In silico identification of a novel Cdc2-like kinase 2 (CLK2) inhibitor in triple negative breast cancer.

Dysregulation of RNA splicing processes is intricately linked to tumorigenesis in various cancers, especially breast cancer. Cdc2-like kinase 2 (CLK2), an oncogenic RNA-splicing kinase pivotal in breast cancer, plays a significant role, particularly in the context of triple-negative breast cancer (TNBC), a subtype marked by substantial medical challenges due to its low survival rates. In this study, we employed a structure-based virtual screening (SBVS) method to identify potential CLK2 inhibitors with novel chemical structures for treating TNBC. Compound 670551 emerged as a novel CLK2 inhibitor with a 50% inhibitory concentration (IC50) value of 619.7 nM. Importantly, Compound 670551 exhibited high selectivity for CLK2 over other protein kinases. Functionally, this compound significantly reduced the survival and proliferation of TNBC cells. Results from a cell-based assay demonstrated that this inhibitor led to a decrease in RNA splicing proteins, such as SRSF4 and SRSF6, resulting in cell apoptosis. In summary, we identified a novel CLK2 inhibitor as a promising potential treatment for TNBC therapy.

Covalent docking-driven virtual screening of extensive small-molecule libraries against Bruton tyrosine kinase for the identification of highly selective and potent novel therapeutic candidates.

Bruton tyrosine kinases (BTKs) play critical roles in various diseases, including chronic lymphatic leukemia (CLL), Waldenström Macroglobulinemia, Marginal Zone Lymphoma, Mantle Cell Lymphoma (MCL), and Graft Versus Host diseases. BTKs are a family of tyrosine kinases involved in B lymphocyte signal transduction, development, and maturation. Their overexpression can lead to cancer as they are essential for the activation of the B Cell Receptor (BCR) signaling pathway. Blocking the activation of BTKs presents a promising approach for treating CLL. This study was centered around the identification of small-molecule therapeutics that have an impact on human BTK. The covalently bound Ibrutinib molecule, recognized for its ability to inhibit BTK, was used as the query molecule. IUPAC text files containing molecular fragments of Ibrutinib were employed to virtually screen five different libraries comprising small-molecules, resulting in the screening of over 2.4 million synthesized compounds. Covalent docking simulations were applied to the selected small-molecules obtained through text mining from databases. Potent hit molecules capable of inhibiting BTKs through virtual screening algorithms were identified, paving the way for novel therapeutic strategies in the treatment of CLL.

Computational Investigation of the Covalent Inhibition Mechanism of Bruton's Tyrosine Kinase by Ibrutinib.

Covalent inhibitors represent a promising class of therapeutic compounds. Nonetheless, rationally designing covalent inhibitors to achieve a right balance between selectivity and reactivity remains extremely challenging. To better understand the covalent binding mechanism, a computational study is carried out using the irreversible covalent inhibitor of Bruton tyrosine kinase (BTK) ibrutinib as an example. A multi-µs classical molecular dynamics trajectory of the unlinked inhibitor is generated to explore the fluctuations of the compound associated with the kinase binding pocket. Then, the reaction pathway leading to the formation of the covalent bond with the cysteine residue at position 481 via a Michael addition is determined using the string method in collective variables on the basis of hybrid quantum mechanical-molecular mechanical (QM/MM) simulations. The reaction pathway shows a strong correlation between the covalent bond formation and the protonation/deprotonation events taking place sequentially in the covalent inhibition reaction, consistent with a 3-step reaction with transient thiolate and enolates intermediate states. Two possible atomistic mechanisms affecting deprotonation/protonation events from the thiolate to the enolate intermediate were observed: a highly correlated direct pathway involving proton transfer to the Cα of the acrylamide warhead from the cysteine involving one or a few water molecules and a more indirect pathway involving a long-lived enolate intermediate state following the escape of the proton to the bulk solution. The results are compared with experiments by simulating the long-time kinetics of the reaction using kinetic modeling.

Screening and Characterization of Allosteric Small Molecules Targeting Bruton's Tyrosine Kinase.

Bruton's Tyrosine Kinase (BTK) is a nonreceptor tyrosine kinase that belongs to the TEC family. Mutations in the BTK gene cause X-linked agammaglobulinemia (XLA) leading to an arrest in B-cell development. BTK is also a drug target for B-cell lymphomas that rely on an intact B-cell receptor signaling cascade for survival. All FDA approved drugs for BTK target the ATP binding site of the catalytic kinase domain, leading to potential adverse events due to off-target inhibition. In addition, acquired resistance mutations occur in a subset of patients, rendering available BTK inhibitors ineffective. Therefore, allosteric sites on BTK should be explored for drug development to target BTK more specifically and in combination with active site inhibitors. Virtual screening against nonactive site pockets and in vitro experiments resulted in a series of small molecules that bind to BTK outside of the active site. We characterized these compounds using biochemical and biophysical techniques and narrowed our focus to compound "C2". C2 activates full-length BTK and smaller multidomain BTK fragments but not the isolated kinase domain, consistent with an allosteric mode of action. Kinetic experiments reveal a C2-mediated decrease in Km and an increase in kcat leading to an overall increase in the catalytic efficiency of BTK. C2 is also capable of activating the BTK XLA mutants. These proof-of-principle data reveal that BTK can be targeted allosterically with small molecules, providing an alternative to active site BTK inhibitors.

Protocol to characterize extracellular c-Src tyrosine kinase function through substrate interaction and phosphorylation.

Cellular Src tyrosine kinase (c-Src) exists in the secretomes of several human cancers (extracellular, e-Src). Phosphoproteomics has demonstrated the existence of 114 potential extracellular e-Src substrates in addition to Tissue Inhibitor of Metalloproteinases 2. Here, we present a protocol to characterize secreted tyrosine-phosphorylated substrates as a result of c-Src expression and secretion. We describe steps for collecting cell secretomes and extracts, performing antibody treatment and Ni-NTA pull-down, and detecting protein-protein interaction and substrate Y-phosphorylation. This protocol is adaptable for studies examining the function of other extracellular kinases. For complete details on the use and execution of this protocol, please refer to Backe et al. (2023)1 and Sánchez-Pozo et al. (2018).2.

Molecular Dynamics and Machine Learning Give Insights on the Flexibility-Activity Relationships in Tyrosine Kinome.

Tyrosine kinases are a subfamily of kinases with critical roles in cellular machinery. Dysregulation of their active or inactive forms is associated with diseases like cancer. This study aimed to holistically understand their flexibility-activity relationships, focusing on pockets and fluctuations. We studied 43 different tyrosine kinases by collecting 120 µs of molecular dynamics simulations, pocket and residue fluctuation analysis, and a complementary machine learning approach. We found that the inactive forms often have increased flexibility, particularly at the DFG motif level. Noteworthy, thanks to these long simulations combined with a decision tree, we identified a semiquantitative fluctuation threshold of the DGF+3 residue over which the kinase has a higher probability to be in the inactive form.

Domain Architecture of the Nonreceptor Tyrosine Kinase Ack1.

The nonreceptor tyrosine kinase (NRTK) Ack1 comprises a distinct arrangement of non-catalytic modules. Its SH3 domain has a C-terminal to the kinase domain (SH1), in contrast to the typical SH3-SH2-SH1 layout in NRTKs. The Ack1 is the only protein that shares a region of high homology to the tumor suppressor protein Mig6, a modulator of EGFR. The vertebrate Acks make up the only tyrosine kinase (TK) family known to carry a UBA domain. The GTPase binding and SAM domains are also uncommon in the NRTKs. In addition to being a downstream effector of receptor tyrosine kinases (RTKs) and integrins, Ack1 can act as an epigenetic regulator, modulate the degradation of the epidermal growth factor receptor (EGFR), confer drug resistance, and mediate the progression of hormone-sensitive tumors. In this review, we discuss the domain architecture of Ack1 in relation to other protein kinases that possess such defined regulatory domains.

Discovery of the First Examples of Threonine Tyrosine Kinase PROTAC Degraders.

The first examples of threonine tyrosine kinase (TTK) PROTACs were designed and synthesized. Two of the most potent molecules, 8e and 8j, demonstrated strong TTK degradation in COLO-205 human colorectal cancer cells with DC50 values of 1.7 and 3.1 nM, respectively. Proteasome-mediated degradation by the compounds could last for approximately 8 h after washout. The degraders 8e and 8j demonstrated improved antiproliferative activities comparing with the structurally similar inhibitor counterparts 8q and 8r. Degraders 8e and 8j also demonstrated reasonable PK profiles and exhibited potent target degradation and in vivo anticancer efficacy in a xenograft mouse model of COLO-205 human colorectal cancer cells upon i.p. administration.

CEP97 phosphorylation by Dyrk1a is critical for centriole separation during multiciliogenesis.

Proper cilia formation in multiciliated cells (MCCs) is necessary for appropriate embryonic development and homeostasis. Multicilia share many structural characteristics with monocilia and primary cilia, but there are still significant gaps in our understanding of the regulation of multiciliogenesis. Using the Xenopus embryo, we show that CEP97, which is known as a negative regulator of primary cilia formation, interacts with dual specificity tyrosine phosphorylation regulated kinase 1A (Dyrk1a) to modulate multiciliogenesis. We show that Dyrk1a phosphorylates CEP97, which in turn promotes the recruitment of Polo-like kinase 1 (Plk1), which is a critical regulator of MCC maturation that functions to enhance centriole disengagement in cooperation with the enzyme Separase. Knockdown of either CEP97 or Dyrk1a disrupts cilia formation and centriole disengagement in MCCs, but this defect is rescued by overexpression of Separase. Thus, our study reveals that Dyrk1a and CEP97 coordinate with Plk1 to promote Separase function to properly form multicilia in vertebrate MCCs.

De novo design of tyrosine and serine kinase-driven protein switches.

Kinases play central roles in signaling cascades, relaying information from the outside to the inside of mammalian cells. De novo designed protein switches capable of interfacing with tyrosine kinase signaling pathways would open new avenues for controlling cellular behavior, but, so far, no such systems have been described. Here we describe the de novo design of two classes of protein switch that link phosphorylation by tyrosine and serine kinases to protein-protein association. In the first class, protein-protein association is required for phosphorylation by the kinase, while in the second class, kinase activity drives protein-protein association. We design systems that couple protein binding to kinase activity on the immunoreceptor tyrosine-based activation motif central to T-cell signaling, and kinase activity to reconstitution of green fluorescent protein fluorescence from fragments and the inhibition of the protease calpain. The designed switches are reversible and function in vitro and in cells with up to 40-fold activation of switching by phosphorylation.

Studies of Interaction Mechanism between Pyrido [3,4-d] Pyrimidine Inhibitors and Mps1.

Monopolar spindle 1 (Mps1), a dual-specific kinase, is related to the proper execution of chromosome biorientation and mitotic checkpoint signaling. The overexpression of Mps1 promotes the occurrence of cancer or the survival of aneuploid cancer cells, in other words, the reduction of Mps1 will severely reduce the viability of human cancer cells. Therefore, Mps1 is a potential target for cancer treatment. Recently, a series of novel pyrido [3,4-d] pyrimidine derivatives targeting Mps1 with high biological activity were synthesized. The crystal structure of Mps1 in complex with pyrido [3,4-d] pyrimidine derivatives was also reported, but there were no specific mechanism studies for this series of small molecule inhibitors. In this study, complexes binding modes were probed by molecular docking and further validated by molecular dynamics simulations and the molecular mechanics/generalized Born surface area (MM/GBSA) method. The results indicated that the van der Waals interactions and the nonpolar solvation energies were responsible to the basis for favorable binding free energies, all inhibitors interacted with residues I531, V539, M602, C604, N606, I607, L654, I663, and P673 of Mps1. By analyzing the hydrogen bonds, we found the residues G605 and K529 in Mps1 formed stable hydrogen bonds with compounds, it was more conducive to activities of Mps1 inhibitors. According to the above analysis, we further designed five new compounds. We found that compounds IV and V were better potential Mps1 inhibitors through docking and ADMET prediction. The obtained new insights not only were helpful in understanding the binding mode of inhibitors in Mps1, but also provided important references for further rational design of Mps1 inhibitors.

Simultaneous CK2/TNIK/DYRK1 inhibition by 108600 suppresses triple negative breast cancer stem cells and chemotherapy-resistant disease.

Triple negative breast cancer (TNBC) remains challenging because of heterogeneous responses to chemotherapy. Incomplete response is associated with a greater risk of metastatic progression. Therefore, treatments that target chemotherapy-resistant TNBC and enhance chemosensitivity would improve outcomes for these high-risk patients. Breast cancer stem cell-like cells (BCSCs) have been proposed to represent a chemotherapy-resistant subpopulation responsible for tumor initiation, progression and metastases. Targeting this population could lead to improved TNBC disease control. Here, we describe a novel multi-kinase inhibitor, 108600, that targets the TNBC BCSC population. 108600 treatment suppresses growth, colony and mammosphere forming capacity of BCSCs and induces G2M arrest and apoptosis of TNBC cells. In vivo, 108600 treatment of mice bearing triple negative tumors results in the induction of apoptosis and overcomes chemotherapy resistance. Finally, treatment with 108600 and chemotherapy suppresses growth of pre-established TNBC metastases, providing additional support for the clinical translation of this agent to clinical trials.

Structural and Functional Insights into the Biofilm-Associated BceF Tyrosine Kinase Domain from Burkholderia cepacia.

BceF is a bacterial tyrosine kinase (BY-kinase) from Burkholderia cepacia, a Gram-negative bacterium accountable for respiratory infections in immunocompromised and cystic fibrosis patients. BceF is involved in the production of exopolysaccharides secreted to the biofilm matrix and promotes resistant and aggressive infections. BY-kinases share no homology with mammalian kinases, and thereby offer a means to develop novel and specific antivirulence drugs. Here, we report the crystal structure of the BceF kinase domain at 1.85 Å resolution. The isolated BceF kinase domain is assembled as a dimer in solution and crystallized as a dimer in the asymmetric unit with endogenous adenosine-diphosphate bound at the active sites. The low enzymatic efficiency measured in solution may be explained by the partial obstruction of the active sites at the crystallographic dimer interface. This study provides insights into self-assembly and the specific activity of isolated catalytic domains. Several unique variations around the active site compared to other BY-kinases may allow for structure-based design of specific inhibitors to target Burkholderia cepacia virulence.

In-Silico Evaluation of Genetic Alterations in Ovarian Carcinoma and Therapeutic Efficacy of NSC777201, as a Novel Multi-Target Agent for TTK, NEK2, and CDK1.

Ovarian cancer is often detected at the advanced stages at the time of initial diagnosis. Early-stage diagnosis is difficult due to its asymptomatic nature, where less than 30% of 5-year survival has been noticed. The underlying molecular events associated with the disease's pathogenesis have yet to be fully elucidated. Thus, the identification of prognostic biomarkers as well as developing novel therapeutic agents for targeting these markers become relevant. Herein, we identified 264 differentially expressed genes (DEGs) common in four ovarian cancer datasets (GSE14407, GSE18520, GSE26712, GSE54388), respectively. We constructed a protein-protein interaction (PPI) interaction network with the overexpressed genes (72 genes) and performed gene enrichment analysis. In the PPI networks, three proteins; TTK Protein Kinase (TTK), NIMA Related Kinase 2 (NEK2), and cyclin-dependent kinase (CDK1) with higher node degrees were further evaluated as therapeutic targets for our novel multi-target small molecule NSC777201. We found that the upregulated DEGs were enriched in KEGG and gene ontologies associated with ovarian cancer progression, female gamete association, otic vesicle development, regulation of chromosome segregation, and therapeutic failure. In addition to the PPI network, ingenuity pathway analysis also implicate TTK, NEK2, and CDK1 in the elevated salvage pyrimidine and pyridoxal pathways in ovarian cancer. The TTK, NEK2, and CDK1 are over-expressed, demonstrating a high frequency of genetic alterations, and are associated with poor prognosis of ovarian cancer cohorts. Interestingly, NSC777201 demonstrated anti-proliferative and cytotoxic activities (GI50 = 1.6 µM~1.82 µM and TGI50 = 3.5 µM~3.63 µM) against the NCI panels of ovarian cancer cell lines and exhibited a robust interaction with stronger affinities for TTK, NEK2, and CDK1, than do the standard drug, paclitaxel. NSC777201 displayed desirable properties of a drug-like candidate and thus could be considered as a novel small molecule for treating ovarian carcinoma.

K63-linked ubiquitination of DYRK1A by TRAF2 alleviates Sprouty 2-mediated degradation of EGFR.

Dual specificity tyrosine phosphorylation regulated kinase 1A, DYRK1A, functions in multiple cellular pathways, including signaling, endocytosis, synaptic transmission, and transcription. Alterations in dosage of DYRK1A leads to defects in neurogenesis, cell growth, and differentiation, and may increase the risk of certain cancers. DYRK1A localizes to a number of subcellular structures including vesicles where it is known to phosphorylate a number of proteins and regulate vesicle biology. However, the mechanism by which it translocates to vesicles is poorly understood. Here we report the discovery of TRAF2, an E3 ligase, as an interaction partner of DYRK1A. Our data suggest that TRAF2 binds to PVQE motif residing in between the PEST and histidine repeat domain (HRD) of DYRK1A protein, and mediates K63-linked ubiquitination of DYRK1A. This results in translocation of DYRK1A to the vesicle membrane. DYRK1A increases phosphorylation of Sprouty 2 on vesicles, leading to the inhibition of EGFR degradation, and depletion of TRAF2 expression accelerates EGFR degradation. Further, silencing of DYRK1A inhibits the growth of glioma cells mediated by TRAF2. Collectively, these findings suggest that the axis of TRAF2-DYRK1A-Sprouty 2 can be a target for new therapeutic development for EGFR-mediated human pathologies.

Identification of Flavonoids as Putative ROS-1 Kinase Inhibitors Using Pharmacophore Modeling for NSCLC Therapeutics.

Non-small cell lung cancer (NSCLC) is a lethal non-immunogenic malignancy and proto-oncogene ROS-1 tyrosine kinase is one of its clinically relevant oncogenic markers. The ROS-1 inhibitor, crizotinib, demonstrated resistance due to the Gly2032Arg mutation. To curtail this resistance, researchers developed lorlatinib against the mutated kinase. In the present study, a receptor-ligand pharmacophore model exploiting the key features of lorlatinib binding with ROS-1 was exploited to identify inhibitors against the wild-type (WT) and the mutant (MT) kinase domain. The developed model was utilized to virtually screen the TimTec flavonoids database and the retrieved drug-like hits were subjected for docking with the WT and MT ROS-1 kinase. A total of 10 flavonoids displayed higher docking scores than lorlatinib. Subsequent molecular dynamics simulations of the acquired flavonoids with WT and MT ROS-1 revealed no steric clashes with the Arg2032 (MT ROS-1). The binding free energy calculations computed via molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) demonstrated one flavonoid (Hit) with better energy than lorlatinib in binding with WT and MT ROS-1. The Hit compound was observed to bind in the ROS-1 selectivity pocket comprised of residues from the ß-3 sheet and DFG-motif. The identified Hit from this investigation could act as a potent WT and MT ROS-1 inhibitor.

Next-Generation Kinase Inhibitors Targeting Specific Biomarkers in Non-Small Cell Lung Cancer (NSCLC): A Recent Overview.

Lung cancer causes many deaths globally. Mutations in regulatory genes, irregularities in specific signal transduction events, or alterations of signalling pathways are observed in cases of non-small cell lung cancer (NSCLC). Over the past two decades, a few kinases have been identified, validated, and studied as biomarkers for NSCLC. Among them, EGFR, ALK, ROS1, MET, RET, NTRK, and BRAF are regarded as targetable biomarkers to cure and/or control the disease. In recent years, the US Food and Drug Administration (FDA) approved more than 15 kinase inhibitors targeting these NSCLC biomarkers. The kinase inhibitors significantly improved the progression-free survival (PFS) of NSCLC patients. Challenges still remain for metastatic diseases and advanced NSCLC cases. New discoveries of potent kinase inhibitors and rapid development of modern medical technologies will help to control NSCLC cases. This article provides an overview of the discoveries of various types of kinase inhibitors against NSCLC, along with medicinal chemistry aspects and related developments in next-generation kinase inhibitors that have been reported in recent years.

5-Methoxybenzothiophene-2-Carboxamides as Inhibitors of Clk1/4: Optimization of Selectivity and Cellular Potency.

Clks have been shown by recent studies to be promising targets for cancer therapy, as they are considered key regulators in the process of pre-mRNA splicing, which in turn affects every aspect of tumor biology. In particular, Clk1 and -4 are overexpressed in several human tumors. Most of the potent Clk1 inhibitors reported in the literature are non-selective, mainly showing off-target activity towards Clk2, Dyrk1A and Dyrk1B. Herein, we present new 5-methoxybenzothiophene-2-carboxamide derivatives with unprecedented selectivity. In particular, the introduction of a 3,5-difluoro benzyl extension to the methylated amide led to the discovery of compound 10b (cell-free IC50 = 12.7 nM), which was four times more selective for Clk1 over Clk2 than the previously published flagship compound 1b. Moreover, 10b showed an improved growth inhibitory activity with T24 cells (GI50 = 0.43 µM). Furthermore, a new binding model in the ATP pocket of Clk1 was developed based on the structure-activity relationships derived from new rigidified analogues.

Spectrum of Mechanisms of Resistance to Crizotinib and Lorlatinib in ROS1 Fusion-Positive Lung Cancer.

PURPOSE: Current standard initial therapy for advanced, ROS proto-oncogene 1, receptor tyrosine kinase fusion (ROS1)-positive (ROS1+) non-small cell lung cancer (NSCLC) is crizotinib or entrectinib. Lorlatinib, a next-generation anaplastic lymphoma kinase/ROS1 inhibitor, recently demonstrated efficacy in ROS1+ NSCLC, including in crizotinib-pretreated patients. However, mechanisms of lorlatinib resistance in ROS1+ disease remain poorly understood. Here, we assessed mechanisms of resistance to crizotinib and lorlatinib. EXPERIMENTAL DESIGN: Biopsies from patients with ROS1 + NSCLC progressing on crizotinib or lorlatinib were profiled by genetic sequencing. RESULTS: From 55 patients, 47 post-crizotinib and 32 post-lorlatinib biopsies were assessed. Among 42 post-crizotinib and 28 post-lorlatinib biopsies analyzed at distinct timepoints, ROS1 mutations were identified in 38% and 46%, respectively. ROS1 G2032R was the most commonly occurring mutation in approximately one third of cases. Additional ROS1 mutations included D2033N (2.4%) and S1986F (2.4%) post-crizotinib and L2086F (3.6%), G2032R/L2086F (3.6%), G2032R/S1986F/L2086F (3.6%), and S1986F/L2000V (3.6%) post-lorlatinib. Structural modeling predicted ROS1L2086F causes steric interference to lorlatinib, crizotinib, and entrectinib, while it may accommodate cabozantinib. In Ba/F3 models, ROS1L2086F, ROS1G2032R/L2086F, and ROS1S1986F/G2032R/L2086F were refractory to lorlatinib but sensitive to cabozantinib. A patient with disease progression on crizotinib and lorlatinib and ROS1 L2086F received cabozantinib for nearly 11 months with disease control. Among lorlatinib-resistant biopsies, we also identified MET amplification (4%), KRAS G12C (4%), KRAS amplification (4%), NRAS mutation (4%), and MAP2K1 mutation (4%). CONCLUSIONS: ROS1 mutations mediate resistance to crizotinib and lorlatinib in more than one third of cases, underscoring the importance of developing next-generation ROS1 inhibitors with potency against these mutations, including G2032R and L2086F. Continued efforts are needed to elucidate ROS1-independent resistance mechanisms.

Pharmacophore-based designing of putative ROS-1 targeting agents for NSCLC.

Non-small cell lung cancer (NSCLC) is a fatal non-immunogenic malignancy, and proto-oncogene receptor tyrosine kinase (ROS-1) is one of its clinically relevant biomarkers. In this context, herein, we report a series of benzimidazol-2-amine derivatives which were synthesized on the basis of the pharmacophore of ROS-1 and evaluated for anti-proliferative activity. For this, the in silico receptor-ligand pharmacophore model of ROS-1, previously published by our own group, was utilized to screen out an in-house database of small molecule heterocycles. Docking analysis of the selected compounds was carried out within the active site of wild-type (WT) ROS-1 as well as Gly2032Arg mutant ROS-1 protein, which confirmed the retention of conserved interaction between selected molecules and hinge region amino acids Glu2027 and Met2029. Docking was followed by molecular dynamics simulations for the stability of the complexes and calculation of the MM-GBSA score for binding affinity. Finally, compounds were synthesized and the anti-proliferative potential of compounds was evaluated using the A549 cell line. Compounds 3a and 3b presented significant GI50 values between 23.0 and 25.4 µM, among all the tested compounds.