM) | Structure | FA IC50 (μM) | ||
---|---|---|---|---|---|
a IC50 values determined by non-linear regression of at least three independent experiments (see ESI). Errors are 95% confidence intervals (CI). Fmoc, 9-fluorenylmethylcarbonyl. | |||||
1 |
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0.17 [0.15–0.19] | 6 |
![]() |
3.38 [2.56–4.47] |
2 |
![]() |
0.36 [0.29–0.45] | 7 |
![]() |
4.39 [3.95–4.87] |
3 |
![]() |
0.62 [0.47–0.80] | 8 |
![]() |
7.03 [6.20–7.97] |
4 |
![]() |
1.01 [0.89–1.15] | 9 |
![]() |
>100 |
5 |
![]() |
1.14 [0.84–1.52] | 10 |
![]() |
>100 |
The NoxaB peptide (AAQLRRIGDKVNLRQKLLN) was employed as a positive control and to ensure the FA assay was performing adequately. Surprisingly, of the ten compounds prepared eight (80%) demonstrated binding in our assay, with an IC50 < 100 μM. Additionally, three of these compounds (1–3) demonstrated IC50 values less than 1 μM Selectivity for the Mcl-1/Noxa was evaluated by also examining the compounds in assays for Bcl-2/Bim, Bcl-xL/Bim, and Nrf2/Keap1. The ten compounds demonstrated excellent selectivity, with no appreciable inhibitory effect in these assays.
This very high hit rate demonstrates the power of peptide-directed ligand design for PPI modulators. Analysis of the compounds shows that four compounds were identified by experimental peptide-directed binding (1, 2, 4 & 6),24 with four new Mcl-1 binders identified with the in silico approach (3, 5, 7 & 8). Three of the top four binders (1, 2 & 4) were identified by the experimental peptide-directed binding, suggesting both experimental and computational methods are likely to find the most efficacious compounds. However, 1–6 have very similar structures, with the Fmoc-D-propargylglycine unit cyclised with a benzylazide, all of which demonstrated IC50 values <10 μM. The in silico method highlighted four new small molecule fragments which had not been highlighted in the experimental peptide-directed binding methodology24 (azide section of 3, 7, 8, and the alkyne section of 7 and 8). The binding of compounds to Mcl-1 was validated with a thermal shift assay33 using the hydrophobic dye Sypro orange.34 Compound 1–8 demonstrated the ability to increase the melting temperature of Mcl-1 compared to the DMSO vehicle control (Fig. 2B).
The compounds which demonstrated activity in the in vitro protein assays were evaluated in the MTS anti-proliferation assay,35 against pancreatic cancer cells lines. BxPC3 cells, which are dependent on Mcl-1, MiaPaCa2 cells, which are dependent on Mcl-1 and Bcl-2, and AsPC-1 cells, which do not overexpress Mcl-1,36–39 were treated with compound 1–8 (Table 2). Compound 5–8 showed no antiproliferative effect in all evaluated cell lines. Compound 1 and 2 demonstrated antiproliferative effects on all evaluated cell lines. Interestingly, compound 3 and 4 demonstrated efficacies in BxPC3 and MiaPaCa-2 cells, but no effect on Mcl-1 independent AsPC-1 cells, perhaps suggesting an Mcl-1 selective mode of action. 3 and 4 also suggest a para substituent of a certain size on the phenyl portion may offer selectivity. Compound 9 and 10 were highlighted by the ADME evaluation as unlikely to demonstrate cellular activity (clog
P of 0.55 and −0.03 respectively), but were prepared nonetheless due to their high docking ranking. These results suggest that the subsequent ADME evaluation will offer increased efficiency if heeded.
BxPC3 (μM) | MiaPaCa-2 (μM) | AsPC-1 (μM) | |
---|---|---|---|
a IC50 values determined by non-linear regression of at least three independent experiments (see ESI). Errors are 95% confidence intervals (CI). Table 2: IC50 results obtained for compound 3 and 5 to selected cancerous cell lines in the presence of jacalin or bovine serum albumin (BSA) at 10 μM. Errors are 95% confidence intervals. | |||
1 | 9.16 [5.97–14.88] | 7.20 [6.15–8.45] | 1.73 [1.42–2.14] |
2 | 1.48 [1.20–1.87] | 1.12 [1.36–1.94] | 4.401 [3.86–5.02] |
3 | 2.74 [1.56–5.00] | 7.09 [5.50–9.09] | >100 μM |
4 | 1.35 [1.23–1.49] | 1.14 [1.10–1.19] | >100 μM |
A tetramethylrhodamine ethyl ester (TMRE) assay was employed to evaluate if compounds generated mitochondrial membrane depolarization, one of the first signs of Mcl-1 inhibition (Fig. 2C).40,41 BxPC-3 cells were incubated with compound 1–4 or positive control, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), followed by TMRE. TMRE readily accumulates in the mitochondria resulting in a fluorescent signal. Depolarized mitochondria result in reduced fluorescence. Compound 1–4 showed significantly reduced fluorescence after 2 h of incubation, indicating depolarized mitochondria membrane and apoptosis.
Docking poses predicted by induced fit docking suggest compounds are able to bind in the key P2 and P3 pockets. Poses for compound 1 and 2 (Fig. 3), are representative of binding poses generated for compound 1–8. The aromatic groups create contacts with Phe270 in P2 and Phe228 in P3, and interaction with Arg263 either through a water bridge interaction or hydrogen bonding. In line with the design of peptide-small molecule hybrids, the azide fragment which interacts with Arg263 in the hybrids, also interacts with Arg263 in the predicted pose for compound 1. This motif is repeated in the majority of the binding poses, but is not necessarily the predicted lowest energy conformer. Voisin-Chiret and co-workers recently highlighted the importance of the P2/P3 binding pockets and Arg263 for binding of non-peptidic ligands.42 Representative 2D ligand interaction diagrams are presented in the ESI.†
Footnote |
† Electronic supplementary information (ESI) available: Full experimental descriptions of synthesis and biological experiments, and supporting figures. See DOI: 10.1039/d0cb00148a |
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