Kübra Açıkalın Coşkun

Background/Objectives: Benzimidazole and hydrazone scaffolds are widely investigated in anticancer drug discovery because of their structural versatility and capacity to interact with multiple cancer-related targets. This study aimed to synthesize a series of trifluoromethyl-substituted benzimidazole-hydrazone derivatives, evaluate their antiproliferative activity and cancer-cell selectivity, and explore their potential interactions with breast cancer-associated molecular targets. Methods: A series of benzimidazole-hydrazone derivatives were prepared through a four-step synthetic route using both conventional and microwave-assisted procedures. The structures of the synthesized compounds were characterized using spectroscopic and analytical methods. Antiproliferative activity was evaluated in estrogen receptor-positive MCF-7 human breast cancer cells using the MTT assay after 48 h of exposure. Selected active compounds were additionally tested against non-tumorigenic hTERT cells to determine their selectivity indices. In silico analyses were performed against estrogen receptor alpha in antagonist and selective estrogen receptor degrader conformations, bromodomain-containing protein 4, and the mTOR kinase domain. Results: Microwave irradiation substantially reduced reaction times from 12–24 h to 4–6 min and increased isolated yields by 7–24 percentage points compared with conventional conditions. Compounds 4, 7, 9, and 11 reduced MCF-7 cell viability below 50% at 20 μM. Compound 7 showed the highest antiproliferative activity, with an IC₅₀ value of 7.5 ± 0.8 μM, and the greatest selectivity toward MCF-7 cells over hTERT cells, with a selectivity index of 12.66. Molecular docking, MD simulation, and MM/GBSA calculations predicted that compound 7 would effectively bind to the investigated targets, particularly estrogen receptor alpha, as well as the BRD4 and mTOR kinase domains. Conclusions: Compound 7 represents a selective, mid-micromolar benzimidazole-hydrazone lead against MCF-7 breast cancer cells. Its predicted molecular interactions warrant further target-based, mechanistic, pharmacokinetic, and in vivo evaluation.

Background: Heat shock protein 90 (HSP90) is a molecular chaperone that stabilizes numerous oncogenic proteins and supports tumor survival. Small molecules targeting HSP90 offer a novel approach to overcome drug resistance and immune suppression in breast cancer. Methods: A novel thiazolyl benzodiazepine (TB) containing a hydrazone moiety was evaluated in breast cancer cell lines (ER⁺ MCF-7, TNBC MDA-MB-231, and HER2⁺ SK-BR-3). Cytotoxicity was assessed using the CCK-8 assay, followed by PCR sequencing, flow cytometry, RT-qPCR, protein profiling, and HSP90 binding assays. Results: TB showed the strongest activity in MCF-7 cells (IC₅₀ = 7.21 µM) compared to MDA-MB-231 (IC₅₀ = 28.07 µM) and SK-BR-3 (IC₅₀ = 12.8 µM) cells. Mechanistic studies showed that TB binds to HSP90 (Kd = 3.10 µM), leading to disruption of the oncogenic signal. TB induced G2/M cell cycle arrest, promoted apoptosis via Bax and Caspase-3 activation, and suppressed cancer stem cell markers (NANOG, OCT4, SOX2). Additionally, TB activated immune-related pathways via ERK/MAPK signaling and upregulated genes such as SMAD2, SMAD3, and JUN.Conclusions: TB functions as an HSP90 inhibitor with dual anticancer and immunomodulatory properties in Estrogen Receptor-Positive (ER⁺) breast cancer cells. These findings suggest that TB represents a promising scaffold for the development of multi-targeted breast cancer therapies.

Background: Breast cancer is the most common cancer among women. Although doxorubicin (DOX) is widely used in its treatment, its dose-dependent toxicity and the development of drug resistance reduce its therapeutic efficacy. Therefore, this study aims to identify a novel anticancer agent that is more effective than DOX, inhibits cancer cell growth, and is less toxic to healthy cells. Methods: The cytotoxic effects of DOX and 2S-series molecules were evaluated on human (MDA-MB-231) and mouse (4T1) TNBC breast cancer cell lines and healthy breast epithelial (hTERT) cells using MTT assays at 48 and 72 h to screen functional similarities and possible differences upon drug/inhibitor treatment. Apoptosis and cell cycle analysis were analyzed by flow cytometry. Gene expression profiles were assessed by qPCR, and binding interactions with Hsp90 were examined via molecular docking. Results: 2S-5 exhibited IC₅₀ values of 6.21 µM (MDA-MB-231) and 7.04 µM (4T1), while 2S-13 showed IC₅₀ values of 7.73 µM and 8.21 µM, respectively. Both compounds demonstrated selective cytotoxicity against cancer cells. Gene expression and pathway analysis revealed that 2S-13 modulated the PI3K-Akt, MAPK, apoptosis, and HIF-1 pathways, showing broader modulation than DOX. Conclusions: 2S-13 appears to be a promising drug candidate, particularly in the MDA-MB-231 cell line. However, the current findings are limited to in vitro models. Further in vivo studies and pharmacokinetic analyses are required to validate its therapeutic potential, assess long-term efficacy and safety, and explore its resistance profile and molecular mechanisms in more detail.

Background: Theophylline, which is biologically important and found in tea, coffee, and cocoa beans, can be synthesized chemically or by direct extraction and concentration from natural sources. Theophylline derivatives have garnered attention in recent years for their potential therapeutic effects on Mycobacterium tuberculosis, antihistaminic, anti-inflammatory, and anticancer. Also, trifluoromethyl (CF₃) group has also been widely used in drug and agrochemical design. Methods: In this study, a series of new theophylline derivatives containing substituted trifluoromethyl and trifluoromethoxy groups were synthesized. The structures of these new compounds were confirmed by NMR, FT-IR, and elemental analyses. Additionally, the anticancer activities of the molecules were analyzed against VEGFR-2, CYP P450, and estrogen receptor by molecular docking method. Furthermore, in vitro biological effects of the compounds were comprehensively evaluated in cancer (A549 and HeLa) and normal (BEAS-2B) cells. Cell viability was assessed by MTT assay, and selectivity index (SI) values were calculated to determine tumor-specific toxicity. Results: N(7)-substituted theophyllines were prepared by the reaction of 1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione (theophylline) and trifluoromethyl substituted benzyl halide compounds. The synthesized N(7)-substituted theophyllines were obtained as white powder in high yield. The structure of synthesized compounds was confirmed by various spectroscopic techniques such as ¹H, ¹³C, ¹⁹F NMR, and FT-IR spectroscopy, and elemental analysis. The highest interaction was recorded as −5.69 kcal/mol for 3-CF₃ substituted against VEGFR-2 structure while the best binding affinity was determined for 4-OCF₃ substituted with −6.69 kcal/mol against Human Cytochrome P450 with in silico analysis. The in vitro anticancer activities of the molecules were also evaluated against A549 and HeLa cells, and displayed considerably higher cytotoxicity with 2-CF₃, 3-CF_3, and 4-CF₃ substituted molecules in Hela and A549 cell line. To elucidate the molecular mechanism, apoptosis-related gene expression changes were analyzed by RT-qPCR in A549 and HeLa cells treated with compound 2-CF₃. Significant upregulation of pro-apoptotic markers and downregulation of anti-apoptotic genes were observed. Consistently, ELISA-based quantification confirmed increased protein levels of Caspase-3, BAX, and Cytochrome C, and decreased BCL-2, validating the apoptotic mechanism at the protein level. Also, the antibacterial and antibiofilm activity details of the molecules were evaluated against DNA Gyrase, and SarA crystal structures by molecular docking method. The highest interaction was recorded as −5.56 kcal/mol for 2-CF₃ substituted with H-bonds with Asn46, Val71, Asp73, and Thr165 against DNA Gyrase crystal structure while 3-CF₃ substituted has the best binding affinity against SarA. The in vitro antimicrobial effects of the molecules were also evaluated. Conclusions: The synthesized molecules may provide insight into the development of potential therapeutic agents to the increasing antimicrobial resistance and biofilm-forming capacity of microorganisms. Additionally, compound 2-CF₃ substituted exhibited promising and selective anticancer activity through apoptosis induction, supported by gene and protein level evidence.

Öz Amaç: Meme kanseri kadınlarda görülen en sık kanser olup görülme sıklığı giderek artmaktadır. Günümüzde meme kanserinin tedavisinde cerrahi tedavi, radyasyon tedavisi ve kemoterapi gibi birçok yöntem vardır. Kemoterapi tedavisinde yaygın olarak kullanılan kemoterapötiklerden birisi olan doksorubisin (DOX) belirli bir dozun üzerinde tehlikeli yan etkilere ve ilaç direncine neden olur. Mevcut tedavilere kıyasla kombinasyon tedaviler son zamanlarda bu sorunların üstesinden gelmek için önem kazanmıştır. Bir fotoduyarlaştırıcı ile uygulanan fotodinamik terapi klinikte birçok kanser türünde uygulanmaktadır. Bu çalışma ile meme kanseri tedavisinde kullanılan DOX’u fotodinamik terapi (PDT) ile aktive edilen metilen mavisi (MB) ile kombinasyon uygulayarak DOX’un uygulanmasında düşük toksisite ile yüksek etkinlik alınması amaçlanmıştır. Yöntem: MCF-7 meme kanseri hücre hattı üzerinde farklı konsantrasyonlarda DOX, MB, MB-PDT, DOX-PDT, MB-PDT+DOX ve DOX+MB-PDT olacak şekilde ayrı ayrı ve kombinasyon tedaviler denenmiştir. Hücre canlılığını belirlemek için MTT testi gerçekleştirilmiş ve hücrelerin %50 canlılık gösterdiği IC50 değerleri hesaplanmıştır. Ayrıca, MB ve DOX arasındaki etkileşimin derecesini belirlemek için kombinasyon indeksi (CI) değeri bulunmuştur. Ayrıca DOX ve fotodinamik terapi kombinasyonunun meme kanseri hücrelerine karşı apoptotik potansiyelini belirlemek için apoptotik ve antiapoptotik belirteçlerin (Bax, Bcl-2) mRNA ekspresyon seviyelerine RT-PCR ile bakılmıştır. Bulgular: MB-PDT ve ardından düşük konsnatrasyonda DOX (428.4 nM ) kombinasyonunun, tek başına DOX'a ve önce DOX ardından MB-PDT’ye kıyasla kanser hücresi ölümünü indüklemede daha iyi bir etkiye sahip olduğunu göstermiştir. Aynı zamanda bu kombine uygulama Bcl-2 gen ekspresyonunu düşürürken, Bax gen ekspresyonunu yükselterek apoptotik potansiyelini göstermiştir. Sonuç: Doksorubisin ve metilen mavisi ile indüklenmiş fotodinamik terapi kombine tedavisi meme kanseri hücreleri üzerinde sinerjik, sitotoksik ve apoptotik etkiler göstererek , umut verici bir yaklaşım sunmuştur.

Toxoplasma gondii is a common intracellular parasite that causes the toxoplasmosis. Heat shock proteins (Hsps) have a critical role in pathogenesis of toxoplasmosis. Hsps are highly conserved proteins in evolution among living organisms. This protein family responsible for a wide range of biological processes such as protein folding, protein translocation, protein aggregation. In the present study, Hsp70, a member of the Hsp family, was isolated from T. gondii and its sequence and motifs were determined by PCR, cloning, sequencing and homology modelling analysis. ATP hydrolysis, luciferase folding, and luciferase aggregation experiments were performed for determination of its chaperone activity while the stability and secondary structure of the Hsp70 were discovered by using biophysical experiments (FTIR, florescence and quenching experiment). In addition, in silico analysis were used to determine the physicochemical characteristics of Hsp70. The results revealed that Hsp70 protein obtained from T. gondii (Hsp70-IPEK1) is similar to Hsp70s from other organisms. Also, the chaperone activity, stability and secondary structure of Hsp70-IPEK1 were determined. Hsp70-IPEK1 together with other chaperones in the presence of nucleotide were dramatically increased protein folding and aggregation. According to these results, it is thought that Hsp70 has a potential to contribute many research areas such as pharmaceutical analysis

Two silver(I) complexes, bis{diethyl[(5-phenyl-1,3,4-oxadiazol-2-yl-κN³:κN⁴-amino) (4-trifluoromethylphenyl)methyl]phosphonate-(tetrafluoroborato-κF)}-di-silver(I) and tetrakis-{diethyl[(5-phenyl-1,3,4-oxadiazol-2-yl-κN³-amino)(4-trifluoromethylphenyl)methyl]phosphonate} silver(I) tetrafluoroborate, were prepared starting from the diethyl[(5-phenyl-1,3,4-oxadiazol-2-yl-amino)(4-trifluoromethylphenyl)methyl]phosphonate (1) ligand and AgBF₄ salt in Ag/ligand ratios of 1/1 and 1/4, respectively. The structure, stoichiometry, and geometry of the silver complexes were fully characterized by elemental analyses, infrared, single-crystal X-ray diffraction studies, multinuclear NMR, and mass spectroscopies. The binuclear complex ([Ag₂(1)₂(BF₄)₂]; 2) crystallizes in the monoclinic asymmetric space group P2₁/c and contains two silver atoms adopting a {AgN₂F} planar trigonal geometry, which are simultaneously bridged by two oxadiazole rings of two ligands, while the mononuclear complex ([Ag(1)₄]BF₄; 3) crystallizes in the non-usual cubic space group Fd-3c in which the silver atom binds to four distinct electronically enriched nitrogen atoms of the oxadiazole ring, in a slightly distorted {AgN₄} tetrahedral geometry. The α-aminophosphonate and the monomeric silver complex were evaluated in vitro against MCF-7 and PANC-1 cell lines. The silver complex is promising as a drug candidate for breast cancer and the pancreatic duct with half-maximal inhibitory concentration (IC₅₀) values of 8.3 ± 1.0 and 14.4 ± 0.6 μM, respectively. Additionally, the interactions of the ligand and the mononuclear complex with Vascular Endothelial Growth Factor Receptor-2 and DNA were evaluated by molecular docking methods.

Protein purification is not a simple task. Yet, overexpression at bacterial systems with recombinant modifications brings further difficulties. Adding a tag, an affinity label, and expressing particular domains of the whole protein, especially hydrophobic sections, make purification a challenging process. Protein folding pattern may perturb N- or C-terminal tag and this terminal preference may lead to poor purification yield. Codon optimization, solvent content and type, ionic conditions, resin types, and self-cleavage of recombinant proteins bring further difficulties to protein expression and purification steps. The chapter overviews problems of protein purification through a small peptide overexpression in bacteria (Recombinant anti-SARS Coronavirus 2 (SARS-Cov-2) Spike protein Receptor Binding Domain (RBD) antibody (Clone Sb#14). The chapter also covers troubleshooting at distinct steps and highlights essential points to solve crucial issues of protein purification.

Cancer is the biggest health problem worldwide due to its high mortality rate. Lymphoma is defined as a group of malignant diseases that is caused by clonal proliferation of lymphocytes and is classified under two major groups: Hodgkin lymphoma and non-Hodgkin lymphoma. Genetic predisposition and some environmental factors constitute risk factors. Symptoms of the disease include unexplained fever, swelling of lymph glands, swollen abdomen, tiredness, loss of appetite, frequent infections, and weight loss. Positron emission tomography (PET) and computed tomography (CT) scans, along with MRI, are widely used for the diagnosis of lymphoma. Advanced blood and lymph node biopsy tests are used to evaluate treatment effect on blood cells and to confirm the diagnosis of lymphoma, respectively. Current treatment options include chemotherapy, radiotherapy, and bone marrow/stem cell transplantation. Development of new treatment options for cancer medications includes small molecules and monoclonal antibodies for immunotherapy. In addition, the discovery of new phytochemical agents used in complementary and alternative medicine adds perspective to the treatment of lymphoma.

Background: HSP70 is a survival factor for tumor cells in transformation and in tumor progression as well as in anti-apoptotic response. Objective: Several inhibitors targeting HSP70 ATPase function displayed off-target affect but PES which targets HSP70 substrate binding domain prevents tumor cell survival prominently. However, PES may not bind HSP70 in the absence of nucleotide. This research aimed to design a unique inhibitor molecule that work both in the presence and absence of nucleotides to amplify inhibition. Methods: A set of chimeric coumarine-pyrazole derivatives determined by in silico techniques and synthesized to elucidate their inhibitory effects. Cell viability experiments displayed KBR1307 as the most efficient inhibitor. A set of characterization experiments performed, and results compared to that of PES agent. Binding constant, ATP hydrolysis rate, and percent aggregation determined in the presence and absence of inhibitors. Results: In silico docking experiments showed that only KBR1307 binds the HSP70 substrate binding domain and interacts with cochaperone interface. Binding experiments indicated that KBR1307 binds HSP70 both in the presence and absence of nucleotides, but PES does not. Both inhibitors significantly lower HSP70 ATPase activity and substrate protein disaggregation activity. However, KBR1307 displays a lower IC₅₀ value at the MCF-7 cell line compared to PES. Both inhibitors do not alter HSP70 secondary structure composition and overall stability. Conclusion: KBR1307 effectively inhibits HSP70 compared to PES and provides promising template for novel anticancer drug development.