NK92 cells and peripheral blood NK cells respond oppositely upon dasatinib treatment.

Natural killer (NK) cell is a valuable tool for immunotherapy in cancer treatment, both the cultured cell line NK92 and primary NK cells are widely studied and used in research and clinical trials. Clinical observations witnessed the improvement of patients' NK cells in terms of cell counts and cytotoxic activity upon dasatinib treatment, an approved drug for chronic myeloid leukaemia and Ph+ acute lymphocytic leukaemia. Several studies supported the clinical observations, yet others argued a detrimental effect of dasatinib on NK cells. Due to the complex conditions in different studies, the definite influence of dasatinib on NK92 and primary NK cells remains to be settled. Here, we used a well-defined in vitro system to evaluate the effects of dasatinib on NK92 cells and peripheral blood (PB)-NK cells. By co-culturing NK cells with dasatinib to test the cell counts and target cell-killing activities, we surprisingly found that the chemical influenced oppositely on these two types of NK cells. While dasatinib suppressed NK92 cell proliferation and cytotoxic activity, it improved PB-NK-killing tumour cells. RNA sequencing analysis further supported this finding, uncovering several proliferating and cytotoxic pathways responding invertedly between them. Our results highlighted an intrinsic difference between NK92 and PB-NK cells and may build clues to understand how dasatinib interacts with NK cells in vivo.

Senolytic combination of dasatinib and quercetin protects against diabetic kidney disease by activating autophagy to alleviate podocyte dedifferentiation via the Notch pathway.

The senolytics dasatinib and quercetin (DQ) alleviate age­related disorders. However, limited information is available regarding the effects of DQ on diabetic kidney disease (DKD). The present study aimed to explore the effects of DQ on DKD and its potential molecular mechanism(s). Dasatinib (5 mg/kg) and quercetin (50 mg/kg) were administered to diabetic db/db mice by gavage for 20 weeks. Body weight, urine albumin­creatinine ratio (ACR), serum creatinine (Scr), and blood urea nitrogen (BUN) were recorded at the indicated time periods. Periodic acid­Schiff and Masson's staining were performed to assess the histopathological changes of kidney tissues. Immunohistochemical analysis, immunofluorescence and western blotting were performed to evaluate the expression levels of extracellular matrix (ECM) proteins, autophagic and podocyte differentiation­related proteins. In addition, mouse podocytes were administered with high­glucose, DQ and 3­methyladenine (3­MA), and the expression levels of autophagic and podocyte differentiation­related proteins were measured. Moreover, following overexpression of the Notch intracellular domain (NICD), the expression levels of NICD, autophagic and podocyte differentiation­related proteins were further assessed. DQ significantly reduced the body weight, blood glucose, ACR, Scr and BUN levels and improved the histopathological changes induced in diabetic db/db mice. In addition, DQ caused a significant downregulation of the expression levels of the ECM proteins, improved autophagy and induced an upregulation of the expression levels of podocyte differentiation­related proteins. Administration of 3­MA to mice significantly reduced podocyte differentiation, and overexpression of NICD could reverse the effects of DQ on autophagy and podocyte differentiation in vitro. The present study suggests that DQ protects against DKD by activation of autophagy to alleviate podocyte dedifferentiation via the Notch pathway.

Study on tumour cell-derived hybrid exosomes as dasatinib nanocarriers for pancreatic cancer therapy.

Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer-related death. Therefore, we intend to explore novel strategies against PDAC. The exosomes-based biomimetic nanoparticle is an appealing candidate served as a drug carrier in cancer treatment, due to its inherit abilities. In the present study, we designed dasatinib-loaded hybrid exosomes by fusing human pancreatic cancer cells derived exosomes with dasatinib-loaded liposomes, followed by characterization for particle size (119.9 ± 6.10 nm) and zeta potential (-11.45 ± 2.24 mV). Major protein analysis from western blot techniques reveal the presence of exosome marker proteins CD9 and CD81. PEGylated hybrid exosomes showed pH-sensitive drug release in acidic condition, benefiting drug delivery to acidic cancer environment. Dasatinib-loaded hybrid exosomes exhibited significantly higher uptake rates and cytotoxicity to parent PDAC cells by two-sample t-test or by one-way ANOVA analysis of variance, as compared to free drug or liposomal formulations. The results from our computational analysis demonstrated that the drug-likeness, ADMET, and protein-ligand binding affinity of dasatinib are verified successfully. Cancer derived hybrid exosomes may serve as a potential therapeutic candidate for pancreatic cancer treatment.

Astrocyte senescence-like response related to peripheral nerve injury-induced neuropathic pain.

BACKGROUND: Peripheral nerve damage causes neuroinflammation, which plays a critical role in establishing and maintaining neuropathic pain (NeP). The mechanisms contributing to neuroinflammation remain poorly elucidated, and pharmacological strategies for NeP are limited. Thus, in this study, we planned to explore the possible link between astrocyte senescence and NeP disorders following chronic sciatic nerve injury. METHODS: An NeP animal model was established by inducing chronic constrictive injury (CCI) to the sciatic nerve in adult rats. A senolytic drug combination of dasatinib and quercetin was gavaged daily from the first postoperative day until the end of the study. Paw mechanical withdrawal threshold (PMWT) and paw thermal withdrawal latency (PTWL) were evaluated to assess behaviors in response to pain in the experimental rats. Senescence-associated ß-galactosidase staining, western blot analysis, and immunofluorescence were applied to examine the levels of proinflammatory factors and severity of the senescence-like response in the spinal cord. Lipopolysaccharide (LPS) was administered to induce senescence of spinal astrocytes in primary cultures in vitro, to explore the potential impacts of senescence on the secretion of proinflammatory factors. Furthermore, single-cell RNA sequencing (scRNA-seq) was conducted to identify senescence-related molecular responses in spinal astrocytes under neuropathic pain. RESULTS: Following sciatic nerve CCI, rats exhibited reduced PMWT and PTWL, increased levels of spinal proinflammatory factors, and an enhanced degree of senescence in spinal astrocytes. Treatment with dasatinib and quercetin effectively attenuated spinal neuroinflammation and mitigated the hypersensitivities of the rats subjected to sciatic nerve CCI. Mechanistically, the dasatinib-quercetin combination reversed senescence in LPS-stimulated primary cultured astrocytes and decreased the levels of proinflammatory factors. The scRNA-seq data revealed four potential senescence-related genes in the spinal astrocyte population, and the expression of clusterin (CLU) protein was validated via in vitro experiments. CONCLUSION: The findings indicate the potential role of astrocyte senescence in neuroinflammation following peripheral nerve injury, and suggest that targeting CLU activation in astrocytes might provide a novel therapeutic strategy to treat NeP.

Dasatinib Attenuates Fibrosis in Keloids by Decreasing Senescent Cell Burden.

Keloids are skin tumours caused by aberrant growth of dermal fibroblasts. Cellular senescence contributes to aging and various pathological conditions, including cancer, atherosclerosis, and fibrotic diseases. However, the effects of cellular senescence and senolytic drugs on keloids remain largely unknown. This study investigated senescent fibroblasts in keloids and assessed the effects of dasatinib on these cells. Tissues acquired from keloid removal surgery were analysed for senescence-associated ß-galactosidase-positive cells, p16 expression, and the effects of dasatinib treatment on keloids. Keloid tissue was xenotransplanted into mice, and the effect of intralesional dasatinib injection on keloid growth was observed. The results showed that the numbers of ß-galactosidase-positive and p16-expressing cells were higher in the keloids compared with in the controls. Dasatinib induced selective clearance of senescent cells and decreased procollagen expression in cultured keloid fibroblasts. In this xenotransplant keloid mouse model, intralesional injection of dasatinib reduced gross keloid tissue weight and the expression of both procollagen and p16. In addition, dasatinib-treated keloid fibroblasts conditioned medium reduced procollagen and p16 expression in cultured keloid fibroblasts. In conclusion, these results suggest that an increased number of senescent fibroblasts may play an important role in the pathogenesis of keloids. Therefore, dasatinib could be an alternative treatment for patients with keloids.

Combined use of dasatinib and quercetin alleviates overtraining-induced deficits in learning and memory through eliminating senescent cells and reducing apoptotic cells in rat hippocampus.

Excessive physical exercise (overtraining, OT) charactered by long-term and excessive training results in the damage of multiple vital tissues including hippocampus which plays a critical role in learning and memory. A combination of dasatinib (D) plus quercetin (Q) (D+Q) belongs to senolytic drugs which selectively kill senescent cells in vitro and vivo. In this study, the rats that suffered a five-week excessive swimming training were subjected to the oral administration of D+Q. D+Q alleviated the decline in exercise performance of OT rats during the swimming training, and prevented learning and memory deficits in Morris water maze, Y-maze and novel object recognition tests after excessive swimming training. Analytical results by SA-ß-gal staining and western blotting showed that D+Q significantly reduced senescent cells with repressed expression of senescence-related proteins, p53 and p21, in hippocampus. Nissl and immunohistochemical staining showed that D+Q significantly attenuated neuronal loss caused by apoptosis. Interestingly, we observed elevated level of cleaved caspase 3, an apoptosis executor protein, in p21 positive hippocampus cells by D+Q treatment in immunofluorescent staining, suggesting that senescent cells were induced to apoptosis in D+Q-treated rats. The positive control drug, silibinin, showed similar protective effect against OT, but did not induce the apoptosis of senescent cells, suggesting a difference in the protective mechanisms. These results indicated that D+Q alleviates overtraining-induced deficits in learning and memory through elimination of senescent cells and reduction of apoptotic cell number.

Feasibility and preclinical efficacy of CD7-unedited CD7 CAR T cells for T cell malignancies.

Chimeric antigen receptor (CAR)-mediated targeting of T lineage antigens for the therapy of blood malignancies is frequently complicated by self-targeting of CAR T cells or their excessive differentiation driven by constant CAR signaling. Expression of CARs targeting CD7, a pan-T cell antigen highly expressed in T cell malignancies and some myeloid leukemias, produces robust fratricide and often requires additional mitigation strategies, such as CD7 gene editing. In this study, we show fratricide of CD7 CAR T cells can be fully prevented using ibrutinib and dasatinib, the pharmacologic inhibitors of key CAR/CD3ζ signaling kinases. Supplementation with ibrutinib and dasatinib rescued the ex vivo expansion of unedited CD7 CAR T cells and allowed regaining full CAR-mediated cytotoxicity in vitro and in vivo on withdrawal of the inhibitors. The unedited CD7 CAR T cells persisted long term and mediated sustained anti-leukemic activity in two mouse xenograft models of human T cell acute lymphoblastic leukemia (T-ALL) by self-selecting for CD7-, fratricide-resistant CD7 CAR T cells that were transcriptionally similar to control CD7-edited CD7 CAR T cells. Finally, we showed feasibility of cGMP manufacturing of unedited autologous CD7 CAR T cells for patients with CD7+ malignancies and initiated a phase I clinical trial (ClinicalTrials.gov: NCT03690011) using this approach. These results indicate pharmacologic inhibition of CAR signaling enables generating functional CD7 CAR T cells without additional engineering.

Polymorphonuclear neutrophil activation by Src phosphorylation contributes to HLA-A2 antibody-induced transfusion-related acute lung injury.

Human leukocyte antigen (HLA)-A2 antibody contributes to the pathogenesis of transfusion-related acute lung injury (TRALI) via polymorphonuclear neutrophil (PMN) activation, but the signaling pathways involved this process remain largely undefined. In this study, we sought to study the signaling pathways involved in the pathogenesis of HLA-A2-induced TRALI. Lipopolysaccharide (LPS), and the plasma from the HLA-A2 antibody-positive donors were utilized to establish a rat model of TRALI. Human pulmonary endothelial cells (HPMECs) were in vitro co-cultured with HLA-A2 antibody-treated PMNs and then treated with LPS to induce a cytotoxicity model. The effects of HLA-A2 antibody on HPMEC injury were evaluated in this model. Besides, dasatinib was used to block the Src phosphorylation to explore whether Src involved in the TRALI or HPMEC injury induced by HLA-A2 antibody. The HLA-A2 antibody plus LPS induced TRALI and stimulated PMN activation in rats. HLA-A2 antibody-induced TRALI could be attenuated via depletion of PMN. HLA-A2 antibody activated NF-κB and NLRP3 inflammasome. In addition, HLA-A2 antibody aggravated the HPMEC injuries and the release of PMN surfaces makers, but dasatinib treatment reversed this effect, indicating that HLA-A2 antibody activated PMNs and exacerbated TRALI by stimulating phosphorylation of Src followed by activation of NF-κB and NLRP3 inflammasome, which was validated in vivo. In summary, HLA-A2 induced PMNs by activating NF-κB/NLRP3 inflammasome via phosphorylated-Src elevation, thereby exacerbating TRALI. This study highlights promising target for the treatment of antibody-mediated TRALI.

Chronic hyperinsulinemia promotes human hepatocyte senescence.

OBJECTIVE: Cellular senescence, an irreversible proliferative cell arrest, is caused by excessive intracellular or extracellular stress/damage. Increased senescent cells have been identified in multiple tissues in different metabolic and other aging-related diseases. Recently, several human and mouse studies emphasized the involvement of senescence in development and progression of NAFLD. Hyperinsulinemia, seen in obesity, metabolic syndrome, and other conditions of insulin resistance, has been linked to senescence in adipocytes and neurons. Here, we investigate the possible direct role of chronic hyperinsulinemia in the development of senescence in human hepatocytes. METHODS: Using fluorescence microscopy, immunoblotting, and gene expression, we tested senescence markers in human hepatocytes subjected to chronic hyperinsulinemia in vitro and validated the data in vivo by using liver-specific insulin receptor knockout (LIRKO) mice. The consequences of hyperinsulinemia were also studied in senescent hepatocytes following doxorubicin as a model of stress-induced senescence. Furthermore, the effects of senolytic agents in insulin- and doxorubicin-treated cells were analyzed. RESULTS: Results showed that exposing the hepatocytes to prolonged hyperinsulinemia promotes the onset of senescence by increasing the expression of p53 and p21. It also further enhanced the senescent phenotype in already senescent hepatocytes. Addition of insulin signaling pathway inhibitors prevented the increase in cell senescence, supporting the direct contribution of insulin. Furthermore, LIRKO mice, in which insulin signaling in the liver is abolished due to deletion of the insulin receptor gene, showed no differences in senescence compared to their wild-type counterparts despite having marked hyperinsulinemia indicating these are receptor-mediated effects. In contrast, the persistent hyperinsulinemia in LIRKO mice enhanced senescence in white adipose tissue. In vitro, senolytic agents dasatinib and quercetin reduced the prosenescent effects of hyperinsulinemia in hepatocytes. CONCLUSION: Our findings demonstrate a direct link between chronic hyperinsulinemia and hepatocyte senescence. This effect can be blocked by reducing the levels of insulin receptors or administration of senolytic drugs, such as dasatinib and quercetin.

Induction of Cellular Senescence in Rat Vaginal Fibroblasts and Treatment With Senolytics: An in Vitro Model for the Study of Pelvic Organ Prolapse.

OBJECTIVE: The objective of this study was to develop an in vitro model of cellular senescence using rat vaginal fibroblasts and determine the effects of treatment with senolytics. METHODS: Rat vaginal tissue biopsies were collected. Primary vaginal fibroblasts were isolated and characterized by immunofluorescence. To induce cellular senescence, fibroblasts were treated with etoposide at 3, 10, and 20 mM for 24 hours, followed by treatment with the senolytics dasatinib (1 mM) and/or quercetin (20 mM). After treatment, RNA was extracted and the expression of selected genes was quantified. Immunostaining of senescence markers was also performed. RESULTS: Fibroblasts were confirmed by positive immunostaining for α-smooth muscle actin and vimentin, and negative immunostaining for pan-cytokeratin. Treatment with etoposide resulted in a dose-dependent increase in expression of the senescence-associated secretory phenotype markers MMP-7, MMP-9, and IL-b1 (P < 0.05) compared with controls. Immunostaining showed increased expression of γ-H2A and p21 after treatment with etoposide. Cells treated with dasatinib and quercetin after etoposide treatment had decreased expression of p21, MMP-7, MMP-9, and IL-1b compared with cells treated only with etoposide (P < 0.05). CONCLUSIONS: Upregulation of senescence-associated factors provided evidence that senescence can be induced in vaginal fibroblasts in vitro. Furthermore, treatment with the senolytics dasatinib and quercetin abrogated the senescence phenotype induced by etoposide in rat vaginal fibroblasts. Our findings provide a novel model for the study and development of new therapies targeting the disordered extracellular matrix associated with pelvic organ prolapse.

Investigation on the interaction behavior of afatinib, dasatinib, and imatinib docked to the BCR-ABL protein.

Chronic myeloid leukemia (CML) is a pathological condition associated with the uncontrolled proliferation of white blood cells and respective loss of function. Imatinib was the first drug that could effectively treat this condition, but its use is hindered by the development of mutations of the BCR-ABL protein, which are the cause of resistance. Therefore, dasatinib and afatinib present similarities that can be explored to discover new molecules capable of overcoming the effects of imatinib. Afatinib exhibited electronic and docking behavior, indicating that a replacement with some minor modifications could design a new potential inhibitor. The amide group in each candidate is clearly of pharmacophoric importance, and it needs to concentrate a negative region. Sulfur group presents a good pharmacophoric profile, which was shown by dasatinib results, adding to the influence of the Met318 residue in the target protein active site configuration. This behavior suggests that the sulfur atom and other fragments that have an affinity for the methionine sidechain may provide a significant positive effect when present in TKI molecules such as afatinib or dasatinib.

Review on in vivo profiling of drug metabolites with LC-MS/MS in the past decade.

Metabolite profiling is an indispensable part of drug discovery and development, enabling a comprehensive understanding of the drug's metabolic behavior. Liquid chromatography-mass spectrometry facilitates metabolite profiling by reducing sample complexity and providing high sensitivity. This review discusses the in vivo metabolite profiling involving LC-MS/MS and the utilization of QTOF, QQQ mass analyzers with a particular emphasis on a mass filter. Further, a summary of sample extraction procedures in biological matrices such as plasma, urine, feces, serum and hair as in vivo samples are outlined. toward the end, we present 15 case studies in biological matrices and their LC-MS/MS conditions to understand the metabolic disposition.

Differential impact of BTK active site inhibitors on the conformational state of full-length BTK.

Bruton's tyrosine kinase (BTK) is targeted in the treatment of B-cell disorders including leukemias and lymphomas. Currently approved BTK inhibitors, including Ibrutinib, a first-in-class covalent inhibitor of BTK, bind directly to the kinase active site. While effective at blocking the catalytic activity of BTK, consequences of drug binding on the global conformation of full-length BTK are unknown. Here, we uncover a range of conformational effects in full-length BTK induced by a panel of active site inhibitors, including large-scale shifts in the conformational equilibria of the regulatory domains. Additionally, we find that a remote Ibrutinib resistance mutation, T316A in the BTK SH2 domain, drives spurious BTK activity by destabilizing the compact autoinhibitory conformation of full-length BTK, shifting the conformational ensemble away from the autoinhibited form. Future development of BTK inhibitors will need to consider long-range allosteric consequences of inhibitor binding, including the emerging application of these BTK inhibitors in treating COVID-19.

Treatments for blood cancers, such as leukemia and lymphoma, rely heavily on chemotherapy, using drugs that target a vulnerable aspect of the cancer cells. B-cells, a type of white blood cell that produces antibodies, require a protein called Bruton's tyrosine kinase, or BTK for short, to survive. The drug ibrutinib (Imbruvica) is used to treat B-cell cancers by blocking BTK. The BTK protein consists of several regions. One of them, known as the kinase domain, is responsible for its activity as an enzyme (which allows it to modify other proteins by adding a 'tag' known as a phosphate group). The other regions of BTK, known as regulatory modules, control this activity. In BTK's inactive form, the regulatory modules attach to the kinase domain, blocking the regulatory modules from interacting with other proteins. When BTK is activated, it changes its conformation so the regulatory regions detach and become available for interactions with other proteins, at the same time exposing the active kinase domain. Ibrutinib and other BTK drugs in development bind to the kinase domain to block its activity. However, it is not known how this binding affects the regulatory modules. Previous efforts to study how drugs bind to BTK have used a version of the protein that only had the kinase domain, instead of the full-length protein. Now, Joseph et al. have studied full-length BTK and how it binds to five different drugs. The results reveal that ibrutinib and another drug called dasatinib both indirectly disrupt the normal position of the regulatory domains pushing BTK toward a conformation that resembles the activated state. By contrast, the three other compounds studied do not affect the inactive structure. Joseph et al. also examined a mutation in BTK that confers resistance against ibrutinib. This mutation increases the activity of BTK by disrupting the inactive structure, leading to B cells surviving better. Understanding how drug resistance mechanisms can work will lead to better drug treatment strategies for cancer. BTK is also a target in other diseases such as allergies or asthma and even COVID-19. If interactions between partner proteins and the regulatory domain are important in these diseases, then they may be better treated with drugs that maintain the regulatory modules in their inactive state. This research will help to design drugs that are better able to control BTK activity.

Production of drug metabolites by human FMO3 in Escherichia coli.

BACKGROUND: In the course of drug discovery and development process, sufficient reference standards of drug metabolites are required, especially for preclinical/clinical or new therapeutic drugs. Whole-cell synthesis of drug metabolites is of great interest due to its low cost, low environmental impact and specificity of the enzymatic reaction compared to chemical synthesis. Here, Escherichia coli (E. coli) JM109 cells over-expressing the recombinant human FMO3 (flavin-containing monooxygenase isoform 3) were used for the conversions of clomiphene, dasatinib, GSK5182 and tozasertib to their corresponding N-oxide metabolites. RESULTS: The effects of NADPH regeneration, organic solvents as well as C-terminal truncations of human FMO3 were investigated. Under the optimized conditions, in excess of 200 mg/L of N-oxide metabolite of each of the four drugs could be produced by whole-cell catalysis within 24 h. Of these, more than 90% yield conversions were obtained for the N-oxidation of clomiphene and dasatinib. In addition, FMO3 shows high regio-selectivity in metabolizing GSK5182 where only the (Z) isomer is monooxygenated. CONCLUSIONS: The study shows the successful use of human FMO3-based whole-cell as a biocatalyst for the efficient synthesis of drug metabolites including regio-selective reactions involving GSK5182, a new candidate against type 2 diabetes mellitus.

Nonparametric Analysis of Thermal Proteome Profiles Reveals Novel Drug-binding Proteins.

Detecting the targets of drugs and other molecules in intact cellular contexts is a major objective in drug discovery and in biology more broadly. Thermal proteome profiling (TPP) pursues this aim at proteome-wide scale by inferring target engagement from its effects on temperature-dependent protein denaturation. However, a key challenge of TPP is the statistical analysis of the measured melting curves with controlled false discovery rates at high proteome coverage and detection power. We present nonparametric analysis of response curves (NPARC), a statistical method for TPP based on functional data analysis and nonlinear regression. We evaluate NPARC on five independent TPP data sets and observe that it is able to detect subtle changes in any region of the melting curves, reliably detects the known targets, and outperforms a melting point-centric, single-parameter fitting approach in terms of specificity and sensitivity. NPARC can be combined with established analysis of variance (ANOVA) statistics and enables flexible, factorial experimental designs and replication levels. An open source software implementation of NPARC is provided.

Dasatinib-induced nephrotic syndrome: a case of phenoconversion.

We present the case of a 33-year-old chronic myeloid leukemia (CML) female patient, in whom the occurrence of nephrotic syndrome, during the treatment with tyrosine kinase activity inhibitors (TKIs), was potentially influenced by transient phenoconversion. Seven years after the CML diagnosis in 2004 and complete response, the patient experienced pain in the mandible and extremities. After this, imatinib was replaced by nilotinib, but generalized maculopapular rash was presented and successfully treated with antihistamines. The therapy was then discontinued due to planned pregnancy, and the patient experienced a relapse of CML with BCR-ABL/ABL1 transcripts of 18.9%. Dasatinib was introduced, and CML was in remission. Two years later, urine protein levels (6.19 g/L) and erythrocyte sedimentation rate were elevated (ESR=90 mm/3.6 ks). The patient was diagnosed with nephrotic syndrome. With dasatinib dose reduction, urine protein level returned to the reference range. In order to determine the best genotype-guided therapy, the patient underwent pharmacogenomic testing, showing a homozygous CYP3A4 genotype *1/*1, associated with extensive metabolizer (EM) enzyme phenotype, typical for normal rates of drug metabolism for TKIs. However, this was inconsistent with nephrotic syndrome occurrence. A possible explanation would be CYP3A4 EM genotype coding a poor metabolizer enzyme phenotype, leading to the drug accumulation in the patient's blood. This transient phenoconversion can be explained by inflammation with elevated ESR during nephrotic syndrome. This case shows that a broader approach that recognizes genetic, clinical, and epigenomic factors is required for a quality decision on the personalized therapy regimen.

Molecular Engineering-Based Aptamer-Drug Conjugates with Accurate Tunability of Drug Ratios for Drug Combination Targeted Cancer Therapy.

Polytherapy (or drug combination cancer therapy (DCCT)), targeting multiple mechanisms associated with tumor proliferation, can efficiently maximize therapeutic efficacy, decrease drug dosage, and reduce drug resistance. However, most DCCT strategies cannot coordinate the specific delivery of a drug combination in an accurately tuned ratio into cancer cells. To address these limitations, the present work reports the engineering of circular bivalent aptamer-drug conjugates (cb-ApDCs). The cb-ApDCs exhibit high stability, specific recognition, excellent cellular uptake, and esterase-triggered release. Furthermore, the drug ratios in cb-ApDCs can be tuned for an enhanced synergistic effect without the need for complex chemistry. Therefore, cb-ApDCs provide a promising platform for the development of DCCT strategies for different drug combinations and ratios.

A Near-IR Fluorescent Dasatinib Derivative That Localizes in Cancer Cells.

Kinase inhibitors (KIs) have had a huge impact on clinical treatment of various cancers, but they are far from perfect medicines. In particular, their efficacies are limited to certain cancer types and, in many cases, provide only temporary remission. This paper explores the possibility of covalently binding a fluorophore for in vivo optical imaging to the KI dasatinib where the particular fluorophore chosen for this study, a heptamethine cyanine (Cy) derivative, tends to accumulate in tumors. Thus, we hypothesized that the dasatinib-fluorophore conjugate might target tumor cells more effectively than the parent KI, give enhanced suppression of viability, and simultaneously serve as a probe for optical imaging. As far as we are aware, the dasatinib conjugate (1) is the first reported to contain this KI and a probe for near-IR imaging, and it is certainly the first conjugate of a tumor-targeting near-IR dye and a KI of any kind. Conjugate 1 suppressed the viability of liver cancer cells (HepG2) more effectively than dasatinib at the same concentration. In scratch assays, 1 prevented regrowth of the tumor cells. Conjugate 1 is cell permeable, and confocal imaging indicates the fluorescence of those cells is concentrated in the mitochondria than lysosomes. In general, this study suggests there is untapped potential for conjugates of KIs with tumor-targeting near-IR dyes in the development of theranostics for optical imaging and treatment of cancer.

What Makes a Kinase Promiscuous for Inhibitors?

ATP-competitive kinase inhibitors often bind several kinases due to the high conservation of the ATP binding pocket. Through clustering analysis of a large kinome profiling dataset, we found a cluster of eight promiscuous kinases that on average bind more than five times more kinase inhibitors than the other 398 kinases in the dataset. To understand the structural basis of promiscuous inhibitor binding, we determined the co-crystal structure of the receptor tyrosine kinase DDR1 with the type I inhibitors dasatinib and VX-680. Surprisingly, we find that DDR1 binds these type I inhibitors in an inactive conformation typically reserved for type II inhibitors. Our computational and biochemical studies show that DDR1 is unusually stable in this inactive conformation, giving a mechanistic explanation for inhibitor promiscuity. This phenotypic clustering analysis provides a strategy to obtain functional insights not available by sequence comparison alone.

Emergence of a Promising Lead Compound in the Treatment of Triple Negative Breast Cancer: An Insight into Conformational Features and Ligand Binding Landscape of c-Src Protein with UM-164.

UM-164, a potent Src/p38 inhibitor, is a promising lead compound for developing the first targeted therapeutic strategy against triple-negative breast cancer (TNBC). However, lack of understanding of conformational features of UM-164 in complex with Src serves a challenge in the rational design of novel Src dual inhibitors. Herein, we provide an in-depth insight into conformational features of Src-UM-164 using different computational approaches. This involved molecular dynamics (MD) simulation, principal component analysis (PCA), thermodynamics calculations, dynamic cross-correlation (DCCM) analysis, and hydrogen bond formation. Findings from this study revealed that (1) the binding of UM-164 to Src induces a more stable and compact conformation; (2) the binding of UM-164 results in increased correlation among the active site residue; (3) the presence of multiple phenyl rings and fluorinated phenyl group in UM-164 contributes to the steric effect; (4) a relatively high-binding free energy estimated for the Src-UM-164 system is affirmative of its experimental potency; (5) hydrophobic packing contributes significantly to the drug binding in Src-UM-164; and (6) observed increase in H-bond distance of interacting residue atoms and Dasatinib compared to UM-164. Findings from this study can serve as a baseline in the design of novel Src inhibitors with dual inhibitory properties.