Mdm2 inhibitors as a platform for the design of P-glycoprotein inhibitors

Article abstract of DOI:10.1016/j.bmcl.2020.127424

Chemoresistance is thought to be the cause of low treatment efficacy and mortality in more than 90percent of patients with advanced cancer. The activation of drug efflux by P-glycoprotein is the key mechanism of resistance. All known P-gp inhibitors are used only in the combination therapy. We propose a new approach based on the multitarget rational design of drugs, which possess both the antitumor and efflux pump inhibitory activity. In this work, the principle possibility of combining the ability to inhibit P-gp and p53-Mdm2 protein-protein interaction in one structure is considered. The biological activity of a number of known and newly synthesized compounds was evaluated using cell lines with different p53 status. The possibility of using computer modeling for the search for P-glycoprotein inhibitors among Mdm2 inhibitors was analyzed; P-gp interaction site and binding modes of substrates and inhibitors were identified. The results obtained in cells that have the native balance of drug resistance and sensitivity showed the ability of the cells to both actively throw out xenobiotics and to lose this ability using P-gp inhibitors. The data obtained indicate that Mdm2 inhibitors are a promising platform for the development of multitarget drugs that can overcome tumor resistance by inhibiting the P-glycoprotein activity.

Full text of DOI:10.1016/j.bmcl.2020.127424

Journal Pre-proofs
Mdm2 inhibitors as a platform for the design of P-glycoprotein inhibitors
T. Grigoreva, A. Romanova, A. Sagaidak, S. Vorona, D. Novikova, V.
Tribulovich
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S0960-894X(20)30535-7
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https://doi.org/10.1016/j.bmcl.2020.127424
BMCL 127424
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Bioorganic & Medicinal Chemistry Letters
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11 May 2020
6 July 2020
16 July 2020
Please cite this article as: Grigoreva, T., Romanova, A., Sagaidak, A., Vorona, S., Novikova, D., Tribulovich, V.,
Mdm2 inhibitors as a platform for the design of P-glycoprotein inhibitors, Bioorganic & Medicinal Chemistry
Letters (2020), doi: https://doi.org/10.1016/j.bmcl.2020.127424
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Mdm2 inhibitors as a platform for the design of P-glycoprotein inhibitors
Grigoreva T., Romanova A., Sagaidak A., Vorona S., Novikova D., Tribulovich V.
St. Petersburg State Institute of Technology (Technical University),
Moskovsky prospect, 26, St. Petersburg, 190013, Russia
Corresponding author: rozentatiana@gmail.com (T.A. Grigoreva).
Abstract:
Chemoresistance is thought to be the cause of low treatment efficacy and mortality in more
than 90% of patients with advanced cancer. The activation of drug efflux by P-glycoprotein is the key
mechanism of resistance. All known P-gp inhibitors are used only in the combination therapy. We
propose a new approach based on the multitarget rational design of drugs, which possess both the
antitumor and efflux pump inhibitory activity. In this work, the principle possibility of combining the
ability to inhibit P-gp and p53-Mdm2 protein-protein interaction in one structure is considered. The
biological activity of a number of known and newly synthesized compounds was evaluated using cell
lines with different p53 status. The possibility of using computer modeling for the search for P-
glycoprotein inhibitors among Mdm2 inhibitors was analyzed; P-gp interaction site and binding
modes of substrates and inhibitors were identified. The results obtained in cells that have the native
balance of drug resistance and sensitivity showed the ability of the cells to both actively throw out
xenobiotics and to lose this ability using P-gp inhibitors. The data obtained indicate that Mdm2
inhibitors are a promising platform for the development of multitarget drugs that can overcome tumor
resistance by inhibiting the P-glycoprotein activity.
Keywords: multitarget drug design; P-glycoprotein; MDR1; p53-Mdm2; drug resistance; efflux
inhibition; cell-based assay; computer modeling.
The problem of multiple drug resistance (MDR) development is becoming increasingly acute
with the improvement of anticancer therapy methods and effectiveness of first-line therapy.
Proliferation of a small number of survived cancer cells leads to the development of a secondary
tumor that is insusceptible to the initial set of drugs. This is manifested as tumor progression after
stabilization or significant regression. Thus, chemotherapy, which was successful at the first stage,
becomes ineffective. Chemoresistance is thought to be the cause of low treatment efficacy and
mortality in more than 90% of patients with advanced cancer. [1] The phenomenon of
chemoresistance is associated not only with the development of relapses, but also with metastasis. [2]
Drug resistance is associated with impaired functioning of various cellular mechanisms
associated with the transport of substances, DNA repair, apoptosis and others. Overexpression of
transporters that prevent a cytostatic from entering the cell is considered the most important
mechanism of drug resistance. [2-4]

P-glycoprotein (P-gp, MDR1) is a transmembrane efflux transporter of the ATP binding
cassette (ABC) family, which pumps out various xenobiotics from the cell. It captures molecules
from the intracellular side of the membrane and throws them out, which prevents the accumulation of
the drug in the tumor and reduces the therapy effectiveness. P-gp overexpression was established in
leukemia, ovarian and breast cancers, neuroblastomas and a number of other hematological and solid
tumors. It was found that high level of P-gp expression can both be originally specific for cancer cells
and appear during chemotherapy. [2] Overexpression of P-gp in cancers reduces the concentration of
drugs in the cell and allows it to develop resistance to a number of topical cytostatics such as taxanes
(paclitaxel), vinca alkaloids (vinblastine), and anthracyclines (daunorubicin). [5] The use of P-gp
pump blockers allows one to restore the ability of the drug to enter the tumor cell and suppress its
vital activity.
The third generation P-gp inhibitors are under clinical trials. They are developed using modern
methods of drug design and showed good efficiency against P-gp with high specificity and minimum
own toxicity. [6] Zosuquidar (NCT00046930) and valspodar (NCT00003190) could not improve the
treatment of acute myeloid leukemia and epithelial ovarian cancer in phase II and III trials. [7, 8] And
although tariquidar successfully passed phase II trials with docetaxel, [9] the phase III with
vinorelbine and paclitaxel/carboplatin were terminated (NCT00042315, NCT00042302).
Nevertheless, trials of P-gp inhibitors is ongoing as a part of the study on the CNS functioning
(NCT03809234, NCT03809234).
The feature of the use of modern P-glycoprotein inhibitors is their ancillary role; they serve to
increase the effectiveness of the main chemotherapeutic agent (taxanes, vinorelbine, etc.) [7-9]. At
the same time, even if the side effects of the ancillary agent are very low, they are combined with the
side effects of the main drug in any case, and it not only increases the toxic load on the organism, but
also complicates the scheme of drug combination to increase the therapy effectiveness.
In this paper, we propose to look at P-glycoprotein inhibitors from a fundamentally different
point of view: to make them an independent combat unit capable of alone exhibiting targeted
biological activity. As a platform for the rational design of P-glycoprotein inhibitors, we consider
p53-Mdm2 protein-protein interaction (PPI) inhibitors. The choice of the platform is determined by
several reasons:
- these compounds are relevant for a wide range of solid tumors, since the p53-Mdm2
pathway is compromised in more than 50% of all human cancers, [10]
- pharmacophore hypothesis of p53-Mdm2 PPI inhibitors was thoroughly developed (see
Supplementary), there are a lot of data on the structure–activity relationship obtained in recent
years that can be used for the rational design of small molecule compounds, [11-13]
- the ability to inhibit P-glycoprotein was shown for these agents, [14, 15]
- their structure is very variable and allows various modifications, while the structure of the
known p53-Mdm2 PPI inhibitors is not antagonistic to the structure of the known
glycoprotein inhibitors (fig. 1). [16-20]

Fig. 1 – The structure similarity of p53-Mdm2 PPI inhibitors and P-glycoprotein inhibitors:
A) Nutlin-3a, MDM2 inhibitor, [16] and Ningalin B analogues, P-glycoprotein inhibitor series, [17]
B) Isatin based p53-Mdm2 PPI inhibitors, [19] and tariquidar, P-glycoprotein inhibitor [20].
In particular, the structural features suggest the presence of anti-Pgp activity in the
developments of our group – Isatin based p53-Mdm2 PPI inhibitors (fig. 2, compound 3 [19, 21]),
and our optimized compounds of Prof. Hardcastle (fig. 2, compound 4 [12, 22], for synthesis see
Supplementary). Tariquidar was considered as the reference P-gp inhibitor. Nutlin-3a (compound 1)
is a p53 reactivator of the cis-imidazoline class, which was shown to competitively inhibit P-gp. [15]
AMG232 (compound 2), a p53-Mdm2 PPI inhibitor, is currently undergoing phase II clinical trials
against Merkel Cell Carcinoma (ClinicalTrials.gov Identifier: NCT03787602), Acute Myeloid
Leukemia (NCT03787602), while its trials against myeloid leukemia are initiated (NCT04190550).
[23-25]
Fig. 2 – Structures of p53-Mdm2 PPI inhibitors 2, 3, 4.

To evaluate the biological activity of the compounds, the following cell lines were used:
HCT116 (р53+/+), H1299 (p53-/-), and MCF7, which is most often used for P-glycoprotein studies.
All lines were routinely cultivated on Petri dishes (63.5 cm2) at 37°C in a CO2 incubator according to
ATCC recommendations. For experiments, cells were plated in wells of 96-well plates with an
optical bottom in DMEM growth medium (Gibco, United States) and after 48 h cultivation, the
required treatment was performed. As controls, similarly cultured cells were used without the
treatment with the test compounds. All experiments were performed in triplicate; average values
(means ± SEM) are presented.
To assess the effect of the compounds on tumor cell growth, the solutions of the studied
substances in the medium obtained by 200X dilution of stock DMSO solutions were added to the
wells. Evaluation of the results was performed after 48 h according to the confluence of the
monolayer after washing with PBS using the Operetta CLS™ multi-parametric cell imaging system
and Harmony 3.1 software (PerkinElmer, United States) in the brightfield mode. [9] The results are
presented in fig. 3.
Fig. 3 – The effect of compounds on tumor cell growth.
Growth of corresponding cell line in the absence of substances was taken as 100%.
As expected, PPI inhibitors suppressed growth of p53+ cells, AMG232 showed the best results
in terms of both the target effect and selectivity: the difference between the effects in p53+ and p53-
cells was the largest. Tariquidar did not affect the cells of all the studied lines in the entire range of
the considered concentrations up to 10 μM, which confirms the absence of its own therapeutic effect.
Along with the antitumor activity, we were interested in the ability of compounds to inhibit the
transport activity in real time. To this end, we studied the effect of the compounds on the
accumulation of Rhodamine 123, a classical P-glycoprotein substrate, in the cells. [17, 26] Tariquidar
was used as a positive control. The cells plated in the wells of a 96-well plate were cultured to the
confluency of 30%, then solutions of the substances and Rhodamine 123 were added (the
concentration of Rhodamine in the solution was 1 μM). The uniform distribution of the cells in the
wells was controlled using the Operetta CLS™ imager (PerkinElmer). After 40 min, the cells were
washed and the fluorescence intensity of Rhodamine 123 (excitation and emission wavelengths of
488 nm and 515-575 nm, respectively) in different wells was compared using a ClarioStar plate
reader (BMG Labtech).

It was found that in the case of HCT116 line under reproducible experimental conditions, the
effect of tariquidar is already observed at 0.5 μM, and this concentration was further used to conduct
comparative studies of the substances on all cell lines. The results are presented in fig. 4.
Fig. 4 – The effect of the compounds on Rhodamine 123 accumulation in cells.
The fluorescence in corresponding cell lines when treated with 0.5 μM tariquidar was taken as
100%).
Nutlin-3a (compound 1) has significant P-gp inhibitory activity in cells with negative p53
status; similarly, AMG232 (compound 2) is the most active against H1299 cells. This fact can be
explained only by the absence of specific transcriptional activity of p53. Cells with positive p53
status (HCT116, MCF7) show a significant structure – P-gp inhibitory activity relationship for Mdm2
inhibitors. Compound 2, although it is an effective Mdm2 inhibitor, does not have a pronounced
ability to inhibit P-gp, while Mdm2 inhibitors based on indolinone (compound 3) and isoindolinone
(compound 4) are significantly more effective. It should be noted that compound 4, which showed
the inhibition efficacy at the level of the tariquidar, was obtained during optimization study of
isoindolinone derivatives. [12, 27] We showed that the modification led to an increase in the
lipophilicity of the compounds and, consequently, both membrane permeability and target binding.
The data obtained indicate that in the case of P-glycoprotein, an increase in the lipophilicity made it
an inhibitor of P-glycoprotein comparable with tariquidar.
Since the approach involves optimization of the proposed Mdm2 inhibitors, we were interested
in whether computer simulation can be used at this stage. Earlier, we showed that in the case of p53-
Mdm2 PPI, computer simulation can be applied quite successfully. [12, 19, 28, 29] To evaluate the
adequacy for the computer prediction of the biological activity of compounds in the case of P-gp, we
performed docking using CCDC GOLD Suite v5.2.2 software package. [30]
It is known that to release substances from the cell, P-gp changes its conformation from
inward-facing to outward-facing. [31, 32] The X-ray structure of inward-facing P-glycoprotein, PDB
ID: 4Q9H [33] was chosen for the analysis. A region that includes binding sites of known P-gp
inhibitors and substrates was chosen as a site for docking. [33, 34] Bis-Benzimide (Huechst 33258)
was analyzed as a substrate, and tariquidar and zosuquidar were used as inhibitors.
Analysis of the substrate binding (bis-Benzimide, fig. 5, red) and the binding of the known
inhibitor (tariquidar, fig. 5, blue) with P-gp suggests that their key difference lies in the binding mode
of the small molecule compound in the protein cavity. If we represent P-gp in the form of the letter
V, then bis-Benzimide is compactly located between the alpha-helices of one branch of the letter V,
while the tariquidar acts as a spacer between the branches, preventing conformational rearrangements
required for the release of a substance (fig. 5).

Fig. 5 – Simulation of the interaction of small molecules with P-gp.
Substrate (bis-Benzimide) is in red, inhibitor (tariquidar) is in blue.
A) Binding of the substrate and the inhibitor to P-gp is shown schematically,
B) Binding mode of compounds in the P-gp cavity according to the docking results,
C) Energy parameters of the interaction.
The energy parameters of the interaction indicate a stronger binding of the inhibitor compared
with the substrate (fig. 5, C). In accordance with this assumption, Nutlin-3a (compound 1) should
rather act as a substrate. This result is in good agreement with the proposed hypothesis that Nutlin-3a
reduces transport of other substrates due to competitive inhibition of P-gp. [13] Binding mode and
molecular dynamics confirm that Nutlin-3a is not able to interfere with P-gp conformational
transitions to a degree sufficient for inhibition. Compounds 3 and 4, in turn, not only fit between the
V branches, but also bind quit strongly to the target, which confirms their inhibitory ability with
respect to P-gp. Binding modes of Nutlin-3a and compound 4 are represented in Supplementary.
The data obtained indicate that Mdm2 inhibitors are a promising platform for the development
of multitarget drugs that can overcome tumor resistance by inhibiting the P-glycoprotein efflux
transporter. The results obtained in cells that have the native balance of drug resistance and
sensitivity showed the ability of a part of the cell population to both actively throw out xenobiotics
and to lose this ability using P-gp inhibitors. From our point of view, this suggests the effectiveness
of using either single target or multitarget P-gp inhibitors at the early stages of the development of
hyperproliferative diseases to overcome primary resistance and prevent the development of
secondary resistance.

Acknowledgments
The work was supported by the Russian Science Foundation (project no. 19-73-10150). The
authors thank the Institute of Cytology of the Russian Academy of Science and BIOCAD
biotechnology company for providing cell cultures. The authors also thank M. A. Gureev for
calculations performed.
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Computer modeling is suitable for search for P-gp inhibitors among Mdm2 inhibitors
P-gp interaction site and binding modes for substrates/inhibitors are identified
Mdm2 inhibitors are promising platform for multitarget drug development
Rhodamine 123 allows quantitative evaluating P-gp inhibitors on non-resistant cells