Identifying Novel Drug Targets by iDTPnd: A Case Study of Kinase Inhibitors

Current FDA-approved kinase inhibitors cause diverse adverse effects, some of which are due to the mechanism-independent effects of these drugs. Identifying these mechanism-independent interactions could improve drug safety and support drug repurposing. Here, we develop iDTPnd (integrated Drug Target Predictor with negative dataset), a computational approach for large-scale discovery of novel targets for known drugs. For a given drug, we construct a positive structural signature as well as a negative structural signature that captures the weakly conserved structural features of drug-binding sites. To facilitate assessment of unintended targets, iDTPnd also provides a docking-based interaction score and its statistical significance. We confirm the interactions of sorafenib, imatinib, dasatinib, sunitinib, and pazopanib with their known targets at a sensitivity of 52% and a specificity of 55%. We also validate 10 predicted novel targets by using in vitro experiments. Our results suggest that proteins other than kinases, such as nuclear receptors, cytochrome P450, and MHC class I molecules, can also be physiologically relevant targets of kinase inhibitors. Our method is general and broadly applicable for the identification of protein–small molecule interactions, when sufficient drug–target 3D data are available. The code for constructing the structural signatures is available at https://sfb.kaust.edu.sa/Documents/iDTP.zip.     

Tyrosine Kinase Inhibitors and the Thyroid as Both an Unintended and an Intended Target

ObjectiveTo review the association of the tyrosine kinase inhibitor sunitinib with hypothyroidism as well as the mean time to onset, possible mechanisms, reversibility, and mean duration.MethodsWe performed a MEDLINE search of the English-language literature using a combination of words (“sunitinib,” “tyrosine kinase inhibitors,” “thyroid,” and “hypothyroidism”) to identify original studies and reviews on sunitinib and thyroid function.ResultsHypothyroidism was reported in 36% to 46% of patients who took sunitinib in prospective studies. A higher incidence (53% to 85%) was reported in studies containing both retrospective and prospective data. The mean time to onset of hypothyroidism after initiation of sunitinib therapy ranged from 12 to 50 weeks. The risk of development of hypothyroidism appears to increase with the increasing duration of sunitinib therapy, and the condition is likely reversible once therapy has been discontinued.ConclusionBaseline thyroid function tests should be performed before the initiation of sunitinib treatment. Because hypothyroidism can develop early in the course of therapy, thyroid function tests should be monitored frequently throughout the duration of treatment. Possible mechanisms for thyroid dysfunction include impaired thyroid hormone synthesis, a destructive thyroiditis preceding the development of hypothyroidism, and increased thyroid hormone clearance. If hypothyroidism is identified, levothyroxine therapy should be promptly initiated. (Endocr Pract. 2008;14:618-624)     

Postoperative Adjuvant Sorafenib or Sunitinib for Nonmetastatic Renal Cell Carcinoma with Venous Tumor Thrombus: a Prospective Cohort Study

PURPOSE: To evaluate the efficacy and safety of antiangiogenic agents (sorafenib and sunitinib) as postoperative adjuvant therapy in patients with nonmetastatic renal cell carcinoma (RCC) and venous tumor thrombus (VTT). MATERIAL AND METHODS: From March 2006 to January 2016, 147 patients who met the inclusion criteria were enrolled; 27 patients received sorafenib, and 17 patients received sunitinib. After radical nephrectomy and thrombectomy, the duration of maintenance targeted medication treatment was approximately 1 year. The primary objective was to compare disease-free survival (DFS) between each experimental group and control. Secondary end points included overall survival (OS) and toxic effects. RESULTS: The three groups were well balanced in terms of age, body mass index, gender, performance status, medical history, American Society of Anesthesiologists score, surgical approach, and tumor side and size. However, more patients receiving adjuvant therapy had inferior vena cava tumor thrombus. DFS and OS did not differ significantly between groups (P = .459 and .871, respectively). After adjusting for potential confounding factors, results of multivariate analysis proved that postoperative adjuvant therapy was not an independent factor for predicting DFS and OS (P > .05 for both). The subgroup analyses for inferior vena cava tumor thrombus found similar results. The common adverse events were hand-foot syndrome, diarrhea, fatigue, and neutropenia. The adverse effects were mild in both groups, and the incidence was not significantly different between sorafenib and sunitinib. CONCLUSIONS: Adjuvant treatment postoperatively with sorafenib or sunitinib showed no survival benefit relative to control for patients with nonmetastatic RCC and VTT in a prospective cohort study.     

索拉非尼与舒尼替尼单药治疗晚期肝细胞癌的效果分析

目的:对比索拉非尼(Sorafenib)和舒尼替尼(Sunitinib)单药治疗晚期肝细胞癌的疗效及安全性。方法:对我院2004年1月-2010年10月收治的44例晚期肝细胞肝癌患者的临床资料进行回顾分析。根据不同给药方式,将患者分为两组。其中,索拉非尼组32例患者采取口服索拉非尼进行治疗,而舒尼替尼组12例患者给予口服舒尼替尼治疗。观察并比较两组患者的治疗效果及药物不良反应情况。结果:索拉非尼组总生存时间为6.3月,1年生存率为16%,肿瘤进展时间为3个月,疾病控制率为71%;舒尼替尼组总生存时间为4.7月,1年生存率为8%,肿瘤进展时间为3个月,疾病控制率为64%。两组临床效果差异无统计学意义(P=0.2415,0.5706,0.7132)。索拉非尼组患者手足皮肤反应、中性粒细胞减少及肝损伤等主要毒副反应的发生率均低于舒尼替尼组,差异具有统计学意义(P0.05)。结论:索拉非尼治疗晚期肝细胞肝癌的临床效果与舒尼替尼具有很好的一致性,药物不良反应相对较轻,患者依从性较好,值得临床推广应用。     

In silico elucidation of the molecular mechanism defining the adverse effect of selective estrogen receptor modulators

Early identification of adverse effect of preclinical and commercial drugs is crucial in developing highly efficient therapeutics, since unexpected adverse drug effects account for one-third of all drug failures in drug development. To correlate protein–drug interactions at the molecule level with their clinical outcomes at the organism level, we have developed an integrated approach to studying protein–ligand interactions on a structural proteome-wide scale by combining protein functional site similarity search, small molecule screening, and protein–ligand binding affinity profile analysis. By applying this methodology, we have elucidated a possible molecular mechanism for the previously observed, but molecularly uncharacterized, side effect of selective estrogen receptor modulators (SERMs). The side effect involves the inhibition of the Sacroplasmic Reticulum Ca2+ ion channel ATPase protein (SERCA) transmembrane domain. The prediction provides molecular insight into reducing the adverse effect of SERMs and is supported by clinical and in vitro observations. The strategy used in this case study is being applied to discover off-targets for other commercially available pharmaceuticals. The process can be included in a drug discovery pipeline in an effort to optimize drug leads and reduce unwanted side effects.     

New targeted therapies for thyroid cancer

The increasing incidence of thyroid cancer is associated with a higher number of advanced disease characterized by the loss of cancer differentiation and metastatic spread. The knowledge of the molecular pathways involved in the pathogenesis of thyroid cancer has made possible the development of new therapeutic drugs able to blockade the oncogenic kinases (BRAF V600E, RET/PTC) or signaling kinases [vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptors (PDGFR)] involved in cellular growth and proliferation. Some clinical trials have been conducted showing the ability of targeted therapies (sorafenib, sunitinib, axitinib, imanitib, vandetanib, pazopanib, gefitinib) in stabilizing the course of the disease. Until now, however, no consensus guidelines have been established for patient selection and more data on toxicities and side effects are needed to be collected.     

Cardiovascular Toxicity of Multi-Tyrosine Kinase Inhibitors in Advanced Solid Tumors: A Population-Based Observational Study

Background

Treatment with small molecule tyrosine kinase inhibitors (TKIs) has improved survival in many cancers, yet has been associated with an increased risk of adverse events. Warnings of cardiovascular events are common in drug labels of many TKIs. Despite these warnings, cardiovascular toxicity of patients treated with TKIs remains unclear. Here, we evaluate the cardiovascular outcomes of advanced cancer patients treated with small molecule tyrosine kinase inhibitors.

Methods

A population based cohort study was undertaken involving adults aged >18 years in Ontario, Canada, diagnosed with any advanced malignancy between 2006 and 2012. Data were extracted from linked administrative governmental databases. Adults with advanced cancer receiving TKIs were identified and followed throughout the time period. The main outcomes of interest were rates of hospitalization for ischemic heart disease (acute myocardial infarction and angina) or cerebrovascular accidents and death.

Results

1642 patients with a mean age of 62.5 years were studied; 1046 were treated with erlotinib, 166 with sorafenib and 430 with sunitinib. Over the 380 day median follow-up period (range 6-1970 days), 1.1% of all patients had ischemic heart events, 0.7% had cerebrovascular accidents and 72.1% died. Rates of cardiovascular events were similar to age and gender-matched individuals without cancer. In a subgroup analysis of treatment patients with a prior history of ischemic heart disease, 3.3% had ischemic heart events while 1.2% had cerebrovascular accidents.

Conclusions

TKIs do not appear to increase the cause-specific hazard of ischemic heart disease and cerebrovascular accidents compared to age and gender-matched individuals without advanced cancer.     

Evolution of an adenocarcinoma in response to selection by targeted kinase inhibitors

Background

Adenocarcinomas of the tongue are rare and represent the minority (20 to 25%) of salivary gland tumors affecting the tongue. We investigated the utility of massively parallel sequencing to characterize an adenocarcinoma of the tongue, before and after treatment.

Results

In the pre-treatment tumor we identified 7,629 genes within regions of copy number gain. There were 1,078 genes that exhibited increased expression relative to the blood and unrelated tumors and four genes contained somatic protein-coding mutations. Our analysis suggested the tumor cells were driven by the RET oncogene. Genes whose protein products are targeted by the RET inhibitors sunitinib and sorafenib correlated with being amplified and or highly expressed. Consistent with our observations, administration of sunitinib was associated with stable disease lasting 4 months, after which the lung lesions began to grow. Administration of sorafenib and sulindac provided disease stabilization for an additional 3 months after which the cancer progressed and new lesions appeared. A recurring metastasis possessed 7,288 genes within copy number amplicons, 385 genes exhibiting increased expression relative to other tumors and 9 new somatic protein coding mutations. The observed mutations and amplifications were consistent with therapeutic resistance arising through activation of the MAPK and AKT pathways.

Conclusions

We conclude that complete genomic characterization of a rare tumor has the potential to aid in clinical decision making and identifying therapeutic approaches where no established treatment protocols exist. These results also provide direct in vivo genomic evidence for mutational evolution within a tumor under drug selection and potential mechanisms of drug resistance accrual.     

Evaluation of Tyrosine Kinase Inhibitor Combinations for Glioblastoma Therapy

Glioblastoma multiforme (GBM) is the most common intracranial cancer but despite recent advances in therapy the overall survival remains about 20 months. Whole genome exon sequencing studies implicate mutations in the receptor tyrosine kinase pathways (RTK) for driving tumor growth in over 80% of GBMs. In spite of various RTKs being mutated or altered in the majority of GBMs, clinical studies have not been able to demonstrate efficacy of molecular targeted therapies using tyrosine kinase inhibitors in GBMs. Activation of multiple downstream signaling pathways has been implicated as a possible means by which inhibition of a single RTK has been ineffective in GBM. In this study, we sought a combination of approved drugs that would inhibit in vitro and in vivo growth of GBM oncospheres. A combination consisting of gefitinib and sunitinib acted synergistically in inhibiting growth of GBM oncospheres in vitro. Sunitinib was the only RTK inhibitor that could induce apoptosis in GBM cells. However, the in vivo efficacy testing of the gefitinib and sunitinib combination in an EGFR amplified/ PTEN wild type GBM xenograft model revealed that gefitinib alone could significantly improve survival in animals whereas sunitinib did not show any survival benefit. Subsequent testing of the same drug combination in a different syngeneic glioma model that lacked EGFR amplification but was more susceptible to sunitinib in vitro demonstrated no survival benefit when treated with gefitinib or sunitinib or the gefitinib and sunitinib combination. Although a modest survival benefit was obtained in one of two animal models with EGFR amplification due to gefitinib alone, the addition of sunitinib, to test our best in vitro combination therapy, did not translate to any additional in vivo benefit. Improved targeted therapies, with drug properties favorable to intracranial tumors, are likely required to form effective drug combinations for GBM.     

Sunitinib treatment promotes metastasis of drug-resistant renal cell carcinoma via TFE3 signaling pathway

Receptor tyrosine kinase (RTK) inhibitors, such as sunitinib and sorafenib, remain the first-line drugs for the treatment of mRCC. Acquired drug resistance and metastasis are the main causes of treatment failure. However, in the case of metastasis Renal Cell Cancer (mRCC), which showed a good response to sunitinib, we found that long-term treatment with sunitinib could promote lysosome biosynthesis and exocytosis, thereby triggering the metastasis of RCC. By constructing sunitinib-resistant cell lines in vivo, we confirmed that TFE3 plays a key role in the acquired resistance to sunitinib in RCC. Under the stimulation of sunitinib, TFE3 continued to enter the nucleus, promoting the expression of endoplasmic reticulum (ER) protein E-Syt1. E-Syt1 and the lysosomal membrane protein Syt7 form a heterodimer, which induces ER fragmentation, Ca2⁺ release, and lysosomal exocytosis. Lysosomal exocytosis has two functions: pumping sunitinib out from the cytoplasm, which promotes resistance to sunitinib in RCC, releasing cathepsin B (CTSB) into the extracellular matrix (ECM), which can degrade the ECM to enhance the invasion and metastasis ability of RCC. Our study found that although sunitinib is an effective drug for the treatment of mRCC, once RCC has acquired resistance to sunitinib, sunitinib treatment will promote metastasis.Subject terms: Renal cell carcinoma, Cell invasion, Prognostic markers     

Selecting Tyrosine Kinase Inhibitors for Gastrointestinal Stromal Tumor with Secondary KIT Activation-Loop Domain Mutations

Advanced gastrointestinal stromal tumors (GIST), a KIT oncogene-driven tumor, on imatinib mesylate (IM) treatment may develop secondary KIT mutations to confer IM-resistant phenotype. Second-line sunitinib malate (SU) therapy is largely ineffective for IM-resistant GISTs with secondary exon 17 (activation-loop domain) mutations. We established an in vitro cell-based platform consisting of a series of COS-1 cells expressing KIT cDNA constructs encoding common primary±secondary mutations observed in GISTs, to compare the activity of several commercially available tyrosine kinase inhibitors on inhibiting the phosphorylation of mutant KIT proteins at their clinically achievable plasma steady-state concentration (Css). The inhibitory efficacies on KIT exon 11/17 mutants were further validated by growth inhibition assay on GIST48 cells, and underlying molecular-structure mechanisms were investigated by molecular modeling. Our results showed that SU more effectively inhibited mutant KIT with secondary exon 13 or 14 mutations than those with secondary exon 17 mutations, as clinically indicated. On contrary, at individual Css, nilotinib and sorafenib more profoundly inhibited the phosphorylation of KIT with secondary exon 17 mutations and the growth of GIST48 cells than IM, SU, and dasatinib. Molecular modeling analysis showed fragment deletion of exon 11 and point mutation on exon 17 would lead to a shift of KIT conformational equilibrium toward active form, for which nilotinib and sorafenib bound more stably than IM and SU. In current preclinical study, nilotinib and sorafenib are more active in IM-resistant GISTs with secondary exon 17 mutation than SU that deserve further clinical investigation.     

妊娠期甲状腺疾病诊断及治疗的研究进展

妊娠期妇女体内激素水平会发生变化,使妊娠妇女甲状腺激素水平的测定和判断存在一定的困难,应选择适用于妊娠妇女的甲状腺激素水平特异值,进而正确评估甲状腺功能状态及对母体和胎儿的影响。孕前及妊娠期测定促甲状腺素和游离甲状腺激素有很大的必要性,因为甲状腺疾病以及单纯性甲状腺抗体阳性会导致多种妊娠不良结局,尤其是甲状腺功能减退对胎儿智力发育和认知功能具有非常大的影响。孕期甲状腺激素的监测对评估甲状腺功能状态及疾病预后具有非常大的作用,可以提示临床医师是否给予药物干预及如何调整药量。对于孕期甲状腺激素补充治疗后应达到的目标值以及甲状腺抗体阴性的亚临床甲状腺功能降低的妊娠患者是否给予干预,目前仍有异议。     

Interaction of tyrosine kinase inhibitors with the human multidrug transporter proteins,MDR1 and MRP1

Specific tyrosine kinase inhibitors (TKIs) are rapidly developing clinical tools applied for the inhibition of malignant cell growth and metastasis formation. Most of these newly developed TKI molecules are hydrophobic, thus rapidly penetrate the cell membranes to reach intracellular targets. However, a large number of tumor cells overexpress multidrug transporter membrane proteins, which efficiently extrude hydrophobic drugs and thus may prevent the therapeutic action of TKIs. In the present work, we demonstrate that the most abundant and effective cancer multidrug transporters, MDR1 and MRP1, directly interact with several TKIs under drug development or already in clinical trials. This interaction with the transporters does not directly correlate with the hydrophobicity or molecular structure of TKIs, and shows a large variability in transporter selectivity and affinity. We suggest that performing enzyme- and cell-based multidrug transporter interaction tests for TKIs may greatly facilitate drug development, and allow the prediction of clinical TKI resistance based on this mechanism. Moreover, diagnostics for the expression of specific multidrug transporters in the malignant cells, combined with information on the interactions of the drug transporter proteins with TKIs, should allow a highly effective, individualized clinical treatment for cancer patients.     

Molecular analysis of sunitinib resistant renal cell carcinoma cells after sequential treatment with RAD001 (everolimus) or sorafenib

Sequential application of target drugs is standard procedure after renal cell carcinoma (RCC) patients develop resistance. To optimize the sequence, antitumour effects of the mTOR inhibitor RAD001 or the tyrosine kinase inhibitor (TKI) sorafenib on RCC cells with acquired resistance to the TKI sunitinib was evaluated. RCC cells were exposed to 1 μM sunitinib for 24 hrs (as control) and for 8 weeks (to induce resistance) and then switched to RAD001 (5 nM) or sorafenib (5 μM) for a further 8 weeks. Tumour cell growth, cell cycle progression, cell cycle regulating proteins and intracellular signalling were then investigated. Short‐term application of sunitinib (24 hrs) induced cell growth blockade with accumulation in the G2/M phase. RCC cells became resistant to sunitinib after 8 weeks, demonstrated by accelerated cell growth along with enhanced cdk1, cdk2, loss of p27, activation of Akt, Rictor and Raptor. Switching to sorafenib only slightly reduced growth of the sunitinib resistant RCC cells and molecular analysis indicated distinct cross‐resistance. In contrast, full response was achieved when the cancer cells were treated with RAD001. p19 and p27 strongly increased, phosphorylated Akt, Rictor and Raptor decreased and the tumour cells accumulated in G0/G1. It is concluded that an mTOR‐inhibitor for second‐line therapy could be the strategy of choice after first‐line sunitinib failure.     

Potentiating the Efficacy of Molecular Targeted Therapy for Hepatocellular Carcinoma by Inhibiting the Insulin-Like Growth Factor Pathway

Insulin-like growth factor (IGF) signaling pathway is an important regulatory mechanism of tumorigenesis and drug resistance in many cancers. The present study explored the potential synergistic effects between IGF receptor (IGFR) inhibition and other molecular targeted agents (MTA) in HCC cells. HCC cell lines (Hep3B, PLC5, and SK-Hep1) and HUVECs were tested. The MTA tested included sorafenib, sunitinib, and the IGFR kinase inhibitor NVP-AEW541. The potential synergistic antitumor effects were tested by median dose effect analysis and apoptosis assay in vitro and by xenograft models in vivo. The activity and functional significance of pertinent signaling pathways and expression of apoptosis-related proteins were measured by RNA interference and Western blotting. We found that IGF can activate IGFR and downstream AKT signaling activities in all the HCC cells tested, but the growth-stimulating effect of IGF was most prominent in Hep3B cells. NVP-AEW541 can abrogate IGF-induced activation of IGFR and AKT signaling in HCC cells. IGF can increase the resistance of HCC cells to sunitinib. The apoptosis-inducing effects of sunitinib, but not sorafenib, were enhanced when IGFR signaling activity was inhibited by NVP-AEW541 or IGFR knockdown. Chk2 kinase activation was found contributory to the synergistic anti-tumor effects between sunitinib and IGFR inhibition. Our data indicate that the apoptosis-potentiating effects of IGFR inhibition for HCC may be drug-specific. Combination therapy of IGFR inhibitors with other MTA may improve the therapeutic efficacy in HCC.     

Identifying Resistance Mechanisms against Five Tyrosine Kinase Inhibitors Targeting the ERBB/RAS Pathway in 45 Cancer Cell Lines

Because of the low overall response rates of 10–47% to targeted cancer therapeutics, there is an increasing need for predictive biomarkers. We aimed to identify genes predicting response to five already approved tyrosine kinase inhibitors. We tested 45 cancer cell lines for sensitivity to sunitinib, erlotinib, lapatinib, sorafenib and gefitinib at the clinically administered doses. A resistance matrix was determined, and gene expression profiles of the subsets of resistant vs. sensitive cell lines were compared. Triplicate gene expression signatures were obtained from the caArray project. Significance analysis of microarrays and rank products were applied for feature selection. Ninety-five genes were also measured by RT-PCR. In case of four sunitinib resistance associated genes, the results were validated in clinical samples by immunohistochemistry. A list of 63 top genes associated with resistance against the five tyrosine kinase inhibitors was identified. Quantitative RT-PCR analysis confirmed 45 of 63 genes identified by microarray analysis. Only two genes (ANXA3 and RAB25) were related to sensitivity against more than three inhibitors. The immunohistochemical analysis of sunitinib-treated metastatic renal cell carcinomas confirmed the correlation between RAB17, LGALS8, and EPCAM and overall survival. In summary, we determined predictive biomarkers for five tyrosine kinase inhibitors, and validated sunitinib resistance biomarkers by immunohistochemistry in an independent patient cohort.