Substituted pteridinones, pyrimidines, pyrrolopyrimidines, and purines as p90 ribosomal S6 protein kinase-2 (RSK2) inhibitors: Pharmacophore modeling data.

The RSK2 kinase is a downstream effector of the Ras/Raf/MEK/ERK pathway that is aberrantly active in a range of cancer types and has been recognized an anticancer target. The inhibition of RSK2 kinase activity would disrupt multiple pro-cancer processes; however, there are few RSK2 inhibitors. The data have been obtained for a series of pteridinone-, pyrimidine-, purine-, and pyrrolopyrimidine-based compounds, developed to establish a structure-activity relationship for RSK inhibition. The compounds were docked into the ATP-binding site of the N-terminal domain of the RSK2 kinase using Glide. The binding conformations of these molecules was then used to generate a set of pharmacophore models to determine the structural requirements for RSK2 inhibition. Through the combination of these models, common features (pharmacophores) can be identified that can inform the development of further small molecule RSK inhibitors. The synthesis and evaluation of the pteridinone- and pyrimidine-based compounds was reported in the related articles: Substituted pteridinones as p90 ribosomal S6 protein kinase (RSK) inhibitors: A structure-activity study (Casalvieri et al., 2020) and Molecular docking of substituted pteridinones and pyrimidines to the ATP-binding site of the N-terminal domain of RSK2 and associated MM/GBSA and molecular field datasets (Casalvieri et al., 2020). [1], [2]. The synthesis and evaluation of the purine- and pyrrolopyrimidine-based compounds was reported in the related research article: N-substituted pyrrolopyrimidines and purines as p90 ribosomal S6 protein kinase-2 (RSK2) inhibitors (Casalvieri et al., 2021) [3].

N-Substituted pyrrolopyrimidines and purines as p90 ribosomal S6 protein kinase-2 (RSK2) inhibitors.

The RSK2 kinase is the downstream effector of the Ras/Raf/MEK/ERK pathway, that is often aberrantly activated in acute myeloid leukemia (AML). Recently, we reported a structure-activity study for BI-D1870, the pan-RSK inhibitor, and identified pteridinones that inhibited cellular RSK2 activity that did not result in concomitant cytotoxicity. In the current study, we developed a series of pyrrolopyrimidines and purines to replace the pteridinone ring of BI-D1870, with a range of N-substituents that extend to the substrate binding site to probe complementary interactions, while retaining the 2,6-difluorophenol-4-amino group to maintain interactions with the hinge domain and the DFG motif. Several compounds inhibited cellular RSK2 activity, and we identified compounds that uncoupled cellular RSK2 inhibition from potent cytotoxicity in the MOLM-13 AML cell line. These N-substituted probes have revealed an opportunity to further examine substituents that extend from the ATP- to the substrate-binding site may confer improved RSK potency and selectivity.

Substituted oxindol-3-ylidenes as AMP-activated protein kinase (AMPK) inhibitors.

AMP-activated protein kinase (AMPK) is a central metabolic regulator that promotes cancer growth and survival under hypoxia and plays a role in the maintenance of cancer stem cells. A major challenge to interrogating the potential of targeting AMPK in cancer is the lack of potent and selective small molecule inhibitors. Compound C has been widely used as an AMPK inhibitor, but it lacks potency and has a poor selectivity profile. The multi-kinase inhibitor, sunitinib, has demonstrated potent nanomolar inhibition of AMPK activity and has scope for modification. Here, we have designed and synthesized several series of oxindoles to determine the structural requirements for AMPK inhibition and to improve selectivity. We identified two potent, novel oxindole-based AMPK inhibitors that were designed to interact with the DFG motif in the ATP-binding site of AMPK, this key feature evades interaction with the common recptor tyrosine kinase targets of sunitinib. Cellular engagement of AMPK by these oxindoles was confirmed by the inhibition of phosphorylation of acetyl-CoA carboxylase (ACC), a known substrate of AMPK, in myeloid leukemia cells. Interestingly, although AMPK is highly expressed and activated in K562 cells these oxindole-based AMPK inhibitors did not impact cell viability or result in significant cytotoxicity. Our studies serve as a platform for the further development of oxindole-based AMPK inhibitors with therapeutic potential.

Molecular docking of substituted pteridinones and pyrimidines to the ATP-binding site of the N-terminal domain of RSK2 and associated MM/GBSA and molecular field datasets.

The data have been obtained for a series of substituted pteridinones and pyrimidines that were developed based on BI-D1870 to establish a structure-activity relationship for RSK inhibition. The 19 compounds, 12 of these with R- and S-isomeric forms, were docked into the ATP-binding site of the N-terminal domain of the RSK2 kinase using Schrodinger Glide. The binding conformations of these molecules and their interactions with RSK2 may inform the development of further small molecule RSK inhibitors. The molecular mechanics energies combined with the generalized Born and surface area continuum solvation (MM-BGSA) method was used to estimate the free energy of binding of the small molecules with RSK2. The molecular field characteristics of the docked confirmations of the inhibitors was examined using Cresset Forge software. The synthesis and evaluation of these compounds was reported in the related research article: Substituted pteridinones as p90 ribosomal S6 protein kinase 2 (RSK2) inhibitors: a structure-activity study (Casalvieri et al., 2020).

Substituted pteridinones as p90 ribosomal S6 protein kinase (RSK) inhibitors: A structure-activity study.

The activity of p90 ribosomal S6 kinase 2 (RSK2) has emerged as an attractive target for cancer therapy due to its role in the regulation of diverse cellular processes, such as cell transformation and proliferation. Several pan-RSK inhibitors have been identified with BI-D1870 and the pseudo-analogs LJH685 and LJI308 being the most selective, potent, and frequently used small molecule inhibitors. We designed and synthesized a series of pteridinones and pyrimidines to evaluate the structural features of BI-D1870 that are required for RSK2 inhibition. We have identified inhibitors of RSK2 activity, evaluated their target engagement in cells, and measured their effect on cell viability and cytotoxicity in the MOLM-13 acute myeloid leukemia (AML) cell line. The results of our studies support that RSK2 inhibition can be achieved in MOLM-13 cells without potent cytotoxicity. The structure-activity data from this study will be used as a platform to develop novel RSK2 inhibitors.

Development of Potent Pyrazolopyrimidinone-Based WEE1 Inhibitors with Limited Single-Agent Cytotoxicity for Cancer Therapy.

WEE1 kinase regulates the G2 /M cell-cycle checkpoint, a critical mechanism for DNA repair in cancer cells that can confer resistance to DNA-damaging agents. We previously reported a series of pyrazolopyrimidinones based on AZD1775, a known WEE1 inhibitor, as an initial investigation into the structural requirements for WEE1 inhibition. Our lead inhibitor demonstrated WEE1 inhibition in the same nanomolar range as AZD1775, and potentiated the effects of cisplatin in medulloblastoma cells, but had reduced single-agent cytotoxicity. These results prompted the development of a more comprehensive series of WEE1 inhibitors. Herein we report a series of pyrazolopyrimidinones and identify a more potent WEE1 inhibitor than AZD1775 and additional compounds that demonstrate that WEE1 inhibition can be achieved with reduced single-agent cytotoxicity. These studies support that WEE1 inhibition can be uncoupled from the potent cytotoxic effects observed with AZD1775, and this may have important ramifications in the clinical setting where WEE1 inhibitors are used as chemosensitizers for DNA-targeted chemotherapy.

Selective Targeting of RSK Isoforms in Cancer.

The p90 ribosomal S6 kinase family (RSK1-4) is a group of highly conserved Ser/Thr kinases that act as downstream effectors of the Ras/Raf/MEK/ERK signaling pathway. The RSKs phosphorylate a range of substrates involved in transcription, translation, cell cycle regulation, and cell survival. Although the RSKs have a high degree of sequence homology, their functional differences in cancer are of great interest. Current RSK inhibitors target more than one RSK isoform, and this may limit their efficacy as anticancer agents. Here, we review the structure and function of the RSK kinases, their role in cancer growth and survival, and their potential as modulators of chemoresistance. In addition, we summarize the development of current RSK inhibitors and their limitations.