Submicromolar Concentrations Research Articles

Pro-aromatic Natural Terpenes as Unusual "Slingshot" Antioxidants with Promising Ferroptosis Inhibition Activity.

Ferroptosis is a cell death mechanism based on extensive cellular membrane peroxidation, implicated in neurodegenerative and other diseases. The essential oil component γ-terpinene, a natural monoterpene with a unique highly oxidizable pro-aromatic 1,4-cyclohexadiene skeleton, inhibits peroxidation of polyunsaturated lipid in model heterogeneous systems (micelles and liposomes). Upon H-atom abstraction, an unstable γ-terpinene-derived peroxyl radical is formed, that aromatizes to p-cymene generating HOO⋅ radicals. As HOO⋅ are small and hydrophilic radicals, they quickly diffuse outside the lipid core, blocking the radical chain propagation of polyunsaturated lipids. This unprecedented antioxidant "slingshot" mechanism explains why γ-terpinene shows a protective activity against ferroptosis, being effective at submicromolar concentrations in human neuroblastoma (SH-SY5Y) cells.

Pharmacologically enabling the degradation of NaV1.8 channels to reduce neuropathic pain.

In phase II clinical trials, NaV1.8 channels were identified as viable targets to treat acute pain. Results were modest, however, and NaV1.8 pore blockers must be given systemically, potentially leading to adverse effects, especially during prolonged use. A local, long-lasting approach is desirable, yet local anesthetics are neither specific nor long-lasting. In lieu of a pore blocker approach, we show a pharmacological method targeting the scaffolding and degradation of NaV1.8 channels, which attenuated neuropathic pain behavior in mice. NaV1.8 channels interact with the WW domain-containing scaffold protein called Magi-1. WW domains are typically found in ubiquitin ligases, and NaV1.8 channels are susceptible to degradation by ubiquitin ligases. Here, we show NaV1.8 and MAGI-1 colocalized in human tissues. We demonstrate that a lipidated peptide derived from the NaV1.8 WW binding domain, at sub-micromolar concentrations, inhibited rodent dorsal root ganglion neuronal firing. The peptide reduced NaV1.8 channel immunoreactivity and tetrodotoxin-resistant currents in human dorsal root ganglion neurons. We found that the lipidated peptide attenuated neuropathic pain behaviors in mice for multiple weeks after a single injection. Our results reveal that the NaV1.8-targeted lipidated peptide provides local and sustained analgesia, serving as a viable alternative to NaV1.8 pore blockers.

Resveratrol Inhibits Nucleosome Binding and Catalytic Activity of PARP1

The natural polyphenol resveratrol is a biologically active compound that interacts with DNA and affects the activity of some nuclear enzymes. Its effect on the interaction between nucleosomes and poly(ADP-ribose) polymerase-1 (PARP1) and on the catalytic activity of PARP1 was studied using Western blotting, spectrophotometry, electrophoretic mobility shift assay, and single particle Förster resonance energy transfer microscopy. Resveratrol inhibited PARP1 activity at micro- and sub-micromolar concentrations, but the inhibitory effect decreased at higher concentrations due to the aggregation of the polyphenol. The inhibition of PARP1 by resveratrol was accompanied by its binding to the enzyme catalytic center and a subsequent decrease in PARP1 affinity to nucleosomal DNA. Concurrent binding of talazoparib to the substrate binding pocket of PARP1, which occurs in the presence of resveratrol, restores the interaction of PARP1 with nucleosomes, suggesting that the binding sites of resveratrol and talazoparib overlap. The data suggest that resveratrol can be classified as a natural inhibitor of PARP1.

2-Thiopyridone-based stilbazoles: synthesis, photo-physical properties and sensing of Hg(II) and Cd(II) ions

A method of preparation of new representatives of donor-acceptor stilbazoles based on pyridine-2-thione containing a cyano group and a trifluoromethyl moiety in the acceptor part of the molecule was developed. The synthesized stilbazoles exhibited pronounced fluorescence, with quantum yields up to about 15% significantly improved compared to traditional thioxonicotinonitriles, which typically lack such properties. The emission turned out to be strongly dependent on the tautomeric equlibrium in the solution that was studied using both computational methods and comprehensive spectroscopic analysis. Solvato(fluoro)chromism and the influence of the substituent on the spectral characteristics of the compounds were also investigated. It was established that the emission maximum could be tuned from 418 to 630 nm covering almost the entire visible spectrum. The design of stilbazoles enhanced their photophysical properties making them suitable candidates for environmental monitoring or biological sensing. As a result a new chemosensor was developed demonstrating high selectivity for the detection of micromolar concentration of mercury(II) ions (LoD 1.34 μM) and submicromolar concentration of cadmium(II) ions (LoD 0.875 μM) in aqueous media.

Mechanism of acid-sensing ion channel modulation by Hi1a.

Acid-sensing ion channels (ASICs) are trimeric cation-selective channels activated by extracellular acidification. Amongst many pathological roles, ASICs are an important mediator of ischemic cell death and hence an attractive drug target for stroke treatment as well as other conditions. A peptide called Hi1a, isolated from Australian funnel web spider venom, inhibits ASIC1a and attenuates cell death in a stroke model up to 8 h after stroke induction. Here, we set out to understand the molecular basis for Hi1a's action. Hi1a is a bivalent toxin with two inhibitory cystine knot domains joined by a short linker. We found that both Hi1a domains modulate human ASIC1a gating with the N-terminal domain impairing channel activation while the C-terminal domain produces a "pro-open" phenotype even at submicromolar concentrations. Interestingly, both domains bind at the same site since a single point mutation, F352A, abolishes functional effects and reduces toxin affinity in surface plasmon resonance measurements. Therefore, the action of Hi1a at ASIC1a appears to arise through a mutually exclusive binding model where either the N or C domain of a single Hi1a binds one ASIC1a subunit. An ASIC1a trimer may bind several inhibitory N domains and one or more pro-open C domains at any one time, accounting for the incomplete inhibition of wild type Hi1a. We also found that the functional differences between these two domains are partially transferred by mutagenesis, affording new insight into the channel function and possible novel avenues of drug design.

Molecular design of −substituted boron difluoride curcuminoids: Tuning luminescence and nonlinear optical properties

Boron β-diketonates are prospective fluorescent dyes for functional organic materials to new technologies in the fields of chemistry, NLO-optics, photonics and bio-imaging. The novel NIR-to-NIR luminophors – curcuminoids of boron difluoride with different substituents at the central carbon atom (at the γ-position) of the chelate ring 1,7-bis(4′-N,N′-dimethylaminophenyl)-4-organyl-gept-1,6-dien-3,5-dionate of boron difluoride (1–5) were synthesized. Luminescence in solutions, crystals and polymer matrices, photobiological properties and NLO properties in polystyrene (PS) and polymethylmethacrylate (PMMA) were studied. The crystal structure has been determined for 1. For 1–5, in PS film, upon excitation by laser (365 nm), two luminescence bands are observed in the blue (430 nm) and red (650–700 nm) regions of the spectrum. For 1–5, two-photon luminescence is recorded in PS and PMMA films. PS 1 films demonstrate high photostability. The synthesized dyes turned out to be almost non-toxic for HCT116 tumor cells both in a long-term dark experiment (72 h) and after photoexcitation in accordance with absorption maxima in the submicromolar concentration range. The compounds were rapidly accumulated by cells within 3 h, demonstrating cytoplasmic distribution and the potential for use as cellular photoimaging agents. The accumulation of dyes with hydrocarbon γ-substitutes (methyl, iso-propyl, phenyl) after 3 and 24 h is more effective compared to dye 2 (commercial product Cranad-2).

Ketamine and Major Ketamine Metabolites Function as Allosteric Modulators of Opioid Receptors.

Ketamine is a glutamate receptor antagonist that was developed over 50 years ago as an anesthetic agent. At subanesthetic doses, ketamine and some metabolites are analgesics and fast-acting antidepressants, presumably through targets other than glutamate receptors. We tested ketamine and its metabolites for activity as allosteric modulators of opioid receptors expressed as recombinant receptors in heterologous systems and with native receptors in rodent brain; signaling was examined by measuring GTP binding, β-arrestin recruitment, MAPK activation, and neurotransmitter release. Although micromolar concentrations of ketamine alone had weak agonist activity at μ opioid receptors, the combination of submicromolar concentrations of ketamine with endogenous opioid peptides produced robust synergistic responses with statistically significant increases in efficacies. All three opioid receptors (μ, δ, and κ) showed synergism with submicromolar concentrations of ketamine and either methionine-enkephalin (Met-enk), leucine-enkephalin (Leu-enk), and/or dynorphin A17 (Dyn A17), albeit the extent of synergy was variable between receptors and peptides. S-ketamine exhibited higher modulatory effects compared with R-ketamine or racemic ketamine, with ∼100% increase in efficacy. Importantly, the ketamine metabolite 6-hydroxynorketamine showed robust allosteric modulatory activity at μ opioid receptors; this metabolite is known to have analgesic and antidepressant activity but does not bind to glutamate receptors. Ketamine enhanced potency and efficacy of Met-enkephalin signaling both in mouse midbrain membranes and in rat ventral tegmental area neurons as determined by electrophysiology recordings in brain slices. Taken together, these findings support the hypothesis that some of the therapeutic effects of ketamine and its metabolites are mediated by directly engaging the endogenous opioid system. SIGNIFICANCE STATEMENT: This study found that ketamine and its major biologically active metabolites function as potent allosteric modulators of μ, δ, and κ opioid receptors, with submicromolar concentrations of these compounds synergizing with endogenous opioid peptides, such as enkephalin and dynorphin. This allosteric activity may contribute to ketamine's therapeutic effectiveness for treating acute and chronic pain and as a fast-acting antidepressant drug.

Disruption of PHF6 Peptide Aggregation from Tau Protein: Mechanisms of Palmatine Chloride in Preventing Early PHF6 Aggregation.

The formation of neurofibrillary tangles (NFTs), composed of tau protein aggregates, is a hallmark of neurodegenerative diseases known as tauopathies, including Alzheimer's disease (AD). NFTs consist of paired helical filaments (PHFs) of tau protein with a dominant β-sheet secondary structure. Within these PHFs, the PHF6 hexapeptide (Val306-Gln-Ile-Val-Tyr-Lys311) has been commonly highlighted as a key site for tau protein nucleation. Palmatine chloride (PC) has been identified as an inhibitor of PHF6 aggregation, capable of reducing aggregation propensity at submicromolar concentrations. In pursuit of novel anti-AD drugs targeting early tau aggregation stages, we conducted an in silico study to elucidate PC's mechanism of action during PHF6 aggregation. Our observations suggest that while PHF6 can still initiate self-aggregation in the presence of PC, PC molecules subtly influence PHF6 aggregation dynamics, favoring smaller aggregates over larger complexes. The study underlined the key roles of aromatic rings in PC binding to different PHF6 aggregates by interacting through π-π stacking with the PHF6 Tyr310 side chain. The presence of aromatic rings in compounds to be able to inhibit the earlier complexation phase seems to be essential. These in silico findings lay a foundation for the design of compounds that could intervene in resolving the neurotoxicity of protein aggregates in AD.

Epertinib counteracts multidrug resistance in cancer cells by antagonizing the drug efflux function of ABCB1 and ABCG2

A significant hurdle in cancer treatment arises from multidrug resistance (MDR), often due to overexpression of ATP-binding cassette (ABC) transporters like ABCB1 and/or ABCG2 in cancer cells. These transporters actively diminish the efficacy of cytotoxic drugs by facilitating ATP hydrolysis-dependent drug efflux and reducing intracellular drug accumulation in cancer cells. Addressing multidrug-resistant cancers poses a significant challenge due to the lack of approved treatments, prompting the exploration of alternative avenues like drug repurposing (also referred to as drug repositioning) of molecularly targeted agents to reverse MDR-mediated by ABCB1 and/or ABCG2 in multidrug-resistant cancer cells. Epertinib, a potent inhibitor of EGFR and HER2 currently in clinical trials for solid tumors, was investigated for its potential to resensitize ABCB1- and ABCG2-overexpressing multidrug-resistant cancer cells to chemotherapeutic agents. Our findings reveal that at sub-toxic, submicromolar concentrations, epertinib restores the sensitivity of multidrug-resistant cancer cells to cytotoxic drugs in a concentration-dependent manner. The results demonstrate that epertinib enhances drug-induced apoptosis in these cancer cells by impeding the drug-efflux function of ABCB1 and ABCG2 without altering their expression. ATPase activity and molecular docking were employed to reveal potential interaction sites between epertinib and the drug-binding pockets of ABCB1 and ABCG2. In summary, our study demonstrates an additional pharmacological capability of epertinib against the activity of ABCB1 and ABCG2. These findings suggest that incorporating epertinib into combination therapy could be advantageous for a specific patient subset with tumors exhibiting high levels of ABCB1 or ABCG2, warranting further exploration.

Ultra-sensitive nitrate-ion detection via transconductance-enhanced graphene ion-sensitive field-effect transistors

Current potentiometric sensing methods are limited to detecting nitrate at parts-per-billion (sub-micromolar) concentrations, and there are no existing potentiometric chemical sensors with ultralow detection limits below the parts-per-trillion (picomolar) level. To address these challenges, we integrate interdigital graphene ion-sensitive field-effect transistors (ISFETs) with a nitrate ion-sensitive membrane (ISM). The work aims to maximize nitrate ion transport through the nitrate ISM, while achieving high device transconductance by evaluating graphene layer thickness, optimizing channel width-to-length ratio (RWL), and enlarging total sensing area. The captured nitrate ions by the nitrate ISM induce surface potential changes that are transduced into electrical signals by graphene, manifested as the Dirac point shifts. The device exhibits Nernst response behavior under ultralow concentrations, achieving a sensitivity of 28 mV/decade and establishing a record low limit of detection of 0.041 ppt (4.8 × 10−13 M). Additionally, the sensor showed a wide linear detection range from 0.1 ppt (1.2 × 10−12 M) to 100 ppm (1.2 × 10−3 M). Furthermore, successful detection of nitrate in tap and snow water was demonstrated with high accuracy, indicating promising applications to drinking water safety and environmental water quality control.

Unveiling Hierarchical Self-Assembly of Triazolylferrocenyl Dendrimers: Producing Non-Traditional Intrinsically Green Fluorescent Vesosomes for Nanotheranostics.

Dendrimers and supramolecular chemistry continue to fascinate researchers due to the endless unrevealed potential of their combination. This study investigates the self-assembly process of a series of hydrophobic triazolylferrocenyl dendrimers in aqueous medium. Deep investigation through NMR spectroscopy, absorption UV-vis spectroscopy along with theoretical simulations demonstrates that the ferrocenyl moieties interact intramolecularly and intermolecularly driving the self-assembly process. Data obtained by DLS, NTA, SEM, TEM, and EF-TEM demonstrate that these dendrimers, in water, spontaneously self-assemble through a hierarchical process. The dendrimers first self-assemble into uniform nanovesicles, which in turn self-assemble into larger vesosomes. The resulting vesosomes emit green non-traditional intrinsic fluorescence, which is a property that emerged from the self-assembled architectures. The vesosomes are efficiently uptaken by cancer cells and induce significant cytotoxic activity against the cancer cell line MCF-7, up to the submicromolar concentration. Positive dendritic effects are identified in the fluorescence intensity and in the cytotoxic activity of the vesosomes, which follow the trend G0-9Fc < G1-27Fc < G2-81Fc. This work showcases the remarkable potential of combining the two dynamic fields of dendrimers and supramolecular chemistry, which resulted in green fluorescent vesosomes capable of performing the dual role of cell imaging and killing, with potential applications in nanotheranostics.

Computational Design of Pore-Forming Peptides with Potent Antimicrobial and Anticancer Activities.

Peptides that form transmembrane barrel-stave pores are potential alternative therapeutics for bacterial infections and cancer. However, their optimization for clinical translation is hampered by a lack of sequence-function understanding. Recently, we have de novo designed the first synthetic barrel-stave pore-forming antimicrobial peptide with an identified function of all residues. Here, we systematically mutate the peptide to improve pore-forming ability in anticipation of enhanced activity. Using computer simulations, supported by liposome leakage and atomic force microscopy experiments, we find that pore-forming ability, while critical, is not the limiting factor for improving activity in the submicromolar range. Affinity for bacterial and cancer cell membranes needs to be optimized simultaneously. Optimized peptides more effectively killed antibiotic-resistant ESKAPEE bacteria at submicromolar concentrations, showing low cytotoxicity to human cells and skin model. Peptides showed systemic anti-infective activity in a preclinical mouse model of Acinetobacter baumannii infection. We also demonstrate peptide optimization for pH-dependent antimicrobial and anticancer activity.

Revisiting capsaicin and nonivamide: Their analogs exert strong inhibitory activity against cholinesterases

The scientific community has long been interested in capsaicin, and the extensive hunt for AChE and BChE enzyme inhibitors is still ongoing. In this investigation analogs of capsaicin, such as the pharmaceutical nonivamide, which is preferred in clinical settings for the topical treatment of pain, were explored in the search for appropriate inhibitors. Thus, to test their inhibitory effect on AChE and BChE, we synthesized a short series of derivatives derived from vanillylamide. Consequently, it was discovered that compounds 12, 34, and 35, which have Ki values in the sub-micromolar concentration range, are especially effective inhibitors. Compound 12 demonstrated dual mixed-type (competitive/uncompetitive) inhibitory activity for both enzymes; compound 34 showed selective mixed-type inhibitory activity for AChE, and compound 35 was found to have selective uncompetitive activity for AChE.

Targeted degradation of undruggable proteins using a novel heterobifunctional proteomimetic platform.

47 Background: Of the ~20,000 proteins in the human genome, only a subset can be modulated via traditional pharmacological approaches. “Undruggable” targets currently include proteins with pivotal roles in cancer pathogenesis. Our work aims to introduce a new paradigm for engaging these clinically relevant proteins using a modular platform we term the HYbrid DegRAding Copolymers (HYDRACs). HYDRACs multiplex targeting warheads with degradation inducers and have long circulation times plus high cell uptake. Preliminary studies on MYC and RAS, targets of great pharmaceutical interest, highlight the potential of this platform to revolutionize drug design. Methods: HYDRACs containing MYC or RAS targeting peptides randomly copolymerized with degrons were synthesized using ROMP. Fluorescent and biotin-tags were incorporated for use in uptake and pull-down assays. Viability following treatment with MYC-HYDRACs was performed in multiple cell lines with both cellular (MYC-independent lines) and polymer composition controls. Target engagement was assessed via immunoprecipitation and circular dichroism with protein degradation monitored by WB. Unbiased whole proteome analysis and RNAseq were done to confirm observed effects were selectively on-target. In vivo efficacy and biodistribution was assessed in tumor-bearing mice. Results: HYDRACs show high levels of cell uptake and antiproliferative effects at sub-micromolar concentrations in a formulation- and target-dependent manner. Cells treated with MYC-HYDRACs displayed reduced MYC protein levels, which were rescued by proteasome or neddylation inhibition. Swapping out the degron for various validated E3 ligase recruiters maintained MYC degradation, highlighting the “plug-and-play” nature of this approach. On-target activity was confirmed via RNAseq. Biophysical analysis showed strong HYDRAC-protein interactions, orthogonally supported by the presence of target protein following pull-down of biotin-terminated HYDRACs. Target protein degradation only occurred following treatment with an intact HYDRAC compound. Mice bearing MYC-CaP tumors showed delayed tumor growth following treatment with MYC-HYDRACs, with compound accumulation in the tumor for up to 72 h following a single injection. Generalizability was demonstrated against RAS, with RAS-HYDRACs capable of degrading KRAS across multiple different alleles, acting as a pan-KRAS degrader. Conclusions: We present a novel platform technology that addresses the challenges inherent to peptide delivery approaches. HYDRACs have the potential to dramatically alter the drug discovery landscape, allowing for the development of cancer-relevant target modulators for which there are no current viable therapies. We envision the HYDRAC platform as a generalizable approach to designing degraders of proteins of interest, greatly expanding the therapeutic armamentarium for TPD.

Proteomic Analysis Reveals Physiological Activities of Aβ Peptide for Alzheimer's Disease.

With the rapid progress in deciphering the pathogenesis of Alzheimer's disease (AD), it has been widely accepted that the accumulation of misfolded amyloid β (Aβ) in the brain could cause the neurodegeneration in AD. Although much evidence demonstrates the neurotoxicity of Aβ, the role of Aβ in the nervous system are complex. However, more comprehensive studies are needed to understand the physiological effect of Aβ40 monomers in depth. To explore the physiological mechanism of Aβ, we employed mass spectrometry to investigate the altered proteomic events induced by a lower submicromolar concentration of Aβ. Human neuroblastoma SH-SY5Y cells were exposed to five different concentrations of Aβ1-40 monomers and collected at four time points. The proteomic analysis revealed the time-course behavior of proteins involved in biological processes, such as RNA splicing, nuclear transport and protein localization. Further biological studies indicated that Aβ40 monomers may activate PI3K/AKT signaling to regulate p-Tau, Ezrin and MAP2. These three proteins are associated with dendritic morphogenesis, neuronal polarity, synaptogenesis, axon establishment and axon elongation. Moreover, Aβ40 monomers may regulate their physiological forms by inhibiting the expression of BACE1 and APP via activation of the ERK1/2 pathway. A comprehensive exploration of pathological and physiological mechanisms of Aβ is beneficial for exploring novel treatment.

New Iron(III)-Containing Composite of Salinomycinic Acid with Antitumor Activity—Synthesis and Characterization

In this study we demonstrated for the first time synthetic procedures for composites of salinomycin (SalH) and two-line ferrihydrite. The products were characterized by various methods such as elemental analysis, attenuated total reflectance–Fourier-transform spectroscopy (ATR-FTIR), electron paramagnetic resonance spectroscopy (EPR), powder X-ray diffraction analysis (XRD), electrospray-ionization mass spectrometry (ESI-MS), thermogravimetric analysis with differential thermal analysis (DTA) and mass spectrometry (TG-DTA/MS). The EPR spectra of the isolated compounds consisted of signals associated with both isolated Fe3+ ions and magnetically coupled Fe3+ ions. Powder XRD analyses of the isolated products showed two intense and broad peaks at 9° and 15° 2Θ, corresponding to salinomycinic acid. Broad peaks with very low intensity around 35°, assigned to two-line ferrihydrite, were also registered. Based on the experimental results, we concluded that salinomycin sodium reacted with Fe(III) chloride to form composites consisting of two-line ferrihydrite and salinomycinic acid. One of the composites exerted pronounced antitumor activity in the sub-micromolar concentration range against human cervical cancer (HeLa), non-small-cell lung cancer (A549), colon cancer (SW480), and ovarian teratocarcinoma (CH1/PA1) cells.

Derivatization of Abietane Acids by Peptide-like Substituents Leads to Submicromolar Cytotoxicity at NCI-60 Panel.

Natural compounds, including diterpenoids, play a critical role in various biological processes and are recognized as valuable components in cancer treatment. Isocyanides multicomponent reactions (IsMCRs) are one of the effective methods to obtain adducts at the carboxyl group with a peptide-like substituent. In this study, dehydroabietic acid and levopimaric acid diene adducts as the starting scaffolds were modified by the multicomponent Passerini (P-3CR) and Ugi (U-4CR) reactions to afford α-acyloxycarboxamides and α-acylaminocarboxamides. A group of twenty novel diterpene hybrids was subjected to NCI in vitro assessment, and a consistent structure-activity relationship was established. Eleven of the synthesized derivatives inhibited the growth of cancer cells of 4 to 39 cell lines in one dose assay, and the most active were derivatives 3d, 9d, and 10d holding a fragment of 1a,4a-dehydroquinopimaric acid. They were selected for a five-dose analysis and demonstrated a significant antiproliferative effect towards human cancer cell lines. The outstanding cytotoxic activity was observed for the P-3CR product 3d with growth inhibitory at submicromolar and micromolar concentrations (GI50 = 0.42-3 μM) against the most sensitive cell lines. The U-4CR products 9d and 10d showed selective activity against all leukemia cell lines with GI50 in the range of 1-17 µM and selectivity indexes of 5.49 and 4.72, respectively. Matrix COMPARE analysis using the GI50 vector showed a moderate positive correlation of compound 3d with standard anticancer agents that can influence kinase receptors and epidermal growth factor receptors (EGFRs). The ADMET analysis acknowledges the favorable prognosis using compounds as potential anticancer agents. The obtained results indicate that these new hybrids could be useful for the further development of anticancer drugs, and 1a,4a-dehydroquinopimaric acid derivatives could be recommended for in-depth studies and the synthesis of new antitumor analogs on their basis.

A phase 1 study evaluating the safety, pharmacokinetics, and efficacy of fadraciclib, an oral CDK2/9 inhibitor, in patients with advanced solid tumors and lymphoma.

3125 Background: Fadraciclib (formerly CYC065)is a highly selective inhibitor of CDK2 (IC50= 5 nM) and CDK9 (IC50=26 nM) causing mitotic catastrophe and apoptotic death of cancer cells at sub-micromolar concentrations. In a previous Phase 1 study of intravenous fadraciclib monotherapy, a heavily pretreated endometrial cancer patient with CDNK2A, CDKN2B and PRMT5 loss achieved confirmed CR. This is the first study to evaluate oral fadraciclib monotherapy. Methods: A global Phase 1/2, open-label, dose-escalation study in subjects with advanced solid tumors or lymphoma (NCT04983810). The Phase 1 portion is exploring both schedule and dose of oral fadraciclib monotherapy in 28-day cycles to identify MTD and/or RP2D using a standard 3+3 design. Results: As of January 31, 2024, 45 subjects have received fadraciclib at 8 dose levels (DL1-6a: 50-150 mg, bid given 3-5 days per week for 3-4 out of 4 weeks or DL6b: 125 mg, daily for 4/4 weeks). Heavily pretreated patients with breast, colorectal, gynecological cancers, and T cell lymphoma with ECOG PS 0:1 (9:36) were enrolled. Fadraciclib was well tolerated at dose levels 1 to 5 (up to 100 mg bid). Adverse events reported were nausea (n=31), vomiting (n=23), thrombocytopenia (n=6). Grade 3 DLT of hyperglycemia (1/2 DL6 and 2/6 DL6a) and nausea (1/2 DL6) was observed at doses above 125mg bid M-F, 4/4 weeks. Hyperglycemia was controlled after dose interruption and blood glucose levels returned to normal range after treatment. Preliminary PK analysis suggests dose dependent exposure. At DL5 and above, plasma concentration was above target concentration from CDK2 and CDK9 target engagement studies. Preliminary PD analysis of treated patient samples showed suppression of expression in multiple target genes, including MYC, MCL1, CCNE/cyclin E, CDKN2A, CDKN2B, and PRMT5. Preliminary efficacy data are available for 31 evaluable patients. Two PRs after the first cycle were observed in one patient with CTCL and one with angioimmunoblastic T-Cell Lymphoma (AITL). Twenty patients showed SD. Tumor size reduction was observed in 10 patients. A squamous NSCLC patient harboring a CDK2NB loss of function mutation and prior chemo- and immunotherapy achieved reduction of 22% in the sum of all target lesions after 1 cycle of treatment. Conclusions: Based on data from the CYC065-101 study, fadraciclib appears well tolerated from DL1 to 5. The MTD for the bid dosing schedule is 100mg bid, M-F, 4/4 weeks. The trial is ongoing with evaluation of a daily dosing schedule. Clinical trial information: NCT04983810 .

Structure-Based Rational Design of Constrained Peptides as TIM-3 Inhibitors.

Blocking the immunosuppressive function of T-cell immunoglobulin mucin-3 (TIM-3) is an established therapeutic strategy to maximize the efficacy of immune checkpoint inhibitors for cancer immunotherapy. Currently, effective inhibition of TIM-3 interactions relies on monoclonal antibodies (mAbs), which come with drawbacks such as immunogenicity risk, limited tumor penetration, and high manufacturing costs. Guided by the X-ray cocrystal structures of TIM-3 with mAbs, we report an in silico structure-based rational design of constrained peptides as potent TIM-3 inhibitors. The top cyclic peptide from our study (P2) binds TIM-3 with a K D value of 166.3 ± 12.1 nM as determined by surface plasmon resonance (SPR) screening. Remarkably, P2 efficiently inhibits key TIM-3 interactions with natural TIM-3 ligands at submicromolar concentrations in a panel of cell-free and cell-based assays. The capacity of P2 to reverse immunosuppression in T-cell/cancer cell cocultures, coupled with favorable in vitro pharmacokinetic properties, highlights the potential of P2 for further evaluation in preclinical models of immuno-oncology.

Novel hybrid benzoisothiazole-1,2,3-triazole-4-carboxamides with sub-micromolar toxicity towards human breast carcinoma cells and high affinity to DNA

A pharmacophore hybridization strategy to combine biologically active scaffolds was implemented for design and synthesis of benzoisothiazole-1,2,3-triazole-4-carboxamide conjugates via piperazine linker. In vitro screening led to identification of the compounds 5h and 5j, with para-chlorine- or fluorine- substitution on the phenyl ring, and isopropyl or cyclopropyl substituents in position 5 of 1,2,3-triazole cycle as potent anticancer agents. These compounds demonstrated toxicity towards human breast adenocarcinoma cells of the MCF-7 line in sub-micromolar concentrations with IC50 = 0.76 ± 0.04 μM and 0.90 ± 0.02 μM, respectively, while showing no toxicity in normal cells (IC50 > 100 μM). Colony formation and proliferation of carcinoma MCF-7 cells were effectively inhibited by the derivatives 5h and 5j. These derivatives caused DNA fragmentation of the MCF-7 cells. We suggest that the DNA-damaging action of the compounds 5h and 5j may be related to their high capability of intercalating into DNA molecule that is comparable to such ability of known anticancer agent – the doxorubicin. High affinity of the compounds 5h and 5j for the DNA was confirmed using computational molecular docking in silico.