Discovery of novel dual extracellular regulated protein kinases (Erk) and phosphoinositide 3-kinase (pi3k) inhibitors as a promising strategy for cancer therapy

Article abstract of DOI:10.3390/molecules25235693

Concomitant inhibition of MAPK and PI3K signaling pathways has been recognized as a promising strategy for cancer therapy, which effectively overcomes the drug resistance of MAPK signaling pathway-related inhibitors. Herein, we report the scaffold-hopping generation of a series of 1H-pyrazolo[3,4-d]pyrimidine dual ERK/PI3K inhibitors. Compound 32d was the most promising candidate, with potent inhibitory activities against both ERK2 and PI3Kα which displays superior anti-proliferative profiles against HCT116 and HEC1B cancer cells. Meanwhile, compound 32d possessed acceptable pharmacokinetic profiles and showed more efficacious anti-tumor activity than GDDC-0980 and the corresponding drug combination (BVD-523 + GDDC-0980) in HCT-116 xenograft model, with a tumor growth inhibitory rate of 51% without causing observable toxic effects. All the results indicated that 32d was a highly effective anticancer compound and provided a promising basis for further optimization towards dual ERK/PI3K inhibitors.

Full text of DOI:10.3390/molecules25235693

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molecules
Article
Discovery of Novel Dual Extracellular Regulated
Protein Kinases (ERK) and Phosphoinositide 3-Kinase
(PI3K) Inhibitors as a Promising Strategy for
Cancer Therapy
Lingzhi Zhang 1, Qiurong Ju 1, Jinjin Sun 1, Lei Huang 1, Shiqi Wu 1, Shuping Wang 1, Yin Li 1,
Zhe Guan 1 , Qihua Zhu 1,2,* and Yungen Xu 1,2,
*
1
State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China;
zhanglingzhicpu@163.com (L.Z.); jqr474678912@163.com (Q.J.); fas15850601108@163.com (J.S.);
18361073522@163.com (L.H.); wushiqicpu1@163.com (S.W.); wangsp16@126.com (S.W.);
linny_cpu@126.com (Y.L.); guanz2008@163.com (Z.G.)
Jiangsu Key Laboratory of Drug Design and Optimization, Department of Medicinal Chemistry,
China Pharmaceutical University, Nanjing 210009, China
2
*
Correspondence: zhuqihua@vip.126.com (Q.Z.); xyg@cpu.edu.cn (Y.X.); Tel.: +86-025-86185303 (Y.X.)
Academic Editors: Jiang Wang, Liang-Ren Zhang, Peng Zhan, Qi-Dong You, Tian-Miao Ou
and Xiao-Yun Lu
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 29 October 2020; Accepted: 30 November 2020; Published: 3 December 2020
Abstract: Concomitant inhibition of MAPK and PI3K signaling pathways has been recognized
as a promising strategy for cancer therapy, which effectively overcomes the drug resistance of
MAPK signaling pathway-related inhibitors. Herein, we report the scaffold-hopping generation
of a series of 1H-pyrazolo[3,4-d]pyrimidine dual ERK/PI3K inhibitors. Compound 32d was the
most promising candidate, with potent inhibitory activities against both ERK2 and PI3Kα which
displays superior anti-proliferative profiles against HCT116 and HEC1B cancer cells. Meanwhile,
compound 32d possessed acceptable pharmacokinetic profiles and showed more efficacious anti-tumor
activity than GDDC-0980 and the corresponding drug combination (BVD-523 + GDDC-0980) in
HCT-116 xenograft model, with a tumor growth inhibitory rate of 51% without causing observable
toxic effects. All the results indicated that 32d was a highly effective anticancer compound and
provided a promising basis for further optimization towards dual ERK/PI3K inhibitors.
Keywords: ERK inhibitor; PI3K inhibitor; dual ERK/PI3K inhibitors; cross-talk
1. Introduction
Numerous small molecule kinase inhibitors have become an effective methods for the treatment of
cancer, due to their good selectivity, high potency and low toxicity. However, because of drug resistance,
the clinical benefits of small molecule kinase inhibitors have been greatly limited [1]. The resistance
mechanism can be divided into two categories: one is from the overexpression and resistance mutations
of the target kinase itself (on-target); the other is not directly related to the target itself, but through
alternative signaling pathways to achieve resistance (by-pass) [2]. Therefore, in addition to the
continuous development of new kinase inhibitors targeting mutation sites, the drug resistance could be
also better overcome by inhibiting related bypass signaling pathways. Now, drug combination strategy
is performed to overcome drug resistance by synergy effects of two or more drugs. However, there are
some drawbacks of drug combination, such as incompatible PK, enhanced toxicity, or even causing
drug-induced diseases and threatening life. On the contrary, multi-target drugs not only simultaneously
act on multiple targets to exert a synergistic effect, but also reduce the dosage of drugs, avoiding the
Molecules 2020, 25, 5693; doi:10.3390/molecules25235693
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safety problems caused by the interaction between drugs. In addition, uniform pharmacokinetic
properties are beneficial to precise drug therapy in the human body. In 2019, a clinical trial of the
combination therapy of MEK Inhibitor Binimetinib and phosphatidylinositol 3-kinase (PI3K) inhibitor
Buparlisib was terminated in patients with advanced solid tumors with RAS/RAF alterations because
of adverse events [3]. Therefore, the development of dual MAPK and PI3K pathways inhibitors has
great potential application prospects.
The mitogen-activated protein kinases (MAPK) pathway, often known as the RAS-RAF-MEK-ERK
signal cascade and activated through polypeptide or growth factors that bind to transmembrane
receptor tyrosine kinases (RTKs), exhibits the ability to transmit upstream signals to its downstream
effectors to regulate physiological processions such as cell proliferation, differentiation, survival and
death. As the most frequently mutated signaling pathway in human cancer, targeting the MAPK
pathway has long been considered as a promising strategy for cancer therapy. Now several drugs
targeting this pathway have been approved by the FDA, such as BRAF inhibitors (Vemurafenib and
Dabrafenib [4,5]) and MEK inhibitors (Trametinib, Cobimetinib and Selumetinib [6,7]). Despite the
considerable success of BRAF and MEK inhibitors, with clinical studies carried out, most patients
experience recurrence in less than a year and about 10~15% of patients harboring B-RafV600E are
insensitive to BRAF and MEK inhibitors due to the amplification and mutation of BRAF/MEK and
the negative feedback loops [8], resulting in the limiting of further clinical application. In such cases,
targeting ERK, a downstream key node of BRAF/MEK, has been proposed as a potential strategy
for overcoming acquired drug resistance. Preclinical studies suggest that ERK inhibitors have an
advantage over inhibiting the growth of BRAF/RAS mutated tumor and overcoming BRAF or/and
MEK inhibitor resistance. Representative drugs have ERK-A (1) [9], GDC-0994 (2) [10], BVD-523 (3), etc.
(Figure 1).
Figure 1. Structures of representative ERK inhibitors.
Phosphoinositide 3-kindase (PI3K) is a conserved family of lipid kinases, which is composed of the
catalytic subunit p110 and the regulatory subunit p85 or p101. PI3K can phosphorylate the 3-hydroxyl
of phosphatidylinositol 4,5-bisphosphate (PIP2) to generate phosphatidylinositol 3,4,5-trisphosphate,
PIP3) [11]. As a second messenger, PIP3 plays an important role in cell survival, growth, proliferation
and metabolism. The tumor suppressor gene PTEN (phosphatase and tension homolog deleted on
chromosome ten) can dephosphorylate PIP3 to generate PIP2, which is an antagonist of the catalytic
effect of PI3K [12]. However, it was found that PTEN was the most mutated tumor suppressor
gene [13] in endometrial tumors, central nervous system diseases, skin cancer and prostate cancer.
Cancer gene research has shown that the PI3K signaling pathway is the most susceptible to mutation
in human tumors, and has been a popular target for tumor treatment. Representative drugs have
Alpelisib (4) [14], GDC-0980 (5) [15], 908737 (6) [16], etc. (Figure 2)

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Figure 2. Structures of representative PI3K inhibitors.
Studies have found that there are multiple feedback adjustments between ERK signaling pathway
and PI3K signaling pathway, and combined blockage of ERK and PI3K pathways significantly decreased
cell viability, compared with single target inhibition [17]. The interaction between the PI3K and ERK
pathways is very complex and has not yet been fully understood [14]. Firstly, in both physiological and
pathological conditions, there is a significant level of cross-talk between kinases of these two pathways
(Figure 3). Phosphorylated PI3K could activate RAS via several mechanisms [18], thereby reactivating
the ERK pathway. Meanwhile, inhibition of PDK1, AKT or Rheb could increase RAF activities [19–21].
Besides, the classic feedback loop (S6-IRS1-PI3K) leads to activation of PI3K but also ERK signaling [22].
PI3K and ERK cascade signaling converge on mTORC1, which is the master regulator of ribosome
biogenesis and protein translation. To sum up, when one pathway is inhibited, the other would be
activated by cross-talk or feedback loops, thereby promoting the development of tumors. Therefore,
the design of ERK and PI3K dual inhibitors is expected to better overcome the drug resistance of ERK
pathway-related inhibitors and obtain better clinical benefits. So far, no dual ERK/PI3K inhibitors are
available in the clinic or the market. In this work, we designed a series of compounds derived from
ERK-A as ERK and PI3K dual inhibitors. Further optimization culminated in the identification of a
potent compound 32d exhibiting the best inhibitory activity against both ERK and PI3K enzymes.
Figure 3. The cross-talk between MAPK/ERK and PI3K/Akt pathways.

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2. Results and Discussion
2.1. Drug Design
In the process of investigating existing PI3K inhibitors, we found that a urea-based with
Nitrogen-containing heterocycle was often introduced into PI3K inhibitors, such as compound
and compound 908737 (Figure 2, the structure marked in red). Meanwhile, in the structure of an
ATP competitive ERK inhibitor ERK-A [ ], it also was found to contain a structure similar to the
urea-based with heterocycle. Therefore, we docked the compound ERK-A into PI3K protein (PDB
ID: 4JPS) (Figure 4b) and compared the co-crystal structure of Alpelisib ( , PI3K selective inhibitor)
(Figure 4a). It was found that when a N atom was introduced at the C3 position, it could form two
hydrogen bonding interactions with the Val851 in the PI3K protein skeleton region which is vital
for maintaining the PI3K inhibition activities (Figure 4b,c). Thus, we designed and synthesized the
1H-pyrazolo[3–d]pyrimidine derivatives 16a 16d, as shown in Figure 5. After preliminary kinase
inhibitory activity studies, to our surprise, compound 16b exhibited 27.4% PI3K inhibitory activity
at 1 M while retaining good ERK2 inhibitory activity. To further improve the PI3K inhibitory
activity, we replaced the 1H-pyrazolo[3,4-d]pyrimidine scaffold with the pyrido[3,2-d]pyrimidine or
pyrido[2,3-d]pyrimidine through scaffold-hopping strategy to synthesize 24 and 32a 32m (Figure 5).
4
6
α
4
α
α
α
~
α
µ
~
After docking 32d with the best potency to the co-crystal structure of ERK2 (PDB ID: 5KE0), it was
found that N1 of compound 32d could form a hydrogen bonding interaction with the Lys52 in the
ERK2 protein skeleton region which is vital for maintaining the ERK2 inhibition activities (Figure 4e);
meanwhile, the pyrazole substituent of 32d extended into the solvent accessible area (Figure 4f).
Figure 4. The 3D interactions diagram of complex structures (
) X-ray structure of Alpelisib (green) bound to PI3K ; ( ) ERK-A (purple) docked into X-ray structure
of PI3K . ( 16b (red) docked into X-ray structure of PI3K . ( ) X-ray structure of ERK-A (purple)
bound to ERK 2; ( 32d (orange) docked into X-ray structure of ERK 2; ( ) Overlap of the proposed
a~c: PDB code 4JPS; d~f: PDB code 5KE0).
(a
α b
α
c)
α d
e)
f
binding modes of ERK-A (purple) and 32d (orange) in ERK2. The figure was generated using PyMol
(http://www.pymol.org/).