In Silico Molecular Modeling of Four New Afatinib Derived Molecules Targeting the Inhibition of the Mutated Form of BCR-ABL T315I.

Four afatinib derivatives were designed and modeled. These derivatives were compared to the known tyrosine-kinase inhibitors in treating Chronic Myeloid Leukemia, i.e., imatinib and ponatinib. The molecules were evaluated through computational methods, including docking studies, the non-covalent interaction index, Electron Localization and Fukui Functions, in silico ADMET analysis, QTAIM, and Heat Map analysis. The AFA(IV) candidate significantly increases the score value compared to afatinib. Furthermore, AFA(IV) was shown to be relatively similar to the ponatinib profile when evaluating a range of molecular descriptors. The addition of a methylpiperazine ring seems to be well distributed in the structure of afatinib when targeting the BCR-ABL enzyme, providing an important hydrogen bond interaction with the Asp381 residue of the DFG-switch of BCR-ABL active site residue and the AFA(IV) new chemical entities. Finally, in silico toxicity predictions show a favorable index, with some molecules presenting the loss of the irritant properties associated with afatinib in theoretical predictions.

QM/MM Simulations of Afatinib-EGFR Addition: The Role of ß-Dimethylaminomethyl Substitution.

Acrylamides are the most commonly used warheads of targeted covalent inhibitors (TCIs) directed at cysteines; however, the reaction mechanisms of acrylamides in proteins remain controversial, particularly for those involving protonated or unreactive cysteines. Using the combined semiempirical quantum mechanics (QM)/molecular mechanics (MM) free energy simulations, we investigated the reaction between afatinib, the first TCI drug for cancer treatment, and Cys797 in the EGFR kinase. Afatinib contains a ß-dimethylaminomethyl (ß-DMAM) substitution which has been shown to enhance the intrinsic reactivity and potency against EGFR for related inhibitors. Two hypothesized reaction mechanisms were tested. Our data suggest that Cys797 becomes deprotonated in the presence of afatinib, and the reaction proceeds via a classical Michael addition mechanism, with Asp800 stabilizing the ion-pair reactant state ß-DMAM+/C797- and the transition state of the nucleophilic attack. Our work elucidates an important structure-activity relationship of acrylamides in proteins.

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.

Discovery and optimization of covalent EGFR T790M/L858R mutant inhibitors.

Epidermal growth factor receptor (EGFR) inhibitors have clinical utility in the treatment of non-small cell lung cancer (NSCLC) patients. Despite encouraging clinical efficacy with these agents, many patients develop resistance due to sensitizing (or activating) mutations ultimately leading to disease progression. In the majority of the cases, this resistance is due to the T790M mutation and frequently coexisting L858R. In addition, EGFR wild type receptor inhibition can lead to on target related dose limiting toxicities such as rash and diarrhea. We describe herein the identification of a mutant selective lead compound 12, an irreversible covalent inhibitor of EGFR T790M/L858R resistance mutations with selectivity over the wild type form. Significant tumor growth inhibition in preclinical models was observed with this lead.

Acetylation of the Catalytic Lysine Inhibits Kinase Activity in PI3Kδ.

Covalent inhibition is a powerful strategy to develop potent and selective small molecule kinase inhibitors. Targeting the conserved catalytic lysine is an attractive method for selective kinase inactivation. We have developed novel, selective inhibitors of phosphoinositide 3-kinase δ (PI3Kδ) which acylate the catalytic lysine, Lys779, using activated esters as the reactive electrophiles. The acylating agents were prepared by adding the activated ester motif to a known selective dihydroisobenzofuran PI3Kδ inhibitor. Three esters were designed, including an acetate ester which was the smallest lysine modification evaluated in this work. Covalent binding to the enzyme was characterized by intact protein mass spectrometry of the PI3Kδ-ester adducts. An enzymatic digest coupled with tandem mass spectrometry identified Lys779 as the covalent binding site, and a biochemical activity assay confirmed that PI3Kδ inhibition was a direct result of covalent lysine acylation. These results indicate that a simple chemical modification such as lysine acetylation is sufficient to inhibit kinase activity. The selectivity of the compounds was evaluated against lipid kinases in cell lysates using a chemoproteomic binding assay. Due to the conserved nature of the catalytic lysine across the kinome, we believe the covalent inhibition strategy presented here could be applicable to a broad range of clinically relevant targets.

One Atom Makes All the Difference: Getting a Foot in the Door between SOS1 and KRAS.

KRAS, the most common oncogenic driver in human cancers, is controlled and signals primarily through protein-protein interactions (PPIs). The interaction between KRAS and SOS1, crucial for the activation of KRAS, is a typical, challenging PPI with a large contact surface area and high affinity. Here, we report that the addition of only one atom placed between Y884SOS1 and A73KRAS is sufficient to convert SOS1 activators into SOS1 inhibitors. We also disclose the discovery of BI-3406. Combination with the upstream EGFR inhibitor afatinib shows in vivo efficacy against KRASG13D mutant colorectal tumor cells, demonstrating the utility of BI-3406 to probe SOS1 biology. These findings challenge the dogma that large molecules are required to disrupt challenging PPIs. Instead, a "foot in the door" approach, whereby single atoms or small functional groups placed between key PPI interactions, can lead to potent inhibitors even for challenging PPIs such as SOS1-KRAS.

Photo-induced specific intracellular release EGFR inhibitor from enzyme/ROS-dual sensitive nano-platforms for molecular targeted-photodynamic combinational therapy of non-small cell lung cancer.

Molecular targeted-photodynamic combinational therapy is a promising strategy to enhance antitumor effects; meanwhile, current nanocarriers face challenges of limited selective delivery and release of therapeutic agents to specific tumor sites, which significantly compromises their therapeutic efficacy. Herein, we report active-targeting, enzyme- and ROS-dual responsive nanoparticles (HPGBCA) consisting of CD44-targeting hyaluronic acid (HA) shells and afatinib (AFT)-loaded, ROS-sensitive poly(l-lysine)-conjugated chlorin e6 (Ce6) derivative nanoparticle cores (PGBCA). HPGBCA can actively carry AFT and Ce6 specifically to tumor cells due to the negatively charged HA and CD44-mediated active targeting. Subsequently, hyaluronidase in the endosome will further spur the degradation of the HA shell to prompt exposure of the positively charged PGBCA core for rapid endosomal escape and intracellular delivery of AFT and Ce6. Furthermore, the generation of ROS produced by Ce6 under NIR irradiation can trigger the rapid oxidation of the thioether linker to facilitate the release of AFT into the cytoplasm. In vitro and in vivo studies demonstrated that the released AFT and excessive ROS at the local site can synergistically induce cell apoptosis to enhance the therapeutic efficacy without side effects. Our developed intelligent nanoparticle provides new avenues to achieve on-demand, specific intracellular drug release for improved molecular targeted-photodynamic combination therapeutic efficacy.

Antiviral Activity of Compound L3 against Dengue and Zika Viruses In Vitro and In Vivo.

Dengue virus (DENV) and Zika virus (ZIKV) are mosquito-borne flaviviruses that cause severe illness after infection. Currently, there are no specific or effective treatments against DENV and ZIKV. Previous studies have shown that tyrosine kinase activities and signal transduction are involved in flavivirus replication, suggesting a potential therapeutic strategy for DENV and ZIKV. In this study, we found that compound L3 can significantly reduce viral protein expression and viral titers in HEK-293, MCF-7, HepG2, and Huh-7 cells and exhibits superior therapeutic efficacy against flaviviral infection compared to other tyrosine kinase inhibitors. In addition, compound L3 can decrease endogenous HER2 activation and inhibit the phosphorylation of the HER2 downstream signaling molecules Src and ERK1/2, the levels of which have been associated with viral protein expression in MCF-7 cells. Moreover, silencing HER2 diminished DENV-2 and ZIKV expression in MCF-7 cells, which suggests that HER2 activity is involved in flavivirus replication. Furthermore, in DENV-2-infected AG129 mice, treatment with compound L3 increased the survival rates and reduced the viremia levels. Overall, compound L3 demonstrates therapeutic efficacy both in vitro and in vivo and could be developed as a promising antiviral drug against emerging flaviviruses or for concurrent DENV and ZIKV outbreaks.

The Anti-Tumor Activity of Afatinib in Pancreatic Ductal Adenocarcinoma Cells.

BACKGROUND: Pancreatic Ductal Adenocarcinoma (PDAC) is the most common form of pancreatic cancer and leading causes of pancreatic cancer death because of most PDAC patients with advanced unresectable disease at that time, which is remarkably resistant to all forms of chemotherapy and radiotherapy. OBJECTIVE: PDAC increases the social and patient's family burden. However, the PDAC pathogenesis is not identified. We are trying to uncover the underlying mechanism in the future. METHODS: In our research, the drug-resistant cell line was successfully induced in the vitro by progressive concentrations of Afatinib, which we named it as BxPC3-AR. RESULTS: It has been observed that the effect of autophagy was on the resistance of BxPC3-AR to Afatinib. CONCLUSION: It has been confirmed that autophagy plays a certain role in BxPC3-AR resistance to Afatinib. Our findings provide a new perspective on the role of autophagy in pancreatic ductal adenocarcinoma.

PLGA Porous Microspheres Dry Powders for Codelivery of Afatinib-Loaded Solid Lipid Nanoparticles and Paclitaxel: Novel Therapy for EGFR Tyrosine Kinase Inhibitors Resistant Nonsmall Cell Lung Cancer.

Combination therapy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR TKIs) with other chemotherapeutic agents is a feasible strategy to overcome resistance that often occurs after 9-13 months of EGFR TKIs administration in nonsmall cell lung cancer (NSCLC). In this study, a pulmonary microspheres system that codelivers afatinib and paclitaxel (PTX) is developed for treatment of EGFR TKIs resistant NSCLC. In this system, afatinib is loaded in stearic acid-based solid lipid nanoparticles, then these nanoparticles and PTX are loaded in poly-lactide-co-glycolide-based porous microspheres. These inhaled microspheres systems are characterized including geometric particle size, drug encapsulation efficiency, morphology by scanning electron microscopy, specific surface area, in vitro drug release, and aerodynamic particle size. Cell experiments indicate that afatinib and PTX have a synergistic effect and the codelivery system shows a superior treatment effect in drug-resistant NSCLC cells. The biocompatibility, pharmacokinetic, and tissue distribution experiments in Sprague-Dawley rats show that afatinib and PTX in the system can maintain 96 h of high lung concentration but low concentration in other tissues, with acceptable safety. These results demonstrate that this system may be a prospective delivery strategy for drug combination treatment in cancers developing resistance, especially drug-resistant lung cancer.

Non-small cell lung cancer-targeted, redox-sensitive lipid-polymer hybrid nanoparticles for the delivery of a second-generation irreversible epidermal growth factor inhibitor-Afatinib: In vitro and in vivo evaluation.

Afatinib (Afa), a second-generation irreversible epidermal growth factor inhibitor for the development of non-small cell lung cancer, has low bioavailability and adverse reactions. Nanoscaled drug delivery systems offer promising alternatives to address these defects and improve therapeutic outcomes. In the present study, a Tf contained, redox-sensitive ligand was synthesized and used for the preparation of afatinib loaded, Tf modified redox-sensitive lipid-polymer hybrid nanoparticles (Tf-SS-Afa-LPNs). Subsequently, studies of biological experiments in vitro and in vivo were performed to investigate the therapeutic effect of the system in lung cancer. The results showed that Tf-SS-Afa-LPNs has particle size of 103.5 ± 4.1 nm and zeta potential of -21.2 ± 2.4 mV. Significantly higher drug release was observed in the presence of glutathione (GSH). The area under the plasma concentration - time curve (AUC), peak concentration (Cmax) and terminal half life (T1/2) of Tf-SS-Afa-LPNs were 866.56 mg/L.h, 25.62 ± 3.21 L/kg/h, and 43.25 ± 2.31 h. Tf-SS-Afa-LPNs exhibited the most remarkable in vivo anti-tumor efficiency efficacy, which inhibited the tumor volume from 919 mm3 to 212 mm3. Tf-SS-Afa-LPNs is a promising platform for the lung cancer treatment.

Improving the anticancer effect of afatinib and microRNA by using lipid polymeric nanoparticles conjugated with dual pH-responsive and targeting peptides.

BACKGROUND: The emergence of resistance to chemotherapy or target therapy, tumor metastasis, and systemic toxicity caused by available anticancer drugs hamper the successful colorectal cancer (CRC) treatment. The rise in epidermal growth factor receptor (EGFR; human epidermal growth factor receptor 1; HER1) expression and enhanced phosphorylation of HER2 and HER3 are associated with tumor resistance, metastasis and invasion, thus resulting in poor outcome of anti-CRC therapy. The use of afatinib, a pan-HER inhibitor, is a potential therapeutic approach for resistant CRC. Additionally, miR-139 has been reported to be negatively correlated with chemoresistance, metastasis, and epithelial-mesenchymal transition (EMT) of CRC. Hence, we develop a nanoparticle formulation consisting of a polymer core to carry afatinib or miR-139, which is surrounded by lipids modified with a targeting ligand and a pH-sensitive penetrating peptide to improve the anticancer effect of cargos against CRC cells. RESULTS: Our findings show that this formulation displays a spherical shape with core/shell structure, homogeneous particle size distribution and negative zeta potential. The prepared formulations demonstrate a pH-sensitive release profile and an enhanced uptake of cargos into human colorectal adenocarcinoma Caco-2 cells in response to the acidic pH. This nanoparticle formulation incorporating afatinib and miR-139 exhibits low toxicity to normal cells but shows a better inhibitory effect on Caco-2 cells than other formulations. Moreover, the encapsulation of afatinib and miR-139 in peptide-modified nanoparticles remarkably induces apoptosis and inhibits migration and resistance of Caco-2 cells via suppression of pan-HER tyrosine kinase/multidrug resistance/metastasis pathways. CONCLUSION: This study proposes a multifunctional nanoparticle formulation for targeted modulation of apoptosis/EGFR/HER/EMT/resistance/progression pathways to increase the sensitivity of colon cancer cells to afatinib.

The EGFR Exon 19 Mutant L747-A750>P Exhibits Distinct Sensitivity to Tyrosine Kinase Inhibitors in Lung Adenocarcinoma.

PURPOSE: EGFR exon 19 deletion (Ex19Del) mutations account for approximately 60% of lung cancer-associated EGFR mutations and include a heterogeneous group of mutations. Although they are associated with benefit from tyrosine kinase inhibitors (TKI), the relative inhibitor sensitivity of individual Ex19Del mutations is unknown.Experimental Design: We studied the TKI sensitivity and structural features of common Ex19Del mutations and the consequences for patient outcomes on TKI treatment. RESULTS: We found that the L747-A750>P mutation, which represents about 4% of all Ex19Del mutations, displays unique inhibitor selectivity. L747-A750>P differs from other Ex19Del mutations in not being suppressed completely by erlotinib or osimertinib, yet is completely inhibited by low doses of afatinib. The HCC4006 cell line (with the L747-A750>P mutation) exhibited increased sensitivity to afatinib over erlotinib and osimertinib, and computational modeling suggests explanations for this sensitivity pattern. Clinically, patients with EGFR L747-A750>P mutant tumors showed inferior outcomes when treated with erlotinib than patients with E746-A750 mutant tumors. CONCLUSIONS: These results highlight important differences between specific Ex19Del mutations that may be relevant for optimizing TKI choice for patients.

Tyrosine Kinase Inhibitor Gold Nanoconjugates for the Treatment of Non-Small Cell Lung Cancer.

Gold nanoparticles (AuNPs) have emerged as promising drug delivery candidates that can be leveraged for cancer therapy. Lung cancer (LC) is a heterogeneous disease that imposes a significant burden on society, with an unmet need for new therapies. Chemotherapeutic drugs such as afatinib (Afb), which is clinically approved for the treatment of epidermal growth factor receptor positive LC, is hydrophobic and has low bioavailability leading to spread around the body, causing severe side effects. Herein, we present a novel afatinib-AuNP formulation termed Afb-AuNPs, with the aim of improving drug efficacy and biocompatibility. This was achieved by synthesis of an alkyne-bearing Afb derivative and reaction with azide-functionalized lipoic acid using copper-catalyzed click chemistry, then conjugation to AuNPs via alkylthiol-gold bond formation. The Afb-AuNPs were found to possess up to 3.7-fold increased potency when administered to LC cells in vitro and were capable of significantly inhibiting cancer cell proliferation, as assessed by MTT assay and electric cell-substrate impedance sensing, respectively. Furthermore, when exposed to Afb-AuNPs, human alveolar epithelial type I-like cells, a model of the healthy lung epithelium, maintained viability and were found to release less proinflammatory cytokines when compared to free drug, demonstrating the biocompatibility of our formulation. This study provides a new platform for the development of nontraditional AuNP conjugates which can be applied to other molecules of therapeutic or diagnostic utility, with potential to be combined with photothermal therapy in other cancers.

Quantification of afatinib, alectinib, crizotinib and osimertinib in human plasma by liquid chromatography/triple-quadrupole mass spectrometry; focusing on the stability of osimertinib.

The development and full validation of a sensitive and selective ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method are described for the simultaneous analysis of afatinib, alectinib, crizotinib and osimertinib in human lithium heparinized plasma. Afatinib-d6, crizotinib-d5 and erlotinib-d6 were used as internal standards. Given osimertinib's instability in plasma and whole blood at ambient temperature, samples should be solely processed on ice (T = 0 °C). Chromatographic separation was obtained on an Acquity UPLC ® BEH C18; 2.1 × 50 mm, 1.7 µm column, which was eluted with 0.400 mL/minute flow on a linear gradient, consisting of 10 mM ammonium formate (pH 4.5) and acetonitrile. Calibration curves for all compounds were linear for concentration ranges of 1.00 to 100 ng/mL for afatinib and 10.0 to 1000 ng/mL for alectinib, crizotinib and osimertinib, herewith validating the lower limits of quantification at 1.00 ng/mL for afatinib and 10.0 ng/mL for alectinib, crizotinib and osimertinib. Within-run and between-run precision measurements fell within 10.2%, with accuracy ranging from 89.2 to 110%.

Isolation and structural characterization of degradation products of afatinib dimaleate by LC-Q-TOF/MS/MS and NMR: cytotoxicity evaluation of afatinib and isolated degradation products.

Afatinib is an irreversible tyrosine kinase receptor inhibitor which was approved lately by USFDA for the treatment of metastatic non-small cell lung cancer (NSCLC). AFT was subjected to stress degradation studies under hydrolytic (acid, base and neutral), oxidative, thermal and photolytic conditions to investigate the inherent stability of the drug. The present study describes the simple and rapid HPLC method development for the selective separation of the AFT and its degradation products. The drug and degradation products were separated on Agilent Eclipse plus C18 (150 × 4.6 mm, 5 µ) column with ammonium acetate buffer (10 mM, pH 6.7) in gradient elution mode. The drug was found to be unstable in all the conditions studied. The developed chromatographic method was extended to tandem mass spectrometry (QTOF-MS) for the characterization of the degradation products. A total of 11 unknown degradation products were characterized using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q-TOF/MS/MS). Two major degradation products (DP2 and DP3) were isolated using preparative HPLC and their structures were confirmed by conducting 1H and 13C NMR experiments. The isolated DPs were evaluated for their anticancer potential using non small cell lung cancer cell line A549. The IC50 values for AFT, DP2 and DP3 were found to be 15.02 ± 1.49, 25.00 ± 1.26 and 32.56 ± 0.11 respectively. The in silico toxicity studies were performed employing ProTox-II software for the assessment of toxicity potential of drug and its degradation products. Finally, the developed chromatographic method was validated as per the International Conference on Harmonization guideline Q2 (R1).

Cryoprotectant toxicity in Caenorhabditis elegans.

We have looked at the effects of the cryoprotectant M22 upon viability in the model organism C. elegans. M22 is a well-known vitrification solution which has been successfully used in the laboratory to preserve organs destined for transplantation. M22 reduces survival of C. elegans in a concentration-dependent manner. M22 at concentrations of 10% (v/v) or higher inhibits progeny production and development. A few mutants in the ILS (insulin-like signaling) pathway of C. elegans are more resistant to the toxic effect of M22 compared to wild-type worms. Afatinib, an anti-cancer drug, protects against M22 toxicity. Afatinib by itself does not increase longevity.

Investigation of the binding characteristics between ligands and epidermal growth factor receptor by cell membrane chromatography.

The binding property between a ligand and its receptor is very important for numerous biological processes. In this study, we developed a high epidermal growth factor receptor (EGFR)-expression cell membrane chromatography (CMC) method to investigate the binding characteristics between EGFR and the ligands gefitinib, erlotinib, canertinib, afatinib, and vandetanib. Competitive binding analysis using gefitinib as the marker was used to investigate the interactions that occurred at specific binding sites on EGFR. The ability of displacement was measured from the HEK293-EGFR/CMC column on the binding sites occupied by gefitinib for these ligands, which revealed the following order: gefitinib (KD, 8.49 ± 0.11 × 10-7  M) > erlotinib (KD, 1.07 ± 0.02 × 10-6  M) > canertinib (KD, 1.41 ± 0.07 × 10-6  M) > afatinib (KD, 1.80 ± 0.12 × 10-6  M) > vandetanib (KD, 1.99 ± 0.03 × 10-6  M). This order corresponded with the values estimated by frontal displacement analysis and the scores obtained with molecular docking. Furthermore, thermodynamic analysis indicated that the hydrogen bond or Van der Waals force was the main interaction force in the process of EGFR binding to all 5 ligands. Overall, these results demonstrate that a CMC method could be an effective tool to investigate the binding characteristics between ligands and receptors.