Targeting caspase-1 with small-molecule inhibitors represents a promising strategy for the treatment of inflammatory diseases. Herein, we report the structure-based design, synthesis, and biological evaluation of a new 1H-tetrazole-containing scaffold for caspase-1 inhibitors. This scaffold incorporates a 1H-tetrazole as a carboxylic acid bioisostere and lacks electrophilic functionalities commonly employed to promote covalent modification of the catalytic cysteine. The resulting molecules inhibited caspase-1 with IC50 values ranging from the mid-nanomolar to low-micromolar range, with compounds 6e and 6f emerging as the most potent inhibitors (IC50 = 380 and 360 nM, respectively). Compared with our previously reported 1,5-disubstituted α-aminotetrazole series, compounds 6e and 6f showed an approximately 30-fold improvement in enzymatic potency. Preliminary in vivo evaluation of compound 6e in a dystrophic mouse model suggested a trend toward improved muscle coordination and strength, while no toxicity was observed in murine muscle cells under the tested conditions. Molecular dynamics simulations suggested different binding behaviors, conformational preferences, and interaction patterns for the two enantiomers of compound 6e within the caspase-1 binding pocket. Overall, this work broadens the structural diversity of tetrazole-based caspase-1 inhibitors by introducing a distinct 1H-tetrazole-containing scaffold with enhanced caspase-1 inhibitory activity and provides a promising starting point for further optimization.
Overcoming multidrug resistance (MDR) remains a formidable obstacle in cancer chemotherapy, largely attributable to drug efflux mediated by the P-glycoprotein (P-gp) pump. To address this challenge, we designed and synthesized 27 novel phenylindole derivatives and systematically assessed their MDR-reversal activity in MCF-7/ADR cells. Among these compounds, Ina4 demonstrated potent reversal activity (RF = 229.4), exceeding that of the reference P-gp inhibitors verapamil (RF = 51.0) and cyclosporine A (RF = 103.3), while exhibiting low intrinsic cytotoxicity. Mechanistic investigations, including western blot and Rhodamine 123 (Rh123) accumulation assays, revealed that Ina4 effectively inhibits the efflux function of P-gp without altering its protein expression levels. Furthermore, molecular docking analysis indicated that Ina4 may bind to the active pocket of P-gp, primarily via π-π stacking interactions. Notably, in a 3D tumor spheroid model, co-administration of Ina4 with doxorubicin (DOX) resulted in significant suppression of spheroid growth. Collectively, these findings indicate that Ina4 is a promising P-gp inhibitor.
Optimizing ring-opened scaffolds from the benzisoselenazolone (BISZ) core offers a promising strategy for developing novel anticancer agents. Herein, eleven new ebselen analogues (3a-k) featuring a symmetric bis-open-ring scaffold were synthesized and evaluated for in vitro antiproliferative activity against seven human cancer cell lines. Results shown compound 3d was the most potent across all seven tested panels, including lung carcinoma A549 (IC50 = 3.75 μM), esophageal KYSE-30 (1.55 μM), hepatocellular HCCLM3 (5.57 μM), prostate PC3 (2.77 μM), colorectal HCT116 (14.12 μM), hepatocellular HepG2 (14.33 μM), and breast MDA-MB-231 (8.28 μM). At 6.25 μM, 3d significantly inhibited angiogenesis in transgenic zebrafish, reducing subintestinal vessel area by 25.6% (p < 0.001) and intersegmental vessel number by 15.8% (p < 0.01), suggesting a potential anti-angiogenic activity against cancer. Docking and MD simulations suggested a possible interaction of 3d to MetAP2.
A novel set of imidazo[2,1-b]thiazolediones 4a,b and 5a-d, with anticipated EGFR and IDO1 inhibition activities, was designed and prepared. These novel derivatives were evaluated in the NCI 60 cell line panel in which the superior compounds 5b and 5d were chosen for further evaluation of their five dose cytotoxicity toward the most sensitive cancer cells namely non-small cell lung cancer EKVX and HOP-92, breast HS 578 T, and normal WI-38 cells. The presence of a substituted benzylidene moiety at position-2 of the imidazothiazole scaffold in compounds 5a-d positively influences anticancer activity, with the phenylallylidene moiety at position-6 exhibiting superior cytotoxic effects compared to the 2-thienylidene moiety. Among the examined hybrids, 5d showed significant antiproliferative effect against HS 578 T tumor cell. To explore the underlying cell-death mechanisms, secondary biological evaluations were conducted, including cell-free EGFR and IDO1 enzymatic inhibition, apoptosis assay, and cell cycle analysis. In cell-free biochemical assays, the most active derivatives demonstrated a promising potential dual inhibitory profile against EGFR and IDO1, with compound 5d exhibiting sub-micromolar activity against both target enzymes, providing a plausible biochemical rationale for its potent cell killing. Furthermore, compound 5d induced cell cycle arrest at the G2/M phase and triggered apoptosis in HS 578 T cells, as supported by the up-regulation of Caspase-3 and Bax accompanied by the down-regulation of Bcl-2. In-silico ADMET profiling and molecular docking simulations further supported the favorable drug-like properties and binding modes of the key compounds within the target active sites. Overall, these findings highlight compound 5d as a promising lead candidate for further optimization and cellular mechanistic validation in anti-cancer drug discovery.
A series of benzo[d]imidazole-isatin hybrids (4a-d, 6a-d, and 8a-d) was developed and synthesized as prospective dual inhibitors of VEGFR-2 and c-MET to address tumor growth and resistance-related signaling pathways. The biological assessment demonstrated encouraging dual kinase inhibitory action, with compound 8b identified as the most balanced inhibitor, displaying IC50 values of 92 nM and 63 nM against VEGFR-2 and c-MET, respectively. In-vitro antiproliferative evaluation against MDA-MB-231 and A549 cancer cell lines revealed notable cytotoxicity for compound 8b, yielding IC50 values of 2.37 μM and 1.79 μM, respectively, exceeding the efficacy of the reference medication sunitinib. Additionally, 8b exhibited a favorable selectivity profile for normal MCF-10A cells. Mechanistic studies demonstrated that 8b caused substantial G2/M cell cycle arrest and facilitated apoptosis in MDA-MB-231 cells. Biomarker studies revealed a reduction in VEGF-A, MMP-9, and Bcl-2 levels, alongside an increase in Bax and Caspase-3, suggesting potential anti-angiogenic and pro-apoptotic characteristics in-vitro. Molecular docking analyses provided supporting models for the identified biological activities and exhibited advantageous binding interactions within both kinase active sites. These data collectively suggest that chemical 8b may serve as a promising candidate for further anticancer research.
Superparamagnetic iron oxide nanoparticles (SPIONs) have emerged as promising theranostic agents owing to their magnetic hyperthermia efficiency and capability as magnetic resonance imaging (MRI) contrast agents. However, achieving sufficient intracellular iron accumulation remains a critical challenge for effective therapy. In this study, we report the development of cancer cell membrane-coated SPIONs (CM-SPIONs) to enhance tumor targeting and intracellular delivery. The CM-SPIONs were prepared using a modified Bangham method in the presence of membranes derived from 4T1 triple-negative breast cancer cells. The resulting nanoparticles exhibited significantly enhanced cellular uptake, reaching an intracellular iron concentration of 6 μg [Fe]/106 cells after 24 h incubation, representing a fivefold increase compared to uncoated SPIONs. Fluorescence imaging revealed predominant localization within lysosomes in the perinuclear region, suggesting that the lysosomal compartment may contribute to the observed AMF-induced cytotoxicity. Upon exposure to an alternating magnetic field (AMF) for 45 min, the CM-SPIONs resulted in 78.6% inhibition of cell proliferation, which was markedly higher than uncoated SPIONs. Furthermore, incubation with CM-SPIONs (0.05 mg [Fe]/mL, 3 h) reduced the T2 relaxation time from 2.71 s in the untreated cell suspension to 0.48 s, demonstrating significant MRI contrast enhancement. These results indicate that the CM-SPIONs represent a promising platform integrating magnetic hyperthermia with MRI-based cancer theranostics.
Synthetic lethality has emerged as an attractive therapeutic strategy for cancers harboring tumor suppressor gene alterations, as exemplified by the clinical success of PARP inhibitors in BRCA1/2-deficient cancers. We identified (±)-Z250-1659 as a synthetic lethal compound against BAP1-deficient mesothelioma cells through screening of a chemical library. To determine the active enantiomer, both enantiomers of Z250-1659 were prepared by organic synthesis and biologically evaluated, revealing that (S)-Z250-1659 is primarily responsible for the synthetic lethal activity. Notably, (±)-Z250-1659 exhibited activity nearly comparable to that of synthesized (S)-Z250-1659, despite the limited activity of the (R)-Z250-1659.
To explore how core backbone modifications influence prostate-specific membrane antigen (PSMA) targeting, we synthesized a series of novel peptidomimetics replacing the classic urea linkage of established radiotracers with a carbamate functionality. This panel systematically varied side chain length (aspartic vs. aminoadipic acid), stereocenter configuration (S/S vs. S/R diastereomers), and the radiometal chelator (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid [DOTA] vs. N,N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid [HBED-CC]). In vitro binding assays in LNCaP cells revealed that the carbamate backbone severely compromised PSMA affinity (Ki) for short-chain aspartic acid derivatives, whereas long-chain aminoadipic acid conjugates successfully retained low-nanomolar potencies. Across all variations, S/S configurations displayed superior affinities over S/R counterparts, demonstrating that natural stereochemistry is essential for optimal binding pocket interactions. Although both conjugates showed high in vitro potency, high-temperature radiolabeling of the DOTA derivative with gallium-68 caused thermal degradation via hydrolytic cleavage and intramolecular cyclization of the carbamate backbone. Conversely, the companion HBED-CC conjugate, 37(S/S), was successfully radiolabeled at ambient temperature without structural compromise to yield the intact radiotracer, 40(S/S). In vivo positron emission tomography (PET) imaging and biodistribution studies of 40(S/S) in LNCaP tumor-bearing mice demonstrated high tumor uptake (∼12%ID/g) at 1 h post-injection, rapid renal clearance, and excellent tumor-to-background ratios (tumor-to-muscle: ∼23; tumor-to-bone: ∼36). These findings demonstrate that carbamate backbones can successfully mimic urea linkages in PSMA ligands, provided they are paired with precise side chain lengths and mild radiolabeling conditions.
G-protein coupled estrogen receptors (GPER) represent clinically important target for detection and treatment of breast cancer owing to their high expression. Present study aimed to incorporate and determine the influence of cationic amino acids on the GPER-targeting tetrapeptide, Pro-Leu-Met-Ile (PLMI). Peptide variants were synthesized on solid phase by introducing nuclear localization sequence (KRSKK) at C and N-terminus of PLMI peptide respectively. The three peptides: DOTA-PLMI, DOTA-PLMIKRSKK and DOTA-KRSKKPLMI were radiolabeled with lutetium-177. [177Lu]Lu-DOTA-PLMI, [177Lu]Lu-DOTA-PLMIKRSKK and [177Lu]Lu-DOTA-KRSKKPLMI could be obtained in high radiolabeling yield (> 97%). Cellular studies in SKOV3 (ER-, PR- and GPER+) cells revealed highest binding affinity (Kd: 13.24 ± 0.89 nM) for [177Lu]Lu-DOTA-PLMIKRSKK along with enhanced nuclear localization and cellular internalization. Amongst the three radiopeptides, [177Lu]Lu-DOTA-PLMIKRSKK exhibited significantly reduced cellular uptake (95% inhibition) during blocking studies in SKOV3 cells (ER-, PR- and GPER+) than in MCF7 (ER+, PR+ and GPER+) cells (80%) indicating high GPER-specificity. Cellular internalization was also observed to be higher in SKOV3 cells (89%) in comparison to MCF7 cells (75%). Confocal microscopy confirmed high nuclear localization (∼85%) of dye conjugated peptide (Cf-PLMIKRSKK). Biodistribution studies in healthy mice demonstrated no radioactive accumulation in major organs except kidneys. Present studies thus demonstrate strong influence of cationic amino acids at C-terminal, boosting the biological properties of GPER-targeting PLMI peptide.
Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder requiring multi-target-directed ligands (MTDLs) to simultaneously modulate cholinergic dysfunction and amyloid-β pathology. In this study, a series of hydrazone-linked pyridin-1-ium salts was rationally designed and synthesized by integrating key pharmacophores of acetylcholinesterase (AChE) and β-secretase 1 (BACE-1) inhibitors into a compact scaffold. Biological evaluation revealed potent AChE inhibitory activity for most compounds, with several derivatives outperforming donepezil. Compound 8b exhibited the highest potency (IC50 = 0.026 ± 0.005 μM). Structure-activity relationship analysis indicated that small, moderately polar substituents enhanced AChE inhibition, whereas the introduction of bulkier groups into this series led to a relative improvement in BACE-1 inhibitory activity. Kinetic studies on AChE suggested a mixed-type inhibition mechanism. Molecular docking favored key π-π and π-cation interactions within the AChE gorge, while hydrophobic interactions contributed to BACE-1 binding. Complementing the static docking analysis, all-atom MD simulations further supported persistent residence of compound 8b within both AChE and BACE-1 binding sites, with interaction fingerprints refining the docking model by revealing dominant hydrophobic/π-π contacts in AChE and dynamic π-cation/hydrophobic contacts in BACE-1. In silico ADMET analysis demonstrated favorable drug-like properties. These findings highlight hydrazone-linked pyridin-1-ium salts as promising scaffolds for developing compact multi-targeted agents for AD.
Cardiotoxicity remains a major limitation of current anticancer therapies, highlighting the need for agents that combine antitumor efficacy with intrinsic cardiac safety. Phenotypic screening of a pyrimidine-based compound library identified compound 9v as a potent anti-prostate cancer candidate with dual biological functions. Compound 9v inhibited PC-3 cell proliferation with an IC₅₀ of 3.96 ± 0.18 μM, outperforming 5-fluorouracil, and markedly suppressed colony formation and migration while inducing apoptosis. Mechanistic studies showed that 9v activated the mitochondrial apoptotic pathway by increasing Bax, cleaved caspase-9, and cleaved caspase-3 levels while reducing Bcl-2 expression. Remarkably, 9v exerted bidirectional regulation of ROS homeostasis, promoting ROS accumulation in PC-3 cells but suppressing oxidative stress in H9C2 cardiomyocytes under H₂O₂ challenge. In addition, 9v exhibited weak cytotoxicity toward normal prostate stromal cells, no obvious acute toxicity in mice, and no detectable cardiotoxicity in vitro. Moreover, it exhibited protective effects against oxidative injury in cardiomyocytes, as demonstrated by increased cell viability and decreased LDH and cTnT release. Taken together, these results identify 9v as a promising lead compound with both anti-prostate cancer activity and cardioprotective potential in an in vitro oxidative stress model, providing a basis for the development of multifunctional anticancer agents with improved cardiovascular safety.
Candida represent a leading cause of morbidity and mortality worldwide resulting from opportunistic fungal infections. Increasing antifungal resistance calls for new ways of treating multidrug resistant fungal infections. Antisense oligonucleotides that target essential genes can offer a new treatment option; however, uptake of oligonucleotides presents the problem of passage across cell membrane and/or cell wall layers. Liposomes have been used to deliver pharmaceutical drugs, proteins, and nucleic acids to a variety of bacterial and eukaryotic cells. Here we investigated the utilization of commercially available liposomes to facilitate uptake of antisense oligonucleotides in Candida. Dynamic light scattering analysis determined the method which produced the most uniform commercially available liposome. Flow cytometry revealed which liposome composition facilitated the best delivery of fluorescein-labeled oligonucleotides in planktonic cells of Candida albicans, Candida auris and Candida glabrata. A time dependent uptake of liposomes within Candida biofilms demonstrated delivery of fluorescein-tagged DNA in Candida glabrata by 24 h. Efficient delivery of antisense-EFG1 was further indicated by a corresponding reduction in C. albicans hyphae length and number as well as a reduction in the levels of EFG1 gene expression.
In search for structurally novel antiviral leads against orthopoxvirus, we have previously identified and characterized ciclopirox (CPX) as a strong antiviral hit inhibiting vaccinia virus (VACV) replication. We report herein the antiviral activity-guided structure-activity relationship (SAR) of CPX, probing three structural zones (R1-R3) with the synthesis of 29 analogs. Major findings include that the hydroxyl group as R1 is required for activity, that most R2 modifications confer cytotoxicity, and that R3 changes are largely tolerated. In the end, the SAR identified one analog (5h, EC50 = 0.12 μM) showing substantially improved antiviral activity over CPX (EC50 = 0.51 μM), and numerous analogs exhibiting strong antiviral activities (EC50 = 0.71-1.2 μM) comparable to CPX without significant cytotoxicity. These SAR trends and additional potent analogs identified provide a strong foundation for future optimization efforts.
Thrombin, a versatile protease is a promising target in coagulation cascade for thrombosis related conditions. We designed and synthesized thirty novel furo[2,3-d]pyrimidinone analogs and evaluated them for their antithrombotic potential. Initial in vitro screening revealed that most of the compounds displayed good to moderate level of antithrombin properties. Compounds (7b, IC50 = 0.96 μM and 7g, IC50 = 1.36 μM) displayed the most potent direct thrombin inhibition activity. The results from the ex vivo anticoagulant activity demonstrated compound (7b) having prolonged PT and aPTT in rats relative to the control. Interestingly, compound (7b, BT = 91 s.) exhibited better safety profile than the standard drug dabigatran (BT = 102 s.) in terms of bleeding risk. Molecular docking and dynamics simulation studies identified and validated the binding interactions of potent derivatives with the target protein thrombin.
Globally, breast cancer stands as one of the primary causes of cancer related deaths. Among the types of breast cancer, estrogen receptor- positive (ER+) breast cancer accounts for the highest incidence and mortality worldwide. The present study emphasizes the design, synthesis, and biological evaluation of a series of novel triphenylethylene (TPE) based selective estrogen receptor modulators (SERMs) for their antiproliferative activity against ER+ breast cancer. All the synthesized compounds were well characterized using spectroscopic and chromatographic techniques. Favorable druglike properties indicating acceptable permeability through membrane and oral bioavailability were observed in in-silico physicochemical, ADME, and toxicity profiling. Further, biological evaluation of the synthesized compounds against MCF-7 breast cancer cells demonstrated significant antiproliferative nature of the synthesized compounds. Compounds 5g and 5o showed antiproliferative activity comparable to the standard metabolite 4-hydroxytamoxifen (4-OH TAM) and the IC50 of compounds 5g and 5o were found to be 4.350 ± 0.373 and 3.562 ± 0.466 μM respectively. Subsequently, the cell death pathway was revealed through caspase-3/7 assay and immunoblotting analysis and the compounds were found to cause cell death through activation of apoptosis-related proteins such as cleaved PARP and cleaved caspase-7. The molecular docking studies on the target protein ERα (PDB ID: 3ERT) exhibited favorable docking interactions between the compounds 5g and 5o with key amino acid residues such as Asp351, Thr347, Leu346, and His524. Similar interactions with amino acid residues were observed in the case of 4-OH TAM. Comprehensively, the results of the study highlight the promising anticancer potential of the synthesized compounds 5g and 5o as TPE-based SERM for selective ER+ breast cancer.
