An ensemble machine learning model generates a focused screening library for the identification of CDK8 inhibitors.

The identification of an effective inhibitor is an important starting step in drug development. Unfortunately, many issues such as the characterization of protein binding sites, the screening library, materials for assays, etc., make drug screening a difficult proposition. As the size of screening libraries increases, more resources will be inefficiently consumed. Thus, new strategies are needed to preprocess and focus a screening library towards a targeted protein. Herein, we report an ensemble machine learning (ML) model to generate a CDK8-focused screening library. The ensemble model consists of six different algorithms optimized for CDK8 inhibitor classification. The models were trained using a CDK8-specific fragment library along with molecules containing CDK8 activity. The optimized ensemble model processed a commercial library containing 1.6 million molecules. This resulted in a CDK8-focused screening library containing 1,672 molecules, a reduction of more than 99.90%. The CDK8-focused library was then subjected to molecular docking, and 25 candidate compounds were selected. Enzymatic assays confirmed six CDK8 inhibitors, with one compound producing an IC50 value of ≤100 nM. Analysis of the ensemble ML model reveals the role of the CDK8 fragment library during training. Structural analysis of molecules reveals the hit compounds to be structurally novel CDK8 inhibitors. Together, the results highlight a pipeline for curating a focused library for a specific protein target, such as CDK8.

Identification of selective dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitors and their effects on tau and microtubule.

The overexpression of dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A), commonly observed in neurodegenerative diseases like Alzheimer's disease (AD) and Down syndrome (DS), can induce the formation of neurofibrillary tangles (NFTs) and amyloid plaques. Hence, designing a selective DYRK1A inhibitor would result in a promising small molecule for treating neurodegenerative diseases. Developing selective inhibitors for DYRK1A has been a difficult challenge due to the highly preserved ATP-binding site of protein kinases. In this study, we employed a structure-based virtual screening (SBVS) campaign targeting DYRK1A from a database containing 1.6 million compounds. Enzymatic assays were utilized to verify inhibitory properties, confirming that Y020-3945 and Y020-3957 showed inhibitory activity towards DYRK1A. In particular, the compounds exhibited high selectivity for DYRK1A over a panel of 120 kinases, reduced the phosphorylation of tau, and reversed the tubulin polymerization for microtubule stability. Additionally, treatment with the compounds significantly reduced the secretion of inflammatory cytokines IL-6 and TNF-α activated by DYRK1A-assisted NFTs and Aß oligomers. These identified inhibitors possess promising therapeutic potential for conditions associated with DYRK1A in neurodegenerative diseases. The results showed that Y020-3945 and Y020-3957 demonstrated structural novelty compared to known DYRK1A inhibitors, making them a valuable addition to developing potential treatments for neurodegenerative diseases.

Establishment of extensive artificial intelligence models for kinase inhibitor prediction: Identification of novel PDGFRB inhibitors.

Identifying hit compounds is an important step in drug development. Unfortunately, this process continues to be a challenging task. Several machine learning models have been generated to aid in simplifying and improving the prediction of candidate compounds. Models tuned for predicting kinase inhibitors have been established. However, an effective model can be limited by the size of the chosen training dataset. In this study, we tested several machine learning models to predict potential kinase inhibitors. A dataset was curated from a number of publicly available repositories. This resulted in a comprehensive dataset covering more than half of the human kinome. More than 2,000 kinase models were established using different model approaches. The performances of the models were compared, and the Keras-MLP model was determined to be the best performing model. The model was then used to screen a chemical library for potential inhibitors targeting platelet-derived growth factor receptor-ß (PDGFRB). Several PDGFRB candidates were selected, and in vitro assays confirmed four compounds with PDGFRB inhibitory activity and IC50 values in the nanomolar range. These results show the effectiveness of machine learning models trained on the reported dataset. This report would aid in the establishment of machine learning models as well as in the discovery of novel kinase inhibitors.

In silico identification and biological evaluation of a selective MAP4K4 inhibitor against pancreatic cancer.

Inhibiting a specific target in cancer cells and reducing unwanted side effects has become a promising strategy in pancreatic cancer treatment. MAP4K4 is associated with pancreatic cancer development and correlates with poor clinical outcomes. By phosphorylating MKK4, proteins associated with cell apoptosis and survival are translated. Therefore, inhibiting MAP4K4 activity in pancreatic tumours is a new therapeutic strategy. Herein, we performed a structure-based virtual screening to identify MAP4K4 inhibitors and discovered the compound F389-0746 with a potent inhibition (IC50 120.7 nM). The results of kinase profiling revealed that F389-0746 was highly selective to MAP4K4 and less likely to cause side effects. Results of in vitro experiments showed that F389-0746 significantly suppressed cancer cell growth and viability. Results of in vivo experiments showed that F389-0746 displayed comparable tumour growth inhibition with the group treated with gemcitabine. These findings suggest that F389-0746 has promising potential to be further developed as a novel pancreatic cancer treatment.

Design, synthesis, and biological evaluation of indolin-2-one derivatives as novel cyclin-dependent protein kinase 8 (CDK8) inhibitors.

Cyclin-dependent protein kinase 8 (CDK8) plays important roles in regulating fibrotic growth factors and inflammatory signaling pathways. Long-term chronic inflammation of the lungs can lead to idiopathic pulmonary fibrosis (IPF). Abnormal alveolar epithelial regeneration leads to the release of various fibrotic growth factors and the activation of inflammatory cells. CDK8 regulates profibrotic cytokines broadly implicated in the pathogenesis of fibrosis. Therefore, inhibition of CDK8 is considered a promising strategy for treating IPF. Here, CDK8 inhibitors were designed and optimized using a fragment-based drug design strategy. Testing results revealed that 71% of the synthesized compounds inhibited CDK8 activity better than the original compound E966-0530. Of these compounds, compound 4k exhibited the strongest CDK8 enzyme-inhibiting activity (IC50 =129 nM). Notably, it displayed a 13-fold increase in potency when compared to E966-0530. Experiments on toxicity and inhibition of epithelial-mesenchymal transition (EMT) protein expressions showed that compound 4k can inhibit EMT protein expressions, but with no significant cytotoxicity for alveolar epithelial cells. Compound 4k showed a potent inhibitory effect in cell migration assays. Furthermore, compound 4k significantly inhibited the phosphorylation of p-Smad3 and RNA Pol II, which are critical mediators in the fibrotic response signaling pathway. Compound 4k remarkably reduced TGF-ß1-induced oxidative stress. The above results reveal optimized CDK8 inhibitors with potential use for IPF therapeutic treatment.

O-methylated flavonol as a multi-kinase inhibitor of leukemogenic kinases exhibits a potential treatment for acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML) is a heterogeneous disease with poor overall survival characterized by various genetic changes. The continuous activation of oncogenic pathways leads to the development of drug resistance and limits current therapeutic efficacy. Therefore, a multi-targeting inhibitor may overcome drug resistance observed in AML treatment. Recently, groups of flavonoids, such as flavones and flavonols, have been shown to inhibit a variety of kinase activities, which provides potential opportunities for further anticancer applications. PURPOSE: In this study, we evaluated the anticancer effects of flavonoid compounds collected from our in-house library and investigated their potential anticancer mechanisms by targeting multiple kinases for inhibition in AML cells. METHODS: The cytotoxic effect of the compounds was detected by cell viability assays. The kinase inhibitory activity of the selected compound was detected by kinase-based and cell-based assays. The binding conformation and interactions were investigated by molecular docking analysis. Flow cytometry was used to evaluate the cell cycle distribution and cell apoptosis. The protein and gene expression were estimated by western blotting and qPCR, respectively. RESULTS: In this study, an O-methylated flavonol (compound 11) was found to possess remarkable cytotoxic activity against AML cells compared to treatment in other cancer cell lines. The compound was demonstrated to act against multiple kinases, which play critical roles in survival signaling in AML, including FLT3, MNK2, RSK, DYRK2 and JAK2 with IC50 values of 1 - 2 µM. Compared to our previous flavonoid compounds, which only showed inhibitions against MNKs or FLT3, compound 11 exhibited multiple kinase inhibitory abilities. Moreover, compound 11 showed effectiveness in inhibiting internal tandem duplications of FLT3 (FLT3-ITDs), which accounts for 25% of AML cases. The interactions between compound 11 and targeted kinases were investigated by molecular docking analysis. Mechanically, compound 11 caused dose-dependent accumulation of leukemic cells at the G0/G1 phase and followed by the cells undergoing apoptosis. CONCLUSION: O-methylated flavonol, compound 11, can target multiple kinases, which may provide potential opportunities for the development of novel therapeutics for drug-resistant AMLs. This work provides a good starting point for further compound optimization.

Identification of a dual FLT3 and MNK2 inhibitor for acute myeloid leukemia treatment using a structure-based virtual screening approach.

Fms-like tyrosine kinase 3 (FLT3) is considered a promising therapeutic target for acute myeloid leukemia (AML) in the clinical. However, monotherapy with FLT3 inhibitor is usually accompanied by drug resistance. Dual inhibitors might be therapeutically beneficial to patients with AML due to their ability to overcome drug resistance. Mitogen-activated protein kinase (MAPK)-interacting kinases (MNKs) phosphorylate eukaryotic translation initiation factor 4E (eIF4E), which brings together the RAS/RAF/ERK and PI3K/AKT/mTOR oncogenic pathways. Therefore, dual inhibition of FLT3 and MNK2 might have an additive effect against AML. Herein, a structure-based virtual screening approach was performed to identify dual inhibitors of FLT3 and MNK2 from the ChemDiv database. Compound K783-0308 was identified as a dual inhibitor of FLT3 and MNK2 with IC50 values of 680 and 406 nM, respectively. In addition, the compound showed selectivity for both FLT3 and MNK2 in a panel of 82 kinases. The structure-activity relationship analysis and common interactions revealed interactions between K783-0308 analogs and FLT3 and MNK2. Furthermore, K783-0308 inhibited MV-4-11 and MOLM-13 AML cell growth and induced G0/G1 cell cycle arrest. Taken together, the dual inhibitor K783-0308 showed promising results and can be potentially optimized as a lead compound for AML treatment.

Structure-based virtual screening and biological evaluation of novel small-molecule BTK inhibitors.

Bruton tyrosine kinase (BTK) is linked to multiple signalling pathways that regulate cellular survival, activation, and proliferation. A covalent BTK inhibitor has shown favourable outcomes for treating B cell malignant leukaemia. However, covalent inhibitors require a high reactive warhead that may contribute to unexpected toxicity, poor selectivity, or reduced effectiveness in solid tumours. Herein, we report the identification of a novel noncovalent BTK inhibitor. The binding interactions (i.e. interactions from known BTK inhibitors) for the BTK binding site were identified and incorporated into a structure-based virtual screening (SBVS). Top-rank compounds were selected and testing revealed a BTK inhibitor with >50% inhibition at 10 µM concentration. Examining analogues revealed further BTK inhibitors. When tested across solid tumour cell lines, one inhibitor showed favourable inhibitory activity, suggesting its potential for targeting BTK malignant tumours. This inhibitor could serve as a basis for developing an effective BTK inhibitor targeting solid cancers.

Identification and analysis of a selective DYRK1A inhibitor.

The dysregulation of DYRK1A is implicated in many diseases such as cancer, diabetes, and neurodegenerative diseases. Alzheimer's disease is one of the most common neurodegenerative disease and has elevated interest in DYRK1A research. Overexpression of DYRK1A has been linked to the formation of tau aggregates. Currently, an effective therapeutic treatment that targets DYRK1A is lacking. A specific small-molecule inhibitor would further our understanding of the physiological role of DYRK1A in neurodegenerative diseases and could be presented as a possible therapeutic option. In this study, we identified pharmacological interactions within the DYRK1A active site and performed a structure-based virtual screening approach to identify a selective small-molecule inhibitor. Several compounds were selected in silico for enzymatic and cellular assays, yielding a novel inhibitor. A structure-activity relationship analysis was performed to identify areas of interactions for the compounds selected in this study. When tested in vitro, reduction of DYRK1A dependent phosphorylation of tau was observed for active compounds. The active compounds also improved tau turbidity, suggesting that these compounds could alleviate aberrant tau aggregation. Testing the active compound against a panel of kinases across the kinome revealed greater selectivity towards DYRK1A. Our study demonstrates a serviceable protocol that identified a novel and selective DYRK1A inhibitor with potential for further study in tau-related pathologies.

Discovery of a novel cyclin-dependent kinase 8 inhibitor with an oxindole core for anti-inflammatory treatment.

Chronic inflammation is an underlying cause in a number of diseases. Cyclin-dependent kinase 8 (CDK8) has been implicated as an inflammatory mediator, indicating its potential as an anti-inflammatory target. Herein, we performed structure-based virtual screening (SBVS) to identify novel CDK8 inhibitors. The pharmacological interactions for CDK8 were identified and incorporated into a SBVS protocol. Selected compounds were tested in enzymatic assays, and one compound was confirmed to be a CDK8 inhibitor with a 50% inhibitory concentration (IC50) value of 1684.4 nM. Comparing structural analogs identified a compound, F059-1017, with greater potency (IC50 558.1 nM). When tested in cell lines, the compounds displayed low cytotoxicity. Cellular assays revealed that the identified CDK8 inhibitors can reduce phosphorylation and expression of signaling mediators associated with inflammation. In addition, results of kinase profiling showed that compound F059-1017 is selective towards CDK8. These findings suggest that the new inhibitors have great potential as lead compounds for developing novel anti-inflammatory therapeutics.

Investigation of Selected Flavonoid Derivatives as Potent FLT3 Inhibitors for the Potential Treatment of Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is an aggressive disease with a poor prognosis and a high degree of relapse seen in patients. Overexpression of FMS-like tyrosine kinase 3 (FLT3) is associated with up to 70% of AML patients. Wild-type FLT3 induces proliferation and inhibits apoptosis in AML cells, while uncontrolled proliferation of FLT3 kinase activity is also associated with FLT3 mutations. Therefore, inhibiting FLT3 activity is a promising AML therapy. Flavonoids are a group of phytochemicals that can target protein kinases, suggesting their potential antitumor activities. In this study, several plant-derived flavonoids have been identified with FLT3 inhibitory activity. Among these compounds, compound 40 (5,7,4'-trihydroxy-6-methoxyflavone) exhibited the most potent inhibition against not only FLT3 (IC50 = 0.44 µM) but also FLT3-D835Y and FLT3-ITD mutants (IC50 = 0.23 and 0.39 µM, respectively). The critical interactions between the FLT3 binding site and the compounds were identified by performing a structure-activity relationship analysis. Furthermore, the results of cellular assays revealed that compounds 28, 31, 32, and 40 exhibited significant cytotoxicity against two human AML cell lines (MOLM-13 and MV-4-11), and compounds 31, 32, and 40 resulted in cell apoptosis and G0/G1 cell cycle arrest. Collectively, these flavonoids have the potential to be further optimized as FLT3 inhibitors and provide valuable chemical information for the development of new AML drugs.

Identification of a dual TAOK1 and MAP4K5 inhibitor using a structure-based virtual screening approach.

The STE20 kinase family is a complex signalling cascade that regulates cytoskeletal organisation and modulates the stress response. This signalling cascade includes various kinase mediators, such as TAOK1 and MAP4K5. The dysregulation of the STE20 kinase pathway is linked with cancer malignancy. A small-molecule inhibitor targeting the STE20 kinase pathway has therapeutic potential. In this study, a structure-based virtual screening (SBVS) approach was used to identify potential dual TAOK1 and MAP4K5 inhibitors. Enzymatic assays confirmed three potential dual inhibitors (>50% inhibition) from our virtual screening, and analysis of the TAOK1 and MAP4K5 binding sites indicated common interactions for dual inhibition. Compound 1 revealed potent inhibition of colorectal and lung cancer cell lines. Furthermore, compound 1 arrested cancer cells in the G0/G1 phase, which suggests the induction of apoptosis. Altogether, we show that the STE20 signalling mediators TAOK1 and MAP4K5 are promising targets for drug research.

Biological Evaluation of Selected Flavonoids as Inhibitors of MNKs Targeting Acute Myeloid Leukemia.

Excessive eIF4E phosphorylation by mitogen-activated protein kinase (MAPK)-interacting kinases 1 and 2 (MNK1 and MNK2; collectively, MNKs) has been associated with oncogenesis. The overexpression of eIF4E in acute myeloid leukemia (AML) is related to cancer cell growth and survival. Thus, the inhibition of MNKs and eIF4E phosphorylation are potential therapeutic strategies for AML. Herein, a structure-based virtual screening approach was performed to identify potential MNK inhibitors from natural products. Three flavonoids, apigenin, hispidulin, and luteolin, showed MNK2 inhibitory activity with IC50 values of 308, 252, and 579 nM, respectively. A structure-activity relationship analysis was performed to disclose the molecular interactions. Furthermore, luteolin exhibited substantial inhibitory efficacy against MNK1 (IC50 = 179 nM). Experimental results from cellular assays showed that hispidulin and luteolin inhibited the growth of MOLM-13 and MV4-11 AML cells by downregulating eIF4E phosphorylation and arresting the cell cycle at the G0/G1 phase. Therefore, hispidulin and luteolin showed promising results as lead compounds for the potential treatment for AML.

A site-moiety map and virtual screening approach for discovery of novel 5-LOX inhibitors.

The immune system works in conjunction with inflammation. Excessive inflammation underlies various human diseases, such as asthma, diabetes and heart disease. Previous studies found that 5-lipoxygenase (5-LOX) plays a crucial role in metabolizing arachidonic acid into inflammatory mediators and is a potential therapeutic target. In this study, we performed an in silico approach to establish a site-moiety map (SiMMap) to screen for new 5-LOX inhibitors. The map is composed of several anchors that contain key residues, moiety preferences, and their interaction types (i.e., electrostatic (E), hydrogen-bonding (H), and van der Waals (V) interactions) within the catalytic site. In total, we identified one EH, one H, and five V anchors, within the 5-LOX catalytic site. Based on the SiMMap, three 5-LOX inhibitors (YS1, YS2, and YS3) were identified. An enzyme-based assay validated inhibitory activity of YS1, YS2, and YS3 against 5-LOX with an IC50 value of 2.7, 4.2, and 5.3 µM, respectively. All three inhibitors significantly decrease LPS-induced TNF-α and IL-6 production, which suggests its potential use an anti-inflammatory agent. In addition, the identified 5-LOX inhibitors contain a novel scaffold. The discovery of these inhibitors presents an opportunity for designing specific anti-inflammatory drugs.

A Novel Selective JAK2 Inhibitor Identified Using Pharmacological Interactions.

The JAK2/STAT signaling pathway mediates cytokine receptor signals that are involved in cell growth, survival and homeostasis. JAK2 is a member of the Janus kinase (JAK) family and aberrant JAK2/STAT is involved with various diseases, making the pathway a therapeutic target. The similarity between the ATP binding site of protein kinases has made development of specific inhibitors difficult. Current JAK2 inhibitors are not selective and produce unwanted side effects. It is thought that increasing selectivity of kinase inhibitors may reduce the side effects seen with current treatment options. Thus, there is a great need for a selective JAK inhibitor. In this study, we identified a JAK2 specific inhibitor. We first identified key pharmacological interactions in the JAK2 binding site by analyzing known JAK2 inhibitors. Then, we performed structure-based virtual screening and filtered compounds based on their pharmacological interactions and identified compound NSC13626 as a potential JAK2 inhibitor. Results of enzymatic assays revealed that against a panel of kinases, compound NSC13626 is a JAK2 inhibitor and has high selectivity toward the JAK2 and JAK3 isozymes. Our cellular assays revealed that compound NSC13626 inhibits colorectal cancer cell (CRC) growth by downregulating phosphorylation of STAT3 and arresting the cell cycle in the S phase. Thus, we believe that compound NSC13626 has potential to be further optimized as a selective JAK2 drug.

ACR11 modulates levels of reactive oxygen species and salicylic acid-associated defense response in Arabidopsis.

The ACT domain (aspartate kinase, chorismate mutase and TyrA), an allosteric effector binding domain, is commonly found in amino acid metabolic enzymes. In addition to ACT domain-containing enzymes, plants have a novel family of ACT domain repeat (ACR) proteins, which do not contain any recognizable catalytic domain. Arabidopsis has 12 ACR proteins, whose functions are largely unknown. To study the functions of Arabidopsis ACR11, we have characterized two independent T-DNA insertion mutants, acr11-2 and acr11-3. RNA gel-blot analysis revealed that the expression of wild-type ACR11 transcripts was not detectable in the acr11 mutants. Interestingly, a lesion-mimic phenotype occurs in some rosette leaves of the acr11 mutants. In addition, high levels of reactive oxygen species (ROS), salicylic acid (SA), and callose accumulate in the mutant leaves when grown under normal conditions. The expression of several SA marker genes and the key SA biosynthetic gene ISOCHORISMATE SYNTHASE1 is up-regulated in the acr11 mutants. Furthermore, the acr11 mutants are more resistant to the infection of bacterial pathogen Pseudomonas syringae pathovar tomato DC3000. These results suggest that ACR11 may be directly or indirectly involved in the regulation of ROS and SA accumulation, which in turn modulates SA-associated defense responses and disease resistance in Arabidopsis.

Identification of neuraminidase inhibitors against dual H274Y/I222R mutant strains.

Influenza is an annual seasonal epidemic that has continually drawn public attentions, due to the potential death toll and drug resistance. Neuraminidase, which is essential for the spread of influenza virus, has been regarded as a valid target for the treatment of influenza infection. Although neuraminidase drugs have been developed, they are susceptible to drug-resistant mutations in the sialic-binding site. In this study, we established computational models (site-moiety maps) of H1N1 and H5N1 to determine properties of the 150-cavity, which is adjacent to the drug-binding site. The models reveal that hydrogen-bonding interactions with residues R118, D151, and R156 and van der Waals interactions with residues Q136, D151, and T439 are important for identifying 150-cavitiy inhibitors. Based on the models, we discovered three new inhibitors with IC50 values <10 µM that occupies both the 150-cavity and sialic sites. The experimental results identified inhibitors with similar activities against both wild-type and dual H274Y/I222R mutant neuraminidases and showed little cytotoxic effects. Furthermore, we identified three new inhibitors situated at the sialic-binding site with inhibitory effects for normal neuraminidase, but lowered effects for mutant strains. The results suggest that the new inhibitors can be used as a starting point to combat drug-resistant strains.

Novel Class IIa-Selective Histone Deacetylase Inhibitors Discovered Using an in Silico Virtual Screening Approach.

Histone deacetylases (HDAC) contain eighteen isoforms that can be divided into four classes. Of these isoform enzymes, class IIa (containing HDAC4, 5, 7 and 9) target unique substrates, some of which are client proteins associated with epigenetic control. Class IIa HDACs are reportedly associated with some neuronal disorders, making HDACs therapeutic targets for treating neurodegenerative diseases. Additionally, some reported HDAC inhibitors contain hydroxamate moiety that chelates with zinc ion to become the cofactor of HDAC enzymes. However, the hydroxamate functional group is shown to cause undesirable effects and has poor pharmacokinetic profile. This study used in silico virtual screening methodology to identify several nonhydroxamate compounds, obtained from National Cancer Institute database, which potentially inhibited HDAC4. Comparisons of the enzyme inhibitory activity against a panel of HDAC isoforms revealed these compounds had strong inhibitory activity against class IIa HDACs, but weak inhibitory activity against class I HDACs. Further analysis revealed that a single residue affects the cavity size between class I and class IIa HDACs, thus contributing to the selectivity of HDAC inhibitors discovered in this study. The discovery of these inhibitors presents the possibility of developing new therapeutic treatments that can circumvent the problems seen in traditional hydroxamate-based drugs.

Anchor-based classification and type-C inhibitors for tyrosine kinases.

Tyrosine kinases regulate various biological processes and are drug targets for cancers. At present, the design of selective and anti-resistant inhibitors of kinases is an emergent task. Here, we inferred specific site-moiety maps containing two specific anchors to uncover a new binding pocket in the C-terminal hinge region by docking 4,680 kinase inhibitors into 51 protein kinases, and this finding provides an opportunity for the development of kinase inhibitors with high selectivity and anti-drug resistance. We present an anchor-based classification for tyrosine kinases and discover two type-C inhibitors, namely rosmarinic acid (RA) and EGCG, which occupy two and one specific anchors, respectively, by screening 118,759 natural compounds. Our profiling reveals that RA and EGCG selectively inhibit 3% (EGFR and SYK) and 14% of 64 kinases, respectively. According to the guide of our anchor model, we synthesized three RA derivatives with better potency. These type-C inhibitors are able to maintain activities for drug-resistant EGFR and decrease the invasion ability of breast cancer cells. Our results show that the type-C inhibitors occupying a new pocket are promising for cancer treatments due to their kinase selectivity and anti-drug resistance.

Functional evidence for the critical amino-terminal conserved domain and key amino acids of Arabidopsis 4-HYDROXY-3-METHYLBUT-2-ENYL DIPHOSPHATE REDUCTASE.

The plant 4-HYDROXY-3-METHYLBUT-2-ENYL DIPHOSPHATE REDUCTASE (HDR) catalyzes the last step of the methylerythritol phosphate pathway to synthesize isopentenyl diphosphate and its allyl isomer dimethylallyl diphosphate, which are common precursors for the synthesis of plastid isoprenoids. The Arabidopsis (Arabidopsis thaliana) genomic HDR transgene-induced gene-silencing lines are albino, variegated, or pale green, confirming that HDR is essential for plants. We used Escherichia coli isoprenoid synthesis H (Protein Data Bank code 3F7T) as a template for homology modeling to identify key amino acids of Arabidopsis HDR. The predicted model reveals that cysteine (Cys)-122, Cys-213, and Cys-350 are involved in iron-sulfur cluster formation and that histidine (His)-152, His-241, glutamate (Glu)-242, Glu-243, threonine (Thr)-244, Thr-312, serine-379, and asparagine-381 are related to substrate binding or catalysis. Glu-242 and Thr-244 are conserved only in cyanobacteria, green algae, and land plants, whereas the other key amino acids are absolutely conserved from bacteria to plants. We used site-directed mutagenesis and complementation assay to confirm that these amino acids, except His-152 and His-241, were critical for Arabidopsis HDR function. Furthermore, the Arabidopsis HDR contains an extra amino-terminal domain following the transit peptide that is highly conserved from cyanobacteria, and green algae to land plants but not existing in the other bacteria. We demonstrated that the amino-terminal conserved domain was essential for Arabidopsis and cyanobacterial HDR function. Further analysis of conserved amino acids in the amino-terminal conserved domain revealed that the tyrosine-72 residue was critical for Arabidopsis HDR. These results suggest that the structure and reaction mechanism of HDR evolution have become specific for oxygen-evolving photosynthesis organisms and that HDR probably evolved independently in cyanobacteria versus other prokaryotes.