KLSD: A CuratedKinase–Ligand Database MappingSelectivity Landscapes and Polypharmacology

Introduction Small molecule kinase inhibitors are crucial in the treatment of tumors, and the development of novel inhibitors is a primary approach to combat the continuous emergence of drug resistance. Macrocyclization has emerged as a cutting-edge strategy to enhance the potency, selectivity, and pharmacokinetic properties of these inhibitors by altering their biological and physicochemical characteristics compared to their acyclic counterparts. Areas covered The present article provides a comprehensive overview of the recent advancements in macrocyclic small molecule inhibitors and their inhibitory activities against various cancer cells, which have been patented since 2019. Expert opinion To date, small-molecule kinase inhibitors have demonstrated remarkable therapeutic efficacy in clinical settings. Recent patents have primarily focused on addressing challenges associated with resistance mutations. Despite the significant success achieved in developing selective kinase agents, the identification of new targets and emergence of novel mutations necessitate the development of novel small-molecule inhibitors. Macrocyclic compounds possess distinctive conformational constraints, enhanced inhibitor potency and selectivity, as well as favorable pharmacokinetic properties, rendering them safe, efficient, selective, low-toxicity agents with unique structural characteristic.

RNAi Functional Screening of the Tyrosine Kinome Identifies Therapeutic Targets in Acute Myeloid Leukemia Patients.

<div>Abstract<p>The clinical utility of approved EGFR small-molecule kinase inhibitors is plagued both by toxicity against wild-type EGFR and by metastatic progression in the central nervous system, a disease sanctuary site. Here, we report the discovery and preclinical efficacy of GNS-1486 and GNS-1481, two novel small-molecule EGFR kinase inhibitors that are selective for T790M-mutant isoforms of EGFR. Both agents were effective in multiple mouse xenograft models of human lung adenocarcinoma (T790M-positive or -negative), exhibiting less activity against wild-type EGFR than existing approved EGFR kinase inhibitors (including osimertinib). In addition, GNS-1486 showed superior potency against intracranial metastasis of EGFR-mutant lung adenocarcinoma. Our results offer a preclinical proof of concept for new EGFR kinase inhibitors with the potential to improve therapeutic index and efficacy against brain metastases in patients. <i>Cancer Res; 77(5); 1200–11. ©2017 AACR</i>.</p></div>

The clinical utility of approved EGFR small-molecule kinase inhibitors is plagued both by toxicity against wild-type EGFR and by metastatic progression in the central nervous system, a disease sanctuary site. Here, we report the discovery and preclinical efficacy of GNS-1486 and GNS-1481, two novel small-molecule EGFR kinase inhibitors that are selective for T790M-mutant isoforms of EGFR. Both agents were effective in multiple mouse xenograft models of human lung adenocarcinoma (T790M-positive or -negative), exhibiting less activity against wild-type EGFR than existing approved EGFR kinase inhibitors (including osimertinib). In addition, GNS-1486 showed superior potency against intracranial metastasis of EGFR-mutant lung adenocarcinoma. Our results offer a preclinical proof of concept for new EGFR kinase inhibitors with the potential to improve therapeutic index and efficacy against brain metastases in patients. Cancer Res; 77(5); 1200-11. ©2017 AACR.

Chemical proteomics is a rapidly evolving technology that allows for drug target identification for small molecule inhibitors. Here we present an approach utilizing a chloroalkane (CA) moiety capture handle, which can be chemically attached to small molecules and used to pull-down protein binding partners using the HaloTag technology. CA-modified compounds are cell permeable and have minimal impact on potency, allowing for phenotypic assays of the derivatized compound to be recapitulated. Here we present a study looking at the targets of two compounds - a Brutons Tyrosine Kinase (BTK) small molecule inhibitor and a Phospho-inositol 3 Kinase (PI3K) small molecule inhibitor. BTK is a non-receptor tyrosine kinase expressed in most hematopoietic cells except T cells, it is essential for B cell receptor (BCR) signaling in B cells and as such is the therapeutic focus in many autoimmune diseases, including rheumatoid arthritis (RA) and lupus. Upon binding to growth factor receptor, PI3K is activated which initiates a signaling cascade involving AKT and mTOR that promotes cell cycle progression and cell proliferation. Mutations of the catalytic subunit of PI3KA are observed in several types of cancers. Inhibition of PI3K is therefore an important therapeutic focus. Taselisib, a selective inhibitor of PIK3CA, and G02599124, an inhibitor of BTK, were derivitized with the CA linker. Phenotypic screens compared to the parental compound showed comparable IC50 values. Both CA-compounds showed good binding to HaloTag and were cell permeable in a U2OS stable cell line. Control and test experiments were performed with BTK-CA compound using Jurkat (T cells) and Ramos (B cells) cell lines. Taselib-CA was studied after 1h and 8h treatment in the HCC1954 cell line. Cells were lysed and the CA compounds, together with the bound targets, were rapidly captured onto magnetic resin coated with HaloTag. Unmodified compound was used to competitively elute interacting proteins. Eluted proteins were processed using SDS-PAGE and in-gel digestion. Peptides were analyzed using nanoscale LC-MS/MS coupled with an Orbitrap Velos Pro mass spectrometer. Using this approach we were able to specifically isolate BTK when compared to control experiments in T cells. In the Taselib-CA experiment, we were able to specifically isolate the PIK3CA catalytic subunit and PIK3R1 and PIK3R2 regulatory subunits. Observation of the wild type and mutant forms of PI3KCA will be discussed. Citation Format: Victoria Pham, James Crawford, Steve Saben, Michael Ford, Richard Jones, Danette Daniels, Thomas Kirkland, Marjeta Urh, Jennie Lill. Chemical Proteomic Analysis of BTK and PI3K Using Chloroalkane-Derivatized Small Molecule Inhibitors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-B10.

The FDA has approved 31 small-molecule kinase inhibitors (KIs) for human use as of November 2016, with six having black box warnings for hepatotoxicity (BBW-H) in product labeling. The precise mechanisms and risk factors for KI-induced hepatotoxicity are poorly understood. Here, the 31 KIs were tested in isolated rat liver mitochondria, an in vitro system recently proposed to be a useful tool to predict drug-induced hepatotoxicity in humans. The KIs were incubated with mitochondria or submitochondrial particles at concentrations ranging from therapeutic maximal blood concentrations (Cmax) levels to 100-fold Cmax levels. Ten endpoints were measured, including oxygen consumption rate, inner membrane potential, cytochrome c release, swelling, reactive oxygen species, and individual respiratory chain complex (I–V) activities. Of the 31 KIs examined only three including sorafenib, regorafenib and pazopanib, all of which are hepatotoxic, caused significant mitochondrial toxicity at concentrations equal to the Cmax, indicating that mitochondrial toxicity likely contributes to the pathogenesis of hepatotoxicity associated with these KIs. At concentrations equal to 100-fold Cmax, 18 KIs were found to be toxic to mitochondria, and among six KIs with BBW-H, mitochondrial injury was induced by regorafenib, lapatinib, idelalisib, and pazopanib, but not ponatinib, or sunitinib. Mitochondrial liability at 100-fold Cmax had a positive predictive power (PPV) of 72% and negative predictive power (NPV) of 33% in predicting human KI hepatotoxicity as defined by product labeling, with the sensitivity and specificity being 62% and 44%, respectively. Similar predictive power was obtained using the criterion of Cmax ≥1.1 µM or daily dose ≥100 mg. Mitochondrial liability at 1–2.5-fold Cmax showed a 100% PPV and specificity, though the NPV and sensitivity were 32% and 14%, respectively. These data provide novel mechanistic insights into KI hepatotoxicity and indicate that mitochondrial toxicity at therapeutic levels can help identify hepatotoxic KIs.

In recent years, protein kinases have been one of the most pursued drug targets. These determined efforts have resulted in ever increasing numbers of small-molecule kinase inhibitors reaching to the market, offering novel treatment options for patients with distinct diseases. One essential component related to the activation and normal functionality of a protein kinase is the regulatory spine (R-spine). The R-spine is formed of four conserved residues named as RS1–RS4. One of these residues, RS3, located in the C-terminal part of αC-helix, is usually accessible for the inhibitors from the ATP-binding cavity as its side chain is lining the hydrophobic back pocket in many protein kinases. Although the role of RS3 has been well acknowledged in protein kinase function, this residue has not been actively considered in inhibitor design, even though many small-molecule kinase inhibitors display interactions to this residue. In this minireview, we will cover the current knowledge of RS3, its relationship with the gatekeeper, and the role of RS3 in kinase inhibitor interactions. Finally, we comment on the future perspectives how this residue could be utilized in the kinase inhibitor design.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by inflammation leading to joint damage and systemic complications. Angiogenesis promotes inflammation and contributes to RA progression. This study evaluated potential anti-angiogenic effects of several compounds including small-molecule kinase inhibitors, such as sunitinib (pan-kinase inhibitor), tofacitinib (JAK-inhibitor), NIKi (NF-κB-inducing kinase inhibitor), and the integrin-targeting peptide fluciclatide, using a scratch assay and 3D spheroid-based models of angiogenesis. For all drugs tested in the low micromolar range (1-25 μM), sunitinib (as positive anti-angiogenetic control) showed marked inhibition of endothelial cell (EC) migration and sprouting, effectively reducing both scratch closure and sprout formation. Tofacitinib exhibited marginal effectiveness in the scratch assay, but performed better in the 3D models (55% inhibition), whereas NIKi showed around 50% anti-angiogenic effects in both models. Fluciclatide changed EC morphology rather than migration, and only when stimulated with synovial fluid in spheroid model did it show inhibitory effects (at ≥2.5 µM), with none below this dosage. These results highlight the potential of NIKi and tofacitinib for angiogenesis inhibition and of fluciclatide for safe diagnostic targeting of microdose in RA, as well as the need for advanced screening models that mimic RA's complex inflammatory pro-angiogenic environment.

The phase I study by Storniolo et al in this issue of Journal of Clinical Oncology, which evaluates the combination of lapatinib (a selective small-molecule ErbB1 and ErbB2 tyrosine kinase inhibitor) and trastuzumab (an antibody blocking the extracellular domain of ErbB2) in patients with advanced ErbB2-positive breast cancer, is the first published study to our knowledge examining dual-agent molecular targeting of the ErbB2 receptor. The study demonstrated that there was clinical activity in a group of heavily pretreated patients and—using a toxicity end point—determined an optimally tolerated regimen for this combination of agents. The epidermal growth factor family of receptors plays a key role in regulating growth, survival, and resistance to chemotherapy in many epithelial malignancies. Approximately 25% of breast cancers overexpress ErbB2 or demonstrate ErbB2 gene amplification; ErbB2 is a key mediator of tumor cell growth and survival, and a predictor of poor outcome. Potential strategies to block peptide growth factor signaling pathways include (1) blocking or neutralizing the growth factor or its production, (2) blocking the receptor-binding site or prevention of receptor dimerization, (3) blocking the receptor tyrosine kinase domain, and (4) blocking downstream signaling pathways. The mechanism of action of trastuzumab is multifactorial and includes (1) antibody-dependent cellular cytotoxity, (2) modulation of ErbB2 signaling, (3) internalization of ErbB2 cell-surface protein, (4) reduction in proteolytic cleavage of the ErbB2 extracellular domain, and (5) interference with DNA damage repair. Small-molecule inhibitors such as lapatinib have an advantage over antibodies because they inhibit multiple signaling pathways at plasma concentrations achieved in the clinic. This is relevant because the survival of epithelial tumors is often dependent on the presence of redundant signaling pathways. Combining anti-ErbB2 antibodies with small-molecule tyrosine kinase inhibitors is an appealing strategy because both therapies exert their antitumor effects via distinct mechanisms of action and target ErbB2 at different sites. Combining distinct classes of inhibitors may not only potentiate cellular cytotoxicity, but may also assist in overcoming inherent or acquired resistance to one class of inhibitors. Mechanisms of resistance identified to date include activating mutations in the domain of the adenosine triphosphate–binding pocket for small-molecule tyrosine kinase inhibitors and the presence of truncated receptors for monoclonal antibodies. Dual-agent molecular targeting has been examined in preclinical models using the ErbB1 pathway as well as the ErbB2 pathway. With respect to the ErbB1 pathway, Huang et al and Matar et al studied the effect of cetuximab (an antibody that binds to the extracellular domain of ErbB1) in combination with either gefitinib or erlotinib (small-molecule inhibitors of the tyrosine kinase domain or ErbB1) against a panel of human cancer cell lines in culture and in tumor xenografts. The combination of cetuximab plus gefitinib or erlotinib enhances growth inhibition, in culture and in vivo, over that observed with either agent alone. Phosphorylation inhibition of downstream effector molecules (mitogen-activated protein kinase and AKT), and apoptosis was enhanced with the combination. In lung and head and neck tumor cells highly resistant to cetuximab, gefitinib, and erlotinib, but not cetuximab, retained the capacity to inhibit these tumor cells and were also able to further inhibit the activation of downstream effectors of ErbB1 signaling. When tested in vivo, single-agent therapy resulted in transient tumor regression only at the highest doses, whereas suboptimal doses of gefitinib and cetuximab in combination resulted in complete and permanent regression of large tumors. Contradictory results were obtained by Fishel et al who determined that gefitinib and cetuximab, when used in combination, were antagonistic when tested against two ErbB1expressing epidermoid cell lines. The combination caused less apoptosis than gefitinib alone, and this appeared to be the result of overexpression of functional ErbB1 associated with the combination. It is likely that these discordant results were a result of the particular cell lines studied. Most relevant to the study being reviewed are the preclinical models examining dual targeting of the ErbB2 pathway. Xia et al treated ErbB2-overexpressing breast cancer cell lines with a combination of lapatinib and either trastuzumab or a polyclonal anti-ErbB2 antibody. The combination treatments led to enhanced tumor cell apoptosis, whereas treatment with the same doses of lapatinib, trastuzumab, or polyclonal anti-ErbB2 antibody alone led to minimal or no increase in apoptosis. They demonstrated that the enhanced tumor cell apoptosis after combination ErbB2-targeted therapies was most closely associated with downregulation of survivin protein. Konecny et al studied the combination of lapatinib and trastuzumab in a panel of human breast cancer cell lines. Synergistic activity was seen when the combination was tested in ErbB2-overexpressing cell lines. Of JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 26 NUMBER 20 JULY 1

Clinical implications of food–drug interactions with small-molecule kinase inhibitors

Outcomes of Patients with Richter Transformation without Prior Chemoimmunotherapy for CLL/SLL: An International Multicenter Retrospective Study

Targeting kinase signaling pathways with constrained peptide scaffolds.

Anti-programmed cell death-1 (anti-PD-1) blockade in tumor therapy results in an expansion of CD8+ T cells and can restore the function of exhausted CD8+ T cells. We identified GSK-3 as a key upstream kinase of regulating PD-1 expression. Inhibition of GSK-3 in CD8+ T cells increased Tbx21 (Tbet), which suppressed Pdcd1 (PD-1) transcription (Taylor et al., Immunity 2016). In controlling spontaneous pulmonary metastasis of B16 melanoma and EL-4 lymphoma cells, GSK-3 blockade is as effective as anti-PD-1 blocking antibody (Taylor et al., Cancer Research 2018). Given the clinical importance in devising strategies to overcome tumor resistance to immune checkpoint blockade, we assessed whether the combined use of GSK-3 small-molecule inhibitor (SMI) and anti-PD-1 can overcome resistance to anti-PD-1 monotherapy. Indeed, GSK-3 SMI and anti-PD-1 synergized in therapy against solid B16 tumors that are resistant to monotherapy, leading to delayed tumor growth. Further, the synergy of GSK-3 SMI and anti-PD-1 therapy has been successfully confirmed by using adoptive transfer of OT1 cells into B16-OVA tumor-bearing mice. High-dimensional profiling of tumor-infiltrated lymphocytes (TILs) using mass cytometry (CyTOF) showed that the combination therapy gave an increase in cellularity of CD8+ effector memory T cells, which correlated to better tumor control. Transcriptome analysis of these CD8+ TILs highlighted the increased cytotoxicity as shown by significant changes of granzymes. Our findings outline a next-generation approach of potentiating PD-1 antibody therapy with SMIs of GSK-3 that synergizes as therapy by boosting the cellularity and cytotoxicity of tumor infiltrated CD8+ T cells. Citation Format: Janna Krueger, Alison Taylor, Alexandra Kazanova, Saloua Jeidane, Vinicius Motta, Ian Watson, Christopher E. Rudd. Inhibition of Glycogen Synthase Kinase 3 (GSK-3) synergizes with anti-PD-1 by potentiating tumor-infiltrated CD8+ T-cells [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A59.

Small-molecule inhibitors of Ataxia Telangiectasia and Rad3 related kinase (ATR) sensitize lymphoma cells to UVA radiation

\nHYPOTHESIS: We hypothesize that pediatric high-grade glioma (pedHGG) tumors are sustained by pro-survival and pro-growth kinase signaling pathways that are distinct from adult GBM and normal brain, and can thereby be specifically targeted with small molecule inhibitors. METHODS: We performed a high-throughput chemical genetic screen using the GSK-PKISv1 library of 310 small molecule kinase inhibitors. These chemically diverse compounds have been tested in cell-free kinase inhibition assays measuring 203 unique kinases (37% of the human kinome), providing information on their efficacy against common and orphan kinases. We initially screened 5 high-grade glioma cell lines: 2 supratentorial pedHGG (KNS-42, SU-pcGBM2), 1 brainstem pedHGG (SU-DIPG-IV), and 2 adult supratentorial GBM (U251 MG, GBM43). We also screened immortalized normal human astrocytes to control for nonspecific cytotoxicity. In the initial screen, cells were treated at 1 μM for 24h, then measured for changes in viability, cell cycle, and apoptosis. Of the initial 310 compounds, 15 were selected for follow-up screening based on evidence of specific killing of pedHGG. To identify mechanisms underlying each compound’s activity, we leveraged the available cell-free kinome data and cross-referenced this against gene expression data from pedHGG and adult GBM tumors. RESULTS: Follow-up screening in 8 cell lines revealed 6 lead compounds that had efficacy against multiple pedHGG lines while sparing normal astrocytes and multiple adult GBM lines. The 6 lead compounds had diverse chemical backbones and patterns of activity across in cell-free kinase assays. The top targets of these compounds include some well-known glioma oncogenes (i.e. PDGFRA, EGFR), as well as novel targets (i.e. ERBB4, PYK2, HIPK1, LTK). CONCLUSIONS: Our chemical genetic approach identified 6 diverse small molecule kinase inhibitors with evidence of specific activity against pedHGG cell lines. Genetic and pharmacological validation of the putative targets of these compounds is underway to prioritize candidates for clinical translation.