RESUMO
Hepatitis C virus (HCV) is a notorious member of the Flaviviridae family of enveloped, positive-strand RNA viruses. Non-structural protein 5A (NS5A) plays a key role in HCV replication and assembly. NS5A is a multi-domain protein which includes an N-terminal amphipathic membrane anchoring alpha helix, a highly structured domain-1, and two intrinsically disordered domains 2-3. The highly structured domain-1 contains a zinc finger (Zf)-site, and binding of zinc stabilizes the overall structure, while ejection of this zinc from the Zf-site destabilizes the overall structure. Therefore, NS5A is an attractive target for anti-HCV therapy by disulfiram, through ejection of zinc from the Zf-site. However, the zinc ejection mechanism is poorly understood. To disclose this mechanism based on three different states, A-state (NS5A protein), B-state (NS5A + Zn), and C-state (NS5A + Zn + disulfiram), we have performed molecular dynamics (MD) simulation in tandem with DFT calculations in the current study. The MD results indicate that disulfiram triggers Zn ejection from the Zf-site predominantly through altering the overall conformation ensemble. On the other hand, the DFT assessment demonstrates that the Zn adopts a tetrahedral configuration at the Zf-site with four Cys residues, which indicates a stable protein structure morphology. Disulfiram binding induces major conformational changes at the Zf-site, introduces new interactions of Cys39 with disulfiram, and further weakens the interaction of this residue with Zn, causing ejection of zinc from the Zf-site. The proposed mechanism elucidates the therapeutic potential of disulfiram and offers theoretical guidance for the advancement of drug candidates.
Assuntos
Antivirais/farmacologia , Dissulfiram/farmacologia , Hepacivirus/efeitos dos fármacos , Proteínas não Estruturais Virais/antagonistas & inibidores , Zinco/farmacologia , Antivirais/síntese química , Antivirais/química , Teoria da Densidade Funcional , Dissulfiram/química , Humanos , Simulação de Dinâmica Molecular , Zinco/químicaRESUMO
Development of multitargeted ligands have demonstrated remarkable efficiency as potential therapeutics for Alzheimer's disease (AD). Herein, we reported a new series of deoxyvasicinone analogues as dual inhibitor of acetylcholinesterase (AChE) and tau aggregation that function as multitargeted ligands for AD. All the multitargeted ligands 11(a-j) and 15(a-g) were designed, synthesized, and validated by 1HNMR, 13CNMR and mass spectrometry. All the synthesized compounds 11(a-j) and 15(a-g) were screened for their ability to inhibit AChE, BACE1, amyloid fibrillation, α-syn aggregation, and tau aggregation. All the screened compounds possessed weak inhibition of BACE-1, Aß42 and α-syn aggregation. However, several compounds were identified as potential hits in the AChE inhibitory screening assay and cellular tau aggregation screening. Among all compounds, 11f remarkably inhibited AChE activity and cellular tau oligomerization at single-dose screening (10 µM). Moreover, 11f displayed a half-maximal inhibitory concentration (IC50) value of 0.91 ± 0.05 µM and half-maximal effective concentration (EC50) value of 3.83 ± 0.51 µM for the inhibition of AChE and cellular tau oligomerization, respectively. In addition, the neuroprotective effect of 11f was determined in tau-expressing SH-SY5Y cells incubated with Aß oligomers. These findings highlighted the potential of 11f to function as a multifunctional ligand for the development of promising anti-AD drugs.
Assuntos
Acetilcolinesterase/metabolismo , Doença de Alzheimer/tratamento farmacológico , Inibidores da Colinesterase/farmacologia , Fármacos Neuroprotetores/farmacologia , Quinazolinas/farmacologia , Proteínas tau/antagonistas & inibidores , Doença de Alzheimer/metabolismo , Inibidores da Colinesterase/síntese química , Inibidores da Colinesterase/química , Relação Dose-Resposta a Droga , Desenho de Fármacos , Humanos , Estrutura Molecular , Fármacos Neuroprotetores/síntese química , Fármacos Neuroprotetores/química , Agregados Proteicos/efeitos dos fármacos , Quinazolinas/síntese química , Quinazolinas/química , Relação Estrutura-Atividade , Proteínas tau/metabolismoRESUMO
V-domain Ig suppressor of T-cell activation (VISTA) is an immune checkpoint that affects the ability of T-cells to attack tumors. A FRET-based high throughput screening identified NSC622608 as the first small-molecule ligand for VISTA. Investigation of the interaction of NSC622608 with VISTA using STD NMR and molecular modeling enabled the identification of a potential binding site in VISTA for NSC622608. Screening NSC622608 against a library of single-point VISTA mutants revealed the key residues in VISTA interacting with NSC622608. Further structural optimization resulted in a lead with submicromolar VISTA binding affinity. The lead compound blocked VISTA signaling in vitro, enhanced T-cell proliferation, and restored T-cell activation in the presence of VISTA-expressing cancer cell lines. This work would enable future development of small molecules targeting VISTA as immunomodulators and imaging probes.
Assuntos
Antígenos B7/antagonistas & inibidores , Descoberta de Drogas , Inibidores de Checkpoint Imunológico/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Antígenos B7/imunologia , Linhagem Celular , Humanos , Inibidores de Checkpoint Imunológico/química , Ligantes , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/química , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
6â³-[18 F]fluoromaltotriose is a positron emission tomography tracer that can differentiate between bacterial infection and inflammation in vivo. Bacteria-specific uptake of 6â³-[18 F]fluoromaltotriose is attributed to the targeting of maltodextrin transporter in bacteria that is absent in mammalian cells. Herein, we report a new synthesis of 6â³-[18 F]fluoromaltotriose as a key step for its clinical translation. In comparison with the previously reported synthesis, the new synthesis features unambiguous assignment of the fluorine-18 position on the maltotriose unit. The new method utilizes direct fluorination of 2â³,3â³,4â³-tri-O-acetyl-6â³-O-trifyl-α-D-glucopyranosyl-(1-4)-O-2',3',6'-tri-O-acetyl-α-D-glucopyranosyl-(1-4)-1,2,3,6-tetra-O-acetyl-D-glucopyranose followed by basic hydrolysis. Radiolabeling of the new maltotriose triflate precursor proceeds using a single HPLC purification step, which results in shorter reaction time in comparison with the previously reported synthesis. Successful synthesis of 6â³-[18 F]fluoromaltotriose has been achieved in 3.5 ± 0.3% radiochemical yield (decay corrected, n = 7) and radiochemical purity above 95%. The efficient radiosynthesis of 6â³-[18 F]fluoromaltotriose would be critical in advancing this positron emission tomography tracer into clinical trials for imaging bacterial infections.
Assuntos
Infecções Bacterianas/congênito , Infecções Bacterianas/diagnóstico por imagem , Radioisótopos de Flúor , Tomografia por Emissão de Pósitrons , Trissacarídeos/química , Trissacarídeos/síntese química , Animais , Técnicas de Química Sintética , HumanosRESUMO
Switchable luminescent bioprobes whose emission can be turned on as a function of specific enzymatic activity are emerging as important tools in chemical biology. We report a promising platform for the development of label-free and continuous enzymatic assays in high-throughput mode based on the reversible solvent-induced self-assembly of a neutral dinuclear Pt(II) complex. To demonstrate the utility of this strategy, the switchable luminescence of a dinuclear Pt(II) complex was utilized in developing an experimentally simple, fast (10 min), low cost, and label-free turn-on luminescence assay for the endonuclease enzyme DNAse I. The complex displays a near-IR (NIR) aggregation-induced emission at 785 nm in aqueous solution that is completely quenched upon binding to G-quadruplex DNA from the human c-myc oncogene. Luminescence is restored upon DNA degradation elicited by exposure to DNAse I. Correlation between near-IR luminescence intensity and DNAse I concentration in human serum samples allows for fast and label-free detection of DNAse I down to 0.002 U/mL. The Pt(II) complex/DNA assembly is also effective for identification of DNAse I inhibitors, and assays can be performed in multiwell plates compatible with high-throughput screening. The combination of sensitivity, speed, convenience, and cost render this method superior to all other reported luminescence-based DNAse I assays. The versatile response of the Pt(II) complex to DNA structures promises broad potential applications in developing real-time and label-free assays for other nucleases as well as enzymes that regulate DNA topology.
Assuntos
Ensaios Enzimáticos/métodos , Compostos Organoplatínicos/química , Platina/química , Quadruplex G , Luminescência , Estrutura MolecularRESUMO
Probes for monitoring aggregation of amyloid beta (Aß) peptides are crucial to advance understanding of the molecular pathogenesis of Alzheimer's Disease (AD). Here, we report luminescent tricarbonyl rhenium complexes of tetraarylethylene (TAE) ligands featuring bis(benzothiazole) chelating groups in combination with (oligo)thiophene units that have been designed for monitoring amyloid fibrillation. Variation in the number of thiophenes influenced the photophysical properties of these complexes, as well as their binding affinities toward Aß42 fibrils. All complexes displayed submicromolar Kd's for binding Aß42 aggregates accompanied by up to 34-fold enhanced luminescence and red-shifted emission wavelengths. The high binding affinities and desirable photophysical properties of these complexes render them potential alternatives to established fluorescent Aß probes such as thioflavin T. Additionally, the general and modular design approach implemented in this study should facilitate development of second-generation TAE-based diagnostic tools for studying protein aggregation in AD and other neurological diseases.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Benzotiazóis/química , Fragmentos de Peptídeos/metabolismo , Rênio/química , Tiazóis/química , Tiazóis/metabolismo , Peptídeos beta-Amiloides/química , Quelantes/química , Humanos , Ligantes , Luminescência , Fragmentos de Peptídeos/químicaRESUMO
A new series of structurally rigid donepezil analogues was designed, synthesized and evaluated as potential multi-target-directed ligands (MTDLs) against neurodegenerative diseases. The investigated compounds 10-13 displayed dual AChE and BACE-1 inhibitory activities in comparison to donepezil, the FDA-approved drug. The hybrid compound 13 bearing 2-aminoquinoline scaffold exhibited potent AChE inhibition (IC50 value of 14.7â¯nM) and BACE-1 inhibition (IC50 value of 13.1â¯nM). Molecular modeling studies were employed to reveal potential dual binding mode of 13 to AChE and BACE-1. The effect of the investigated compounds on the viability of SH-SY5Y neuroblastoma cells and their ability to cross the blood-brain barrier (BBB) in PAMPA-BBB assay were further studied.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Inibidores da Colinesterase/farmacologia , Donepezila/farmacologia , Desenho de Fármacos , Acetilcolinesterase/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Barreira Hematoencefálica/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Inibidores da Colinesterase/síntese química , Inibidores da Colinesterase/química , Cristalografia por Raios X , Donepezila/síntese química , Donepezila/química , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Deficiencies in DNA mismatch repair (MMR) machinery result in greater incidence of DNA base pair mismatches in many types of cancer cells relative to normal cells. Consequently, luminescent probes capable of signaling the presence of mismatched DNA hold promise as potential cancer diagnostic and therapeutic tools. In this study, a series of cyclometalated platinum(II) complexes with sterically expansive tetraarylethylene ligands were synthesized and examined for selective detection of mismatched DNA. Increased steric bulk of the tetraarylethylene ligands in these complexes was observed to correlate with greater preferential luminescence enhancement in the presence of hairpin DNA oligonucleotides containing a mismatched site compared to well-matched oligonucleotides, with the most effective complex displaying â¼14-fold higher emission upon binding CC mismatched oligonucleotides compared to well-matched oligonucleotides. The results indicate binding to mismatched sites in DNA oligonucleotides occurs through metalloinsertion, and the luminescence response increases as a function of thermodynamic destabilization of the mismatch. Luminescence quenching experiments with Cu(phen)22+ and NaI further indicate mismatch binding from the minor groove, consistent with metalloinsertion. Binding to CC mismatched oligonucleotides was also investigated by isothermal titration calorimetry and UV-melting studies. These results demonstrate the efficacy of tetraarylethylene-based platinum(II) complexes for detection of mismatched DNA and establish a new molecular platform for development of organometallic DNA binding agents.
Assuntos
Dano ao DNA , Etilenos/química , Compostos de Platina/química , Reparo de Erro de Pareamento de DNA , Ligantes , Modelos Moleculares , Estrutura MolecularRESUMO
Multi-target-directed ligands (MTDLs) centered on ß-secretase 1 (BACE-1) inhibition are emerging as innovative therapeutics in addressing the complexity of neurodegenerative diseases. A new series of donepezil analogues was designed, synthesized and evaluated as MTDLs against neurodegenerative diseases. Profiling of donepezil, a potent acetylcholinesterase (hAChE) inhibitor, into BACE-1 inhibition was achieved through introduction of backbone amide linkers to the designed compounds which are capable of hydrogen-bonding with BACE-1 catalytic site. In vitro assays and molecular modeling studies revealed the dual mode of action of compounds 4-6 against hAChE and BACE-1. Notably, compound 4 displayed potent hAChE inhibition (IC50 value of 4.11â¯nM) and BACE-1 inhibition (IC50 value of 18.3â¯nM) in comparison to donepezil (IC50 values of 6.21 and 194â¯nM against hAChE and BACE-1, respectively). Moreover, 4 revealed potential metal chelating property, low toxicity on SH-SY5Y neuroblastoma cells and ability to cross the blood-brain barrier (BBB) in PAMPA-BBB assay which renders 4 a potential lead for further optimization of novel small ligands for the treatment of Alzheimer's disease.
Assuntos
Acetilcolinesterase/química , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Inibidores da Colinesterase/síntese química , Donepezila/química , Desenho de Fármacos , Acetilcolinesterase/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Sítios de Ligação , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Domínio Catalítico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Inibidores da Colinesterase/farmacologia , Inibidores da Colinesterase/uso terapêutico , Donepezila/farmacologia , Donepezila/uso terapêutico , Humanos , Metais/química , Simulação de Dinâmica Molecular , Permeabilidade/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
A new series of fluorene derivatives was designed and synthesized as novel retinoic acid receptor-related orphan receptor gamma t (RORγt) inverse agonists utilizing a molecular hybridization approach. The new compounds 10 - 15 were evaluated for their RORγt activity using biochemical FRET and cellular reporter gene assays. Moreover, the inhibitory activity of the fluorene derivatives 10 - 15 in mouse Th17 cell differentiation assay was assessed. The hybrid compound 15 that combines both fluorene and arylsulfone moieties displayed promising RORγt activity with IC50 values of 68.6 and 99.5 nm in FRET and cellular assays, respectively. In addition, molecular modeling studies were employed to investigate potential binding mode of 15 to RORγt. These results render 15 a potential lead compound for development of therapeutics for Th17-driven autoimmune diseases.
Assuntos
Agonismo Inverso de Drogas , Fluorenos/química , Fluorenos/farmacologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/agonistas , Animais , Barreira Hematoencefálica , Fluorenos/farmacocinética , Transferência Ressonante de Energia de Fluorescência , Genes Reporter , Camundongos , Estrutura Molecular , Relação Estrutura-Atividade , Células Th17Assuntos
Valva Aórtica/diagnóstico por imagem , Endocardite Bacteriana/diagnóstico por imagem , Radioisótopos de Flúor , Imagem Molecular , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Compostos Radiofarmacêuticos , Trissacarídeos , Animais , Valva Aórtica/microbiologia , Proteínas de Bactérias/metabolismo , Modelos Animais de Doenças , Endocardite Bacteriana/microbiologia , Humanos , Proteínas de Membrana Transportadoras , Camundongos , Polissacarídeos/metabolismo , Valor Preditivo dos Testes , Staphylococcus aureus/metabolismoRESUMO
Metastatic melanoma is the most aggressive and lethal form of skin cancer. Emerging evidence suggests that differences in melanoma metabolism relative to nonmalignant cells represent potential targets for improved therapy for melanoma. Specifically, melanoma cells exhibit increased mitochondrial electron transport chain (ETC) activity and concomitant hyperpolarized mitochondrial membrane potential relative to nonmalignant cells. We have synthesized several new fluorescent lipophilic vinylpyridinium cations built from tetraarylethylene scaffolds that target mitochondria via attraction to the hyperpolarized mitochondrial membrane potential. Mitochondria-specific accumulation in melanoma cells relative to normal human fibroblasts was demonstrated using confocal fluorescence microscopy and resulted in the disruption of oxidative metabolism leading to melanoma specific cell death in vitro. Thus, the pyridinium tetraarylethylene platform represents a promising new mitochondrial-targeted delivery vehicle with potential imaging and therapeutic properties.
Assuntos
Antineoplásicos/farmacologia , Melanoma/diagnóstico por imagem , Melanoma/tratamento farmacológico , Compostos de Piridínio/química , Compostos de Piridínio/farmacologia , Trifosfato de Adenosina/metabolismo , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Química Sintética , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Fibroblastos/efeitos dos fármacos , Humanos , Melanoma/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Microscopia Confocal , Microscopia de Fluorescência , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Compostos de Piridínio/síntese químicaRESUMO
EGFR tyrosine kinase has been reported mainly in 40-80% of non-small lung cancers, in addition to colon and breast cancers. In this study, we illustrate the synthesis of a highly potent antitumor agent. The synthesized compound 4 was screened at NCI, USA, for antitumor activity against non-small lung cancer, colon cancer and breast cancer cell lines. Results indicated that this compound is more potent antitumor agent compared to erlotinib against all tested cell lines except breast cancer (MDA-MB-468) cell line. In addition, it was tested initially at a single dose concentration of 100 µM over 11 different kinases. At this concentration, 94.45% inhibition of the enzymatic activity of EGFR kinase was observed, while the inhibition in activity was below 55% in all other kinases. Compound 4 was further tested in a 10-dose IC50 mode and showed IC50 value of 0.239 µM for EGFR kinase. In vivo acute toxicity of this compound was also tested.
Assuntos
Acetamidas/química , Antineoplásicos/química , Benzotiazóis/química , Receptores ErbB/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Acetamidas/síntese química , Acetamidas/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Benzotiazóis/síntese química , Benzotiazóis/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Desenho de Fármacos , Receptores ErbB/química , Feminino , Humanos , Dose Letal Mediana , Masculino , Camundongos , Simulação de Acoplamento Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Relação Estrutura-AtividadeRESUMO
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.
RESUMO
Retinoic acid receptor-related orphan receptor γt (RORγt) is a nuclear receptor found in various tissues that plays a crucial role in the differentiation and proliferation of T helper 17 (Th17) cells, as well as in their generation of the pro-inflammatory cytokine IL-17A. RORγt represents a promising therapeutic target for autoimmune diseases, metabolic disorders, and multiple tumors. Despite extensive research efforts focused on the development of small molecule RORγt modulators, no drug candidates have advanced to phase 3 clinical trials owing to a lack of efficacy or safety margin. This outcome highlights the unmet need to optimize small molecule drug candidates targeting RORγt to develop effective therapies for autoimmune and inflammatory diseases. In this study, we synthesized and evaluated 3-oxo-lithocholic acid amidates as a new class of RORγt modulators. Our evaluation entailed biophysical screening, cellular screening in different platforms, molecular docking, and in vitro pharmacokinetic profiling. The top compound from our study (3-oxo-lithocholic acid amidate, A2) binds to RORγt at an equilibrium dissociation constant (KD) of 16.5 ± 1.34 nM based on microscale thermophoresis (MST). Assessment of the efficacy of A2 in the cellular RORγt reporter luciferase assay revealed a half-maximal inhibitory concentration (IC50) value of 225 ± 10.4 nM. Unlike 3-oxo-lithocholic acid, A2 demonstrated the ability to reduce the IL-17A mRNA expression levels in EL4 cells with RORγt expression using quantitative reverse transcriptase PCR (RT-PCR). Validation of the desirable physicochemical properties and stability of A2 sets the stage for the preclinical evaluation of this new class of RORγt modulators in animal models of autoimmune diseases.
RESUMO
Inducible T cell co-stimulator (ICOS) is a positive immune checkpoint receptor expressed on the surface of activated T cells, which could promote cell function after being stimulated with ICOS ligand (ICOS-L). Although clinical benefits have been reported in the ICOS modulation-based treatment for cancer and autoimmune disease, current modulators are restricted in biologics, whereas ICOS-targeted small molecules are lacking. To fill this gap, we performed an affinity selection mass spectrometry (ASMS) screening for ICOS binding using a library of 15,600 molecules. To the best of our knowledge, this is the first study that utilizes ASMS screening to discover small molecules targeting immune checkpoints. Compound 9 with a promising ICOS/ICOS-L inhibitory profile (IC50 = 29.38 ± 3.41 µM) was selected as the template for the modification. Following preliminary structure-activity relationship (SAR) study and molecular dynamic (MD) simulation revealed the critical role of the ortho-hydroxy group on compound 9 in the ICOS binding, as it could stabilize the interaction via the hydrogen bond formation with residuals on the glycan, and the depletion could lead to an activity lost. This work validates a promising inhibitor for the ICOS/ICOS-L interaction, and we anticipate future modifications could provide more potent modulators for this interaction.
RESUMO
The development of multitargeted therapeutics has evolved as a promising strategy to identify efficient therapeutics for neurological disorders. We report herein new quinolinone hybrids as dual inhibitors of acetylcholinesterase (AChE) and Aß aggregation that function as multitargeted ligands for Alzheimer's disease. The quinoline hybrids (AM1-AM16) were screened for their ability to inhibit AChE, BACE1, amyloid fibrillation, α-syn aggregation, and tau aggregation. Among the tested compounds, AM5 and AM10 inhibited AChE activity by more than 80% at single-dose screening and possessed a remarkable ability to inhibit the fibrillation of Aß42 oligomers at 10 µM. In addition, dose-dependent screening of AM5 and AM10 was performed, giving half-maximal AChE inhibitory concentration (IC50) values of 1.29 ± 0.13 and 1.72 ± 0.18 µM, respectively. In addition, AM5 and AM10 demonstrated concentration-dependent inhibitory profiles for the aggregation of Aß42 oligomers with estimated IC50 values of 4.93 ± 0.8 and 1.42 ± 0.3 µM, respectively. Moreover, the neuroprotective properties of the lead compounds AM5 and AM10 were determined in SH-SY5Y cells incubated with Aß oligomers. This work would enable future research efforts aiming at the structural optimization of AM5 and AM10 to develop potent dual inhibitors of AChE and amyloid aggregation. Furthermore, the in vivo assay confirmed the antioxidant activity of compounds AM5 and AM10 through increasing GSH, CAT, and SOD activities that are responsible for scavenging the ROS and restoring its normal level. Blood investigation illustrated the protective activity of the two compounds against lead-induced neurotoxicity through retaining hematological and liver enzymes near normal levels. Finally, immunohistochemistry investigation revealed the inhibitory activity of ß-amyloid (Aß) aggregation.
Assuntos
Doença de Alzheimer , Neuroblastoma , Quinolonas , Humanos , Doença de Alzheimer/tratamento farmacológico , Acetilcolinesterase/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Inibidores da Colinesterase/farmacologia , Quinolonas/uso terapêutico , Ácido Aspártico Endopeptidases/metabolismo , Neuroblastoma/tratamento farmacológico , Peptídeos beta-Amiloides/química , Relação Estrutura-AtividadeRESUMO
The interaction of the inducible co-stimulator (ICOS) with its ligand (ICOSL) plays key roles in T-cell differentiation and activation of T-cell to B-cell functions. The ICOS/ICOSL pathway is a validated target for T-cell lymphomas induced by the proliferation of T-follicular helper (Tfh) cells. Moreover, the inhibition of ICOS/ICOSL interaction can decrease the enhancement of immunosuppressive regulatory T cells (Tregs) in both hematologic malignancies and solid tumors. However, targeting ICOS/ICOSL interaction is currently restricted to monoclonal antibodies (mAbs) and there are no small molecules in existence that can block ICOS/ICOSL. To fill this gap, we report herein the first time-resolved fluorescence resonance energy transfer (TR-FRET) assay to evaluate the ability of small molecules to inhibit ICOS/ICOSL interaction. Implementation of the developed TR-FRET assay in high-throughput screening (HTS) of a focused chemical library resulted in the identification of AG-120 as a first-in-class inhibitor of ICOS/ICOSL interaction. We further employed docking studies and molecular dynamics (MD) simulations to identify the plausible mechanism of blocking ICOS/ICOSL complex formation by AG-120. Using the structure-activity relationship (SAR) by catalog approach, we identified AG-120-X with an IC50 value of 4.68 ± 0.47 µM in the ICOS/ICOSL TR-FRET assay. Remarkably, AG-120-X revealed a dose-dependent ability to block ICOS/ICOSL interaction in a bioluminescent cellular assay based on co-culturing Jurkat T cells expressing ICOS and CHO-K1 cells expressing ICOSL. This work will pave the way for future drug discovery efforts aiming at the development of small molecule inhibitors of ICOS/ICOSL interaction as potential therapeutics for cancer as well as other diseases.
RESUMO
Lymphocyte activation gene 3 (LAG-3) is a negative immune checkpoint and a key regulator of immune homeostasis with multiple biological activities related to T-cell functions. Fibrinogen-like protein 1 (FGL1) is a major LAG-3 functional ligand that is upregulated in various human cancers. LAG-3 positive T cells bind FGL1 expressed by cancer cells, which inhibits T-cell activation and cytokine secretion via indirect blocking of T cell receptor (TCR) signaling. High expression of LAG-3 and FGL1 in patients with solid tumors is associated with drug resistance and decreased survival in response to FDA-approved immune checkpoint inhibitors. Therefore, targeting the LAG-3/FGL1 pathway represents a promising therapeutic strategy to maximize the number of patients benefiting from checkpoint blockade therapy. However, there are no small molecules in existence that target LAG-3/FGL1 interaction. Herein, we report a time-resolved fluorescence resonance energy transfer (TR-FRET) assay to evaluate the ability of small molecules to inhibit LAG-3/FGL1 interaction. We further demonstrate the implementation of the developed assay in screening chemical libraries of small molecules from the NCI Diversity Set VII, FDA-approved drugs, and a focused library of NF-κB modulators. This work will pave the way for drug discovery efforts focused on therapeutic targeting of LAG-3/FGL1 interaction using small molecules.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Ensaios de Triagem em Larga Escala , Humanos , Descoberta de Drogas , Bibliotecas de Moléculas Pequenas/farmacologia , Ativação Linfocitária , FibrinogênioRESUMO
Lymphocyte activation gene 3 (LAG-3) is a negative immune checkpoint that plays a key role in downregulating the immune response to cancer. Inhibition of LAG-3 interactions allows T cells to regain cytotoxic activity and reduce the immunosuppressive function of regulating T cells. We utilized a combination approach of focused screening and "SAR by catalog" to identify small molecules that function as dual inhibitors of the interactions of LAG-3 with major histocompatibility complex (MHC) class II and fibrinogen-like protein 1 (FGL1). Our top hit compound inhibited both LAG-3/MHCII and LAG-3/FGL1 interactions in biochemical binding assays with IC50 values of 4.21 ± 0.84 and 6.52 ± 0.47 µM, respectively. Moreover, we have demonstrated the ability of our top hit compound to block LAG-3 interactions in cell-based assays. This work will pave the way for future drug discovery efforts aiming at the development of LAG-3-based small molecules for cancer immunotherapy.