RESUMEN
Single-chain fragment variable (scFv) domains play an important role in antibody-based therapeutic modalities, such as bispecifics, multispecifics and chimeric antigen receptor T cells or natural killer cells. However, scFv domains exhibit lower stability and increased risk of aggregation due to transient dissociation ("breathing") and inter-molecular reassociation of the two domains (VL and VH). We designed a novel strategy, referred to as stapling, that introduces two disulfide bonds between the scFv linker and the two variable domains to minimize scFv breathing. We named the resulting molecules stapled scFv (spFv). Stapling increased thermal stability (Tm) by an average of 10°C. In multiple scFv/spFv multispecifics, the spFv molecules display significantly improved stability, minimal aggregation and superior product quality. These spFv multispecifics retain binding affinity and functionality. Our stapling design was compatible with all antibody variable regions we evaluated and may be widely applicable to stabilize scFv molecules for designing biotherapeutics with superior biophysical properties.
Asunto(s)
Anticuerpos , Región Variable de Inmunoglobulina , Región Variable de Inmunoglobulina/química , Fragmentos de InmunoglobulinasRESUMEN
Compounds that bind in the DNA minor groove have provided critical information on DNA molecular recognition, have found extensive uses in biotechnology, and are providing clinically useful drugs against diseases as diverse as cancer and sleeping sickness. This review focuses on the development of clinically useful heterocyclic diamidine minor groove binders. These compounds show that the classical model for minor groove binding in AT DNA sequences must be expanded in several ways: compounds with nonstandard shapes can bind strongly to the groove, water can be directly incorporated into the minor groove complex in an interfacial interaction, compounds can be designed to recognize GC and mixed AT/GC base pair sequences, and stacked dimers can form to recognize specific sequences. © 2023 Wiley Periodicals LLC.
Asunto(s)
ADN , Resonancia por Plasmón de Superficie , Sitios de Unión , ADN/química , ADN/metabolismo , Emparejamiento Base , PentamidinaRESUMEN
PURPOSE: Preclinical characterization of cetrelimab (JNJ-63723283), a fully humanized immunoglobulin G4 kappa monoclonal antibody targeting programmed cell death protein-1 (PD-1), in human cancer models. METHODS: Cetrelimab was generated by phage panning against human and cynomolgus monkey (cyno) PD-1 extracellular domains (ECDs) and affinity maturation. Binding to primate and rodent PD-1 ECDs, transfected and endogenous cell-surface PD-1, and inhibition of ligand binding were measured. In vitro activity was evaluated using cytomegalovirus recall, mixed lymphocyte reaction, staphylococcal enterotoxin B stimulation, and Jurkat-PD-1 nuclear factor of activated T cell reporter assays. In vivo activity was assessed using human PD-1 knock-in mice implanted with MC38 tumors and a lung patient-derived xenograft (PDX) model (LG1306) using CD34 cord-blood-humanized NSG mice. Pharmacodynamics, toxicokinetics, and safety were assessed in cynos following single and/or repeat intravenous dosing. RESULTS: Cetrelimab showed high affinity binding to human (1.72 nM) and cyno (0.90 nM) PD-1 and blocked binding of programmed death-ligand 1 (PD-L1; inhibitory concentration [IC] 111.7 ng/mL) and PD-L2 (IC 138.6 ng/mL). Cetrelimab dose-dependently increased T cell-mediated cytokine production and stimulated cytokine expression. Cetrelimab 10 mg/kg reduced mean MC38 tumor volume in PD-1 knock-in mice at Day 21 (P < 0.0001) versus control. In a PDX lung model, 10 mg/kg cetrelimab (every 5 days for six cycles) increased frequency of peripheral T cells and reduced (P < 0.05) mean tumor volume versus control. Activity was consistent with that of established PD-1 inhibitors. Cetrelimab dosing was well tolerated in cynos and mean drug exposure increase was dose-dependent. CONCLUSION: Cetrelimab potently inhibits PD-1 in vitro and in vivo, supporting its clinical evaluation.
Asunto(s)
Anticuerpos Monoclonales Humanizados , Anticuerpos Monoclonales , Inhibidores de Puntos de Control Inmunológico , Neoplasias , Receptor de Muerte Celular Programada 1 , Animales , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados/uso terapéutico , Citocinas/metabolismo , Modelos Animales de Enfermedad , Humanos , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Macaca fascicularis , Ratones , Neoplasias/tratamiento farmacológico , Receptor de Muerte Celular Programada 1/antagonistas & inhibidoresRESUMEN
B-cell maturation antigen (BCMA), a member of the tumor necrosis factor family of receptors, is predominantly expressed on the surface of terminally differentiated B cells. BCMA is highly expressed on plasmablasts and plasma cells from multiple myeloma (MM) patient samples. We developed a BCMAxCD3 bispecific antibody (teclistamab [JNJ-64007957]) to recruit and activate T cells to kill BCMA-expressing MM cells. Teclistamab induced cytotoxicity of BCMA+ MM cell lines in vitro (H929 cells, 50% effective concentration [EC50] = 0.15 nM; MM.1R cells, EC50 = 0.06 nM; RPMI 8226 cells, EC50 = 0.45 nM) with concomitant T-cell activation (H929 cells, EC50 = 0.21 nM; MM.1R cells, EC50 = 0.1 nM; RPMI 8226 cells, EC50 = 0.28 nM) and cytokine release. This activity was further increased in the presence of a γ-secretase inhibitor (LY-411575). Teclistamab also depleted BCMA+ cells in bone marrow samples from MM patients in an ex vivo assay with an average EC50 value of 1.7 nM. Under more physiological conditions using healthy human whole blood, teclistamab mediated dose-dependent lysis of H929 cells and activation of T cells. Antitumor activity of teclistamab was also observed in 2 BCMA+ MM murine xenograft models inoculated with human T cells (tumor inhibition with H929 model and tumor regression with the RPMI 8226 model) compared with vehicle and antibody controls. The specific and potent activity of teclistamab against BCMA-expressing cells from MM cell lines, patient samples, and MM xenograft models warrant further evaluation of this bispecific antibody for the treatment of MM. Phase 1 clinical trials (monotherapy, #NCT03145181; combination therapy, #NCT04108195) are ongoing for patients with relapsed/refractory MM.
Asunto(s)
Anticuerpos Biespecíficos , Mieloma Múltiple , Animales , Anticuerpos Biespecíficos/farmacología , Antígeno de Maduración de Linfocitos B , Humanos , Activación de Linfocitos , Ratones , Mieloma Múltiple/tratamiento farmacológico , Linfocitos TRESUMEN
BACKGROUND: As a consequence of the discovery of an extracellular component responsible for the progression of tau pathology, tau immunotherapy is being extensively explored in both preclinical and clinical studies as a disease modifying strategy for the treatment of Alzheimer's disease. OBJECTIVE: Describe the characteristics of the anti-phospho (T212/T217) tau selective antibody PT3 and its humanized variant hPT3. METHODS: By performing different immunization campaigns, a large collection of antibodies has been generated and prioritized. In depth, in vitro characterization using surface plasmon resonance, phospho-epitope mapping, and X-ray crystallography experiments were performed. Further characterization involved immunohistochemical staining on mouse- and human postmortem tissue and neutralization of tau seeding by immunodepletion assays. RESULTS AND CONCLUSION: Various in vitro experiments demonstrated a high intrinsic affinity for PT3 and hPT3 for AD brain-derived paired helical filaments but also to non-aggregated phospho (T212/T217) tau. Further functional analyses in cellular and in vivo models of tau seeding demonstrated almost complete depletion of tau seeds in an AD brain homogenate. Ongoing trials will provide the clinical evaluation of the tau spreading hypothesis in Alzheimer's disease.
Asunto(s)
Anticuerpos Monoclonales Humanizados/metabolismo , Anticuerpos Monoclonales/metabolismo , Descubrimiento de Drogas/métodos , Proteínas tau/metabolismo , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales Humanizados/química , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Estructura Terciaria de Proteína , Proteínas tau/químicaRESUMEN
Biosensor-surface plasmon resonance (SPR) technology is now well established as a quantitative approach for the study of nucleic acid interactions in real time, without the need for labeling any components of the interaction. The method provides real-time equilibrium and kinetic characterization for quadruplex DNA interactions and requires small amounts of materials and no external probe. A detailed protocol for quadruplex-DNA interaction analyses with a variety of binding molecules using biosensor-SPR methods is presented. Explanations of the SPR method with basic fundamentals for use and analysis of results are described with recommendations on the preparation of the SPR instrument, sensor chips, and samples. Details of experimental design, quantitative and qualitative data analyses, and presentation are described. Some specific examples of small molecule-DNA quadruplex interactions are presented with results evaluated by both kinetic and steady-state SPR methods.
Asunto(s)
Técnicas Biosensibles , Ácidos Nucleicos/química , Cinética , Resonancia por Plasmón de SuperficieRESUMEN
The tau spreading hypothesis provides rationale for passive immunization with an anti-tau monoclonal antibody to block seeding by extracellular tau aggregates as a disease-modifying strategy for the treatment of Alzheimer's disease (AD) and potentially other tauopathies. As the biochemical and biophysical properties of the tau species responsible for the spatio-temporal sequences of seeding events are poorly defined, it is not yet clear which epitope is preferred for obtaining optimal therapeutic efficacy. Our internal tau antibody collection has been generated by immunizations with different tau species: aggregated- and non-aggregated tau and human postmortem AD brain-derived tau fibrils. In this communication, we describe and characterize a set of these anti-tau antibodies for their biochemical and biophysical properties, including binding, tissue staining by immunohistochemistry, and epitope. The antibodies bound to different domains of the tau protein and some were demonstrated to be isoform-selective (PT18 and hTau56) or phospho-selective (PT84). Evaluation of the antibodies in cellular- and in vivo seeding assays revealed clear differences in maximal efficacy. Limited proteolysis experiments support the hypothesis that some epitopes are more exposed than others in the tau seeds. Moreover, antibody efficacy seems to depend on the structural properties of fibrils purified from tau Tg mice- and postmortem human AD brain.
Asunto(s)
Enfermedad de Alzheimer/patología , Anticuerpos Monoclonales/metabolismo , Encéfalo/metabolismo , Proteínas tau/inmunología , Animales , Mapeo Epitopo , Femenino , Células HEK293 , Humanos , Inmunización Pasiva , Masculino , Ratones , Ratones Noqueados , Mutación/genética , Resonancia por Plasmón de Superficie , Proteínas tau/deficiencia , Proteínas tau/genéticaRESUMEN
In therapeutic antibody discovery and early development, mice and cynomolgus monkey are used as animal models to assess toxicity, efficacy and other properties of candidate molecules. As more candidate antibodies are based on human immunoglobulin (IgG) subclasses, many strategies are pursued to simulate the human system in the test animal. However, translation rate from a successful preclinical trial to an approved drug is extremely low. This may partly be due to differences in interaction of human IgG based candidate molecules to endogenous Fcγ receptors of model animals in comparison to those of human Fcγ receptors. In this study, we compare binding characteristics of human IgG subclasses commonly used in drug development (IgG1, IgG2, IgG4) and their respective Fc silent versions (IgG1σ, IgG2σ, IgG4 PAA) to human, mouse, and cynomolgus monkey Fcγ receptors. To control interactions between Fab and Fc domains, the test IgGs all have the same variable region sequences. We found distinct variations of interaction of human IgG subclasses to model animal Fcγ receptors in comparison to their human counterparts. Particularly, cynomolgus monkey Fcγ receptors showed consistently tighter binding to human IgGs than human Fcγ receptors. Moreover, the presumably Fc silent human IgG4 PAA framework bound to cynomolgus monkey FcγRI with nanomolar affinity while only very weak binding was observed for the human FcγRI. Our results highlighted the need for a thorough in vitro affinity characterization of candidate IgGs against model animal Fcγ receptors and careful design of preclinical studies.
Asunto(s)
Fragmentos Fc de Inmunoglobulinas/metabolismo , Inmunoglobulina G/metabolismo , Isotipos de Inmunoglobulinas/metabolismo , Inmunoterapia/métodos , Receptores de IgG/metabolismo , Animales , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Células HEK293 , Humanos , Inmunoglobulina G/genética , Inmunoglobulina G/uso terapéutico , Región Variable de Inmunoglobulina/genética , Macaca fascicularis , Ratones , Unión Proteica , Investigación Biomédica TraslacionalRESUMEN
OBJECTIVE: Insulin resistance is a key feature of Type 2 Diabetes (T2D), and improving insulin sensitivity is important for disease management. Allosteric modulation of the insulin receptor (IR) with monoclonal antibodies (mAbs) can enhance insulin sensitivity and restore glycemic control in animal models of T2D. METHODS: A novel human mAb, IRAB-A, was identified by phage screening using competition binding and surface plasmon resonance assays with the IR extracellular domain. Cell based assays demonstrated agonist and sensitizer effects of IRAB-A on IR and Akt phosphorylation, as well as glucose uptake. Lean and diet-induced obese mice were used to characterize single-dose in vivo pharmacological effects of IRAB-A; multiple-dose IRAB-A effects were tested in obese mice. RESULTS: In vitro studies indicate that IRAB-A exhibits sensitizer and agonist properties distinct from insulin on the IR and is translated to downstream signaling and function; IRAB-A bound specifically and allosterically to the IR and stabilized insulin binding. A single dose of IRAB-A given to lean mice rapidly reduced fed blood glucose for approximately 2 weeks, with concomitant reduced insulin levels suggesting improved insulin sensitivity. Phosphorylated IR (pIR) from skeletal muscle and liver were increased by IRAB-A; however, phosphorylated Akt (pAkt) levels were only elevated in skeletal muscle and not liver vs. control; immunochemistry analysis (IHC) confirmed the long-lived persistence of IRAB-A in skeletal muscle and liver. Studies in diet-induced obese (DIO) mice with IRAB-A reduced fed blood glucose and insulinemia yet impaired glucose tolerance and led to protracted insulinemia during a meal challenge. CONCLUSION: Collectively, the data suggest IRAB-A acts allosterically on the insulin receptor acting non-competitively with insulin to both activate the receptor and enhance insulin signaling. While IRAB-A produced a decrease in blood glucose in lean mice, the data in DIO mice indicated an exacerbation of insulin resistance; these data were unexpected and suggested the interplay of complex unknown pharmacology. Taken together, this work suggests that IRAB-A may be an important tool to explore insulin receptor signaling and pharmacology.
Asunto(s)
Sitio Alostérico , Anticuerpos Monoclonales/farmacología , Hipoglucemiantes/farmacología , Receptor de Insulina/agonistas , Células 3T3 , Regulación Alostérica , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Glucemia/metabolismo , Línea Celular Tumoral , Humanos , Hipoglucemiantes/química , Hipoglucemiantes/inmunología , Insulina/metabolismo , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/metabolismo , Receptor de Insulina/química , Receptor de Insulina/inmunología , Transducción de SeñalRESUMEN
It is now well established that, although only about 5% of the human genome codes for protein, most of the DNA has some function, such as synthesis of specific, functional RNAs and/or control of gene expression. These functional sequences open immense possibilities in both biotechnology and therapeutics for the use of cell-permeable, small molecules that can bind mixed-base pair sequences of DNA for regulation of genomic functions. Unfortunately very few types of modules have been designed to recognize mixed DNA sequences and for progress in targeting specific genes, it is essential to have additional classes of compounds. Compounds that can be rationally designed from established modules and which can bind strongly to mixed base pair DNA sequences are especially attractive. Based on extensive experience in design of minor-groove agents for AT recognition, a small library of compounds with two AT specific binding modules, connected through linkers which can recognize the G·C base pairs, were prepared. The compound-DNA interactions were evaluated with a powerful array of biophysical methods and the results show that some pyridyl-linked compounds bind with the target sequence with sub-nanomolar KD, with very slow dissociation kinetics and 200 times selectivity over the related sequence without a G·C base pair. Interestingly, a set of compounds with AT module connected by different linkers shows cooperative dimer recognition of related sequences. This type of design approach can be expanded to additional modules for recognition of a wide variety of sequences.
Asunto(s)
Amidinas/química , Bencimidazoles/química , Bibliotecas de Moléculas Pequeñas/química , Emparejamiento Base , Secuencia de Bases , Sitios de Unión/efectos de los fármacos , Técnicas Biosensibles , Dicroismo Circular , Humanos , Estructura Molecular , Bibliotecas de Moléculas Pequeñas/síntesis química , Espectrometría de Masa por Ionización de Electrospray , Resonancia por Plasmón de SuperficieRESUMEN
A hallmark of type 2 diabetes is impaired insulin receptor (IR) signaling that results in dysregulation of glucose homeostasis. Understanding the molecular origins and progression of diabetes and developing therapeutics depend on experimental models of hyperglycemia, hyperinsulinemia, and insulin resistance. We present a novel monoclonal antibody, IRAB-B, that is a specific, potent IR antagonist that creates rapid and long-lasting insulin resistance. IRAB-B binds to the IR with nanomolar affinity and in the presence of insulin efficiently blocks receptor phosphorylation within minutes and is sustained for at least 3 days in vitro. We further confirm that IRAB-B antagonizes downstream signaling and metabolic function. In mice, a single dose of IRAB-B induces rapid onset of hyperglycemia within 6 h, and severe hyperglycemia persists for 2 weeks. IRAB-B hyperglycemia is normalized in mice treated with exendin-4, suggesting that this model can be effectively treated with a GLP-1 receptor agonist. Finally, a comparison of IRAB-B with the IR antagonist S961 shows distinct antagonism in vitro and in vivo. IRAB-B appears to be a powerful tool to generate both acute and chronic insulin resistance in mammalian models to elucidate diabetic pathogenesis and evaluate therapeutics.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Resistencia a la Insulina/fisiología , Receptor de Insulina/metabolismo , Animales , Western Blotting , Línea Celular , Diabetes Mellitus Tipo 2 , Receptor del Péptido 1 Similar al Glucagón/agonistas , Receptor del Péptido 1 Similar al Glucagón/antagonistas & inhibidores , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Humanos , Hiperglucemia/tratamiento farmacológico , Hiperglucemia/metabolismo , Ratones , Ratones Endogámicos C57BL , Péptidos/farmacología , Fosforilación , Unión Proteica , Receptor de Insulina/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacosRESUMEN
DNA minor-groove-binding compounds have limited biological applications, in part due to problems with sequence specificity that cause off-target effects. A model to enhance specificity has been developed with the goal of preparing compounds that bind to two AT sites separated by G·C base pairs. Compounds of interest were probed using thermal melting, circular dichroism, mass spectrometry, biosensor-SPR, and molecular modeling methods. A new minor groove binder that can strongly and specifically recognize a single G·C base pair with flanking AT sequences has been prepared. This multi-site DNA recognition mode offers novel design principles to recognize entirely new DNA motifs.
Asunto(s)
Emparejamiento Base , Derivados del Benceno/química , ADN/química , Secuencia de Bases , Simulación del Acoplamiento Molecular , Estructura MolecularRESUMEN
There is a long history for the bioorganic and biomedical use of N-methyl-pyrrole-derived polyamides (PAs) that are higher homologs of natural products such as distamycin A and netropsin. This work has been pursued by many groups, with the Dervan and Sugiyama groups responsible for many breakthroughs. We have studied PAs since about 1999, partly in industry and partly in academia. Early in this program, we reported methods to control cellular uptake of polyamides in cancer cell lines and other cells likely to have multidrug resistance efflux pumps induced. We went on to discover antiviral polyamides active against HPV31, where SAR showed that a minimum binding size of about 10 bp of DNA was necessary for activity. Subsequently we discovered polyamides active against two additional high-risk HPVs, HPV16 and 18, a subset of which showed broad spectrum activity against HPV16, 18 and 31. Aspects of our results presented here are incompatible with reported DNA recognition rules. For example, molecules with the same cognate DNA recognition properties varied from active to inactive against HPVs. We have since pursued the mechanism of action of antiviral polyamides, and polyamides in general, with collaborators at NanoVir, the University of Missouri-St. Louis, and Georgia State University. We describe dramatic consequences of ß-alanine positioning even in relatively small, 8-ring polyamides; these results contrast sharply with prior reports. This paper was originally presented by JKB as a Keynote Lecture in the 2nd International Conference on Medicinal Chemistry and Computer Aided Drug Design Conference in Las Vegas, NV, October 2013.
RESUMEN
We report here on the discovery and preliminary evaluation of a novel non-macrocyclic low molecular weight quadruplex-stabilizing chemotype. The lead compounds, based on a furan core, show high G-quadruplex stabilisation and selectivity as well as potent in vitro anti-proliferative activity.
Asunto(s)
G-Cuádruplex , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Dicroismo Circular , Furanos/química , HumanosRESUMEN
Quadruplexes DNA are present in telomeric DNA as well as in several cancer-related gene promoters and hence affect gene expression and subsequent biological processes. The conformations of G4 provide selective recognition sites for small molecules and thus these structures have become important drug-design targets for cancer treatment. The DNA G-quadruplex binding pentacyclic acridinium salt RHPS4 (1) has many pharmacological attributes of an ideal telomere-targeting agent but has undesirable off-target liabilities. Notably a cardiovascular effect was evident in a guinea pig model, manifested by a marked and sustained increase in QTcB interval. In accordance with this, significant interaction with the human recombinant ß2 adrenergic receptor, and M1, M2 and M3 muscarinic receptors was observed, together with a high inhibition of the hERG tail current tested in a patch clamp assay. Two related pentacyclic structures, the acetylamines (2) and (3), both show a modest interaction with ß2 adrenergic receptor, and do not significatively inhibit the hERG tail current while demonstrating potent telomere on-target properties comparing closely with 1. Of the two isomers, the 2-acetyl-aminopentacycle (2) more closely mimics the overall biological profile of 1 and this information will be used to guide further synthetic efforts to identify novel variants of this chemotype, to maximize on-target and minimize off-target activities. Consequently, the improvement of toxicological profile of these compounds could therefore lead to the obtainment of suitable molecules for clinical development offering new pharmacological strategies in cancer treatment.
Asunto(s)
Acridinas/química , Acridinas/farmacología , G-Cuádruplex , Telómero/metabolismo , Acridinas/síntesis química , Animales , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Cobayas , Humanos , Ligandos , Telomerasa/antagonistas & inhibidoresRESUMEN
A variety of cyanines provide versatile and sensitive agents acting as DNA stains and sensors and have been structurally modified to bind in the DNA minor groove in a sequence dependent manner. Similarly, we are developing a new set of cyanines that have been designed to achieve highly selective binding to DNA G-quadruplexes with much weaker binding to DNA duplexes. A systematic set of structurally analogous trimethine cyanines has been synthesized and evaluated for quadruplex targeting. The results reveal that elevated quadruplex binding and specificity are highly sensitive to the polymethine chain length, heterocyclic structure and intrinsic charge of the compound. Biophysical experiments show that the compounds display significant selectivity for quadruplex binding with a higher preference for parallel stranded quadruplexes, such as cMYC. NMR studies revealed the primary binding through an end-stacking mode and SPR studies showed the strongest compounds have primary KD values below 100 nM that are nearly 100-fold weaker for duplexes. The high selectivity of these newly designed trimethine cyanines for quadruplexes as well as their ability to discriminate between different quadruplexes are extremely promising features to develop them as novel probes for targeting quadruplexes in vivo.
Asunto(s)
Carbocianinas/química , G-Cuádruplex , Espectroscopía de Resonancia Magnética , Resonancia por Plasmón de Superficie , Telómero/químicaRESUMEN
Rules for polyamide-DNA recognition have proved invaluable for the design of sequence-selective DNA binding agents in cell-free systems. However, these rules are not fully transferrable to predicting activity in cells, tissues or animals, and additional refinements to our understanding of DNA recognition would help biomedical studies. Similar complexities are encountered when using internal ß-alanines as polyamide building blocks in place of N-methylpyrrole; ß-alanines were introduced in polyamide designs to maintain good hydrogen bonding registry with the target DNA, especially for long polyamides or those with several GC bp (P.B. Dervan, A.R. Urbach, Essays Contemp. Chem. (2001) 327-339). Thus, to clarify important subtleties of molecular recognition, we studied the effects of replacing a single pyrrole with ß-alanine in 8-ring polyamides designed against the Ets-1 transcription factor. Replacement of a single internal N-methylpyrrole with ß-alanine to generate a ß/Im pairing in two 8-ring polyamides causes a decrease in DNA binding affinity by two orders of magnitude and decreases DNA binding selectivity, contrary to expectations based on the literature. Measurements were made by fluorescence spectroscopy, quantitative DNA footprinting and surface plasmon resonance, with these vastly different techniques showing excellent agreement. Furthermore, results were validated for a range of DNA substrates from small hairpins to long dsDNA sequences. Docking studies helped show that ß-alanine does not make efficient hydrophobic contacts with the rest of the polyamide or nearby DNA, in contrast to pyrrole. These results help refine design principles and expectations for polyamide-DNA recognition.
Asunto(s)
Ciclooxigenasa 2/química , ADN/química , Papillomavirus Humano 16/química , Nylons/química , Proteína Proto-Oncogénica c-ets-1/química , Pirroles/química , beta-Alanina/química , Secuencia de Bases , Ciclooxigenasa 2/genética , Huella de ADN , Papillomavirus Humano 16/genética , Humanos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Secuencias Invertidas Repetidas/genética , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Nylons/síntesis química , Regiones Promotoras Genéticas , Proteína Proto-Oncogénica c-ets-1/genética , Espectrometría de Fluorescencia , Relación Estructura-Actividad , Resonancia por Plasmón de SuperficieRESUMEN
Based on molecular docking analysis of complexes between flavone and the c-myc G-quadruplex, we designed and screened 30 flavone derivatives containing various side chains that could potentially form interactions with the G-quadruplex grooves. As a proof-of-concept, the highest-scoring flavone derivatives containing cationic pyridinium side chains were synthesized and their interactions with the c-myc G-quadruplex were examined using a PCR-stop assay. The stabilizing effects of the flavone derivatives were found to be selective towards the c-myc G-quadruplex over other biologically relevant G-quadruplex structures, such as the human telomeric sequence (HTS). The interaction between the most potent compound of the series and the c-myc G-quadruplex was examined in depth using UV-Vis titration, molecular modeling and CD spectroscopy. Our results suggest that in addition to stabilizing the c-myc G-quadruplex, the flavone derivatives were capable of inducing the formation of the G-quadruplex structure even in the absence of monovalent cations. The flavone derivatives were found to be potent inhibitors of c-myc promoters within the cellular environment and displayed promising cytotoxic behavior against human cancer cell lines.
Asunto(s)
Flavonas/farmacología , G-Cuádruplex , Genes myc , Supervivencia Celular/efectos de los fármacos , Dicroismo Circular , Regulación hacia Abajo/efectos de los fármacos , Flavonas/química , Células Hep G2 , Humanos , Simulación del Acoplamiento Molecular , Estructura Molecular , Regiones Promotoras GenéticasRESUMEN
Compounds that bind in the DNA minor groove have provided critical information on DNA molecular recognition, have found extensive uses in biotechnology, and are providing clinically useful drugs against diseases as diverse as cancer and sleeping sickness. This review focuses on the development of clinically useful heterocyclic diamidine minor groove binders. These compounds have shown us that the classical model for minor groove binding in AT DNA sequences must be expanded in several ways: compounds with nonstandard shapes can bind strongly to the groove, water can be directly incorporated into the minor groove complex in an interfacial interaction, and the compounds can form cooperative stacked dimers to recognize GC and mixed AT/GC base pair sequences.
Asunto(s)
Cationes Bivalentes/química , ADN Forma B/química , ADN/química , Amidinas/química , Emparejamiento Base , Secuencia de Bases , Bencimidazoles/química , Sitios de Unión , Dimerización , Compuestos Heterocíclicos/química , Modelos Moleculares , Conformación de Ácido Nucleico , Agua/químicaRESUMEN
Design and optimization of quadruplex-specific small molecules is developing into an attractive strategy for anti-cancer therapeutics with some promising candidates in clinical trials. A number of therapeutically favorable features of cyanine molecules can be effectively exploited to develop them as promising quadruplex-targeting agents. Herein, the design, synthesis and evaluation of a series of dimethylindolenine cyanine dyes with varying halogen substitutions are reported. Their interactions with telomeric and c-myc quadruplexes as well as a reference duplex sequence have been evaluated using thermal melting, biosensor-surface plasmon resonance, circular dichroism, isothermal titration calorimetry and mass spectrometry. Thermal melting analysis indicates that these ligands exhibit significant quadruplex stabilization and a very low duplex binding, with the dimethyl incorporation of paramount importance for decreased duplex affinity. Circular dichroism studies showed that the interaction of cyanines with quadruplex structures are primarily through stacking at one or both ends of the terminal tetrads with the two (trimethylammonium)propyl groups interacting in the accessible quadruplex grooves. Surface plasmon resonance and mass spectral studies shows the formation of an initial strong 1:1 complex followed by a significantly weaker secondary binding. Isothermal calorimetry studies show that the interaction of cyanines is predominantly entropy driven. In line with the design principles, this work provides new insights for further developing potent, highly selective cyanines as promising quadruplex-specific agents.