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1.
Cell ; 187(10): 2521-2535.e21, 2024 May 09.
Artículo en Inglés | MEDLINE | ID: mdl-38697107

RESUMEN

Cancer immunotherapy remains limited by poor antigenicity and a regulatory tumor microenvironment (TME). Here, we create "onion-like" multi-lamellar RNA lipid particle aggregates (LPAs) to substantially enhance the payload packaging and immunogenicity of tumor mRNA antigens. Unlike current mRNA vaccine designs that rely on payload packaging into nanoparticle cores for Toll-like receptor engagement in immune cells, systemically administered RNA-LPAs activate RIG-I in stromal cells, eliciting massive cytokine/chemokine response and dendritic cell/lymphocyte trafficking that provokes cancer immunogenicity and mediates rejection of both early- and late-stage murine tumor models. In client-owned canines with terminal gliomas, RNA-LPAs improved survivorship and reprogrammed the TME, which became "hot" within days of a single infusion. In a first-in-human trial, RNA-LPAs elicited rapid cytokine/chemokine release, immune activation/trafficking, tissue-confirmed pseudoprogression, and glioma-specific immune responses in glioblastoma patients. These data support RNA-LPAs as a new technology that simultaneously reprograms the TME while eliciting rapid and enduring cancer immunotherapy.


Asunto(s)
Inmunoterapia , Lípidos , ARN , Microambiente Tumoral , Animales , Perros , Femenino , Humanos , Ratones , Antígenos de Neoplasias/inmunología , Neoplasias Encefálicas/terapia , Neoplasias Encefálicas/inmunología , Vacunas contra el Cáncer/inmunología , Vacunas contra el Cáncer/uso terapéutico , Línea Celular Tumoral , Citocinas/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Glioblastoma/terapia , Glioblastoma/inmunología , Glioma/terapia , Glioma/inmunología , Inmunoterapia/métodos , Ratones Endogámicos C57BL , Neoplasias/terapia , Neoplasias/inmunología , ARN/química , ARN/uso terapéutico , ARN Mensajero/metabolismo , ARN Mensajero/genética , Lípidos/química
2.
Mol Ther ; 31(12): 3441-3456, 2023 Dec 06.
Artículo en Inglés | MEDLINE | ID: mdl-37814449

RESUMEN

Adeno-associated virus (AAV) continues to be the gold standard vector for therapeutic gene delivery and has proven especially useful for treating ocular disease. Intravitreal injection (IVtI) is a promising delivery route because it increases accessibility of gene therapies to larger patient populations. However, data from clinical and non-human primate (NHP) studies utilizing currently available capsids indicate that anatomical barriers to AAV and pre-existing neutralizing antibodies can restrict gene expression to levels that are "sub-therapeutic" in a substantial proportion of patients. Here, we performed a combination of directed evolution in NHPs of an AAV2-based capsid library with simultaneous mutations across six surface-exposed variable regions and rational design to identify novel capsid variants with improved retinal transduction following IVtI. Following two rounds of screening in NHP, enriched variants were characterized in intravitreally injected mice and NHPs and shown to have increased transduction relative to AAV2. Lead capsid variant, P2-V1, demonstrated an increased ability to evade neutralizing antibodies in human vitreous samples relative to AAV2 and AAV2.7m8. Taken together, this study further contributed to our understanding of the selective pressures associated with retinal transduction via the vitreous and identified promising novel AAV capsid variants for clinical consideration.


Asunto(s)
Anticuerpos Neutralizantes , Cápside , Humanos , Ratones , Animales , Dependovirus , Inyecciones Intravítreas , Transducción Genética , Primates/genética , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Vectores Genéticos/genética
3.
Biochemistry ; 62(11): 1755-1766, 2023 06 06.
Artículo en Inglés | MEDLINE | ID: mdl-37172221

RESUMEN

DNA adopts a number of conformations that can affect its binding to other macromolecules. The conformations (A, B, Z) can be sequence- and/or solution-dependent. While AT-rich DNA sequences generally adopt a Canonical B-form structure, GC-rich sequences are more promiscuous. Recognition of GC-rich nucleic acids by small molecules has been much more challenging than the recognition of AT-rich duplexes. Spectrophotometric and calorimetric techniques were used to characterize the binding of neomycin-class aminoglycosides to a GC-rich DNA duplex, G4C4, in various ionic and pH conditions. Our results reveal that binding enhances the thermal stability of G4C4, with thermal enhancement decreasing with increasing pH and/or Na+ concentration. Although G4C4 bound to aminoglycosides demonstrated a mixed A- and B-form conformation, circular dichroism studies indicate that binding induces a conformational shift toward A-form DNA. Isothermal titration calorimetry studies reveal that aminoglycoside binding to G4C4 is linked to the uptake of protons at pH = 7.0 and that this uptake is pH-dependent. Increased pH and/or Na+ concentration results in a decrease in G4C4 affinity for the aminoglycosides. The binding affinities of the aminoglycosides follow the expected hierarchy: neomycin > paromomycin > ribostamycin. The salt dependence of DNA binding affinities of aminoglycosides is consistent with at least two drug NH3+ groups participating in electrostatic interactions with G4C4. These studies further embellish our understanding of the many factors facilitating recognition of GC-rich DNA structures as guided by their optimum charge and shape complementarity for small-molecule amino sugars.


Asunto(s)
Aminoglicósidos , Neomicina , Neomicina/química , Neomicina/metabolismo , Aminoglicósidos/metabolismo , Antibacterianos/química , ADN/química , Termodinámica , Conformación de Ácido Nucleico , Sitios de Unión
4.
Biochemistry ; 56(49): 6434-6447, 2017 Dec 12.
Artículo en Inglés | MEDLINE | ID: mdl-29131946

RESUMEN

Small molecules that modulate biological functions are targets of modern day drug discovery efforts. In a common platform fragment-based drug discovery, two fragments that bind to adjacent sites on a target are identified and are then linked together using different linkers to identify the linkage for optimum activity. What are not known from these studies are the effects these linkers, which typically contain C, H, and O atoms, have on the properties of the individual fragment. Herein, we investigate such effects in a bisbenzimidazole fragment whose derivatives have a wide range of therapeutic applications in nucleic acid recognition, sensing, and photodynamic therapy and as cellular probes. We report a dramatic effect of linker length and composition of alkynyl (clickable) Hoechst 33258 derivatives in target binding and cell uptake. We show that the binding of Hoechst 33258-modeled bisbenzimidazoles (1-9) that contain linkers of varying lengths (3-21 atoms) display length- and composition-dependent variation in B-DNA stabilization using a variety of spectroscopic methods. For a dodecamer DNA duplex, the thermal stabilization varied from 0.3 to 9.0 °C as the linker length increased from 3 to 21 atoms, respectively. Compounds with linker lengths of ≤11 atoms (such as compounds 1 and 5) are localized in the nucleus, while compounds with long linkers (such as compounds 8 and 9) are distributed in the extranuclear space, as well, with possible interactions with extranuclear targets. These findings provide insights into future drug design by revealing how linkers can influence the biophysical and cellular properties of individual drug fragments.


Asunto(s)
Bisbenzimidazol/química , ADN Forma B/química , Sitios de Unión , Cinética , Estructura Molecular , Espectrometría de Fluorescencia
5.
Bioorg Med Chem ; 25(4): 1309-1319, 2017 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-28129992

RESUMEN

Nucleic acids adopt a broad array of hydrogen-bonded structures that enable their diverse roles in the cell; even the familiar DNA double helix displays subtle architectural nuances that are sequence dependent. While there have been many approaches for recognition of B-form nucleic acids, A-form DNA recognition has lagged behind. Here, using a tight binding fluorescein-neomycin (F-neo) conjugate that can probe the electrostatic environment of A-form DNA major groove, we developed a fluorescent displacement assay to be used as a screen for DNA duplex-binding compounds. As opposed to intercalating dyes that can significantly perturb DNA structure, the groove binding F-neo allows the probing of native DNA conformation. In combination with the assay development and probing of DNA grooves, we also report the synthesis and binding of a series of neomycin-anthraquinone conjugates, two units with a known preference for binding GC rich DNA. The assay can be used to identify duplex DNA-binding compounds, as well as probe structural features of a target DNA duplex, and can easily be scaled up for high throughput screening of compound libraries.


Asunto(s)
ADN de Forma A/análisis , Fluoresceína/química , Colorantes Fluorescentes/análisis , Neomicina/química , Simulación del Acoplamiento Molecular , Estructura Molecular
6.
Org Biomol Chem ; 14(6): 2052-6, 2016 Feb 14.
Artículo en Inglés | MEDLINE | ID: mdl-26765486

RESUMEN

Recognition of RNA by high-affinity binding small molecules is crucial for expanding existing approaches in RNA recognition, and for the development of novel RNA binding drugs. A novel neomycin dimer benzimidazole conjugate 5 (DPA 83) was synthesized by conjugating a neomycin-dimer with a benzimidazole alkyne using click chemistry to target multiple binding sites on HIV TAR RNA. Ligand 5 significantly enhances the thermal stability of HIV TAR RNA and interacts stoichiometrically with HIV TAR RNA with a low nanomolar affinity. 5 displayed enhanced binding compared to its individual building blocks including the neomycin dimer azide and benzimidazole alkyne. In essence, a high affinity multivalent ligand was designed and synthesized to target HIV TAR RNA.


Asunto(s)
Aminoglicósidos/farmacología , Bencimidazoles/farmacología , Duplicado del Terminal Largo de VIH/efectos de los fármacos , ARN Viral/antagonistas & inhibidores , Aminoglicósidos/síntesis química , Aminoglicósidos/química , Bencimidazoles/síntesis química , Bencimidazoles/química , Sitios de Unión/efectos de los fármacos , Química Clic , Relación Dosis-Respuesta a Droga , Ligandos , Conformación Molecular , Neomicina/química , Neomicina/farmacología , Relación Estructura-Actividad
7.
Cell Death Discov ; 9(1): 1, 2023 Jan 02.
Artículo en Inglés | MEDLINE | ID: mdl-36588105

RESUMEN

Small-cell lung cancer (SCLC) is an aggressive malignancy with limited therapeutic options. The dismal prognosis in SCLC is in part associated with an upregulation of BCL-2 family anti-apoptotic proteins, including BCL-XL and MCL-1. Unfortunately, the currently available inhibitors of BCL-2 family anti-apoptotic proteins, except BCL-2 inhibitors, are not clinically relevant because of various on-target toxicities. We, therefore, aimed to develop an effective and safe strategy targeting these anti-apoptotic proteins with DT2216 (our platelet-sparing BCL-XL degrader) and AZD8055 (an mTOR inhibitor) to avoid associated on-target toxicities while synergistically optimizing tumor response. Through BH3 mimetic screening, we identified a subset of SCLC cell lines that is co-dependent on BCL-XL and MCL-1. After screening inhibitors of selected tumorigenic pathways, we found that AZD8055 selectively downregulates MCL-1 in SCLC cells and its combination with DT2216 synergistically killed BCL-XL/MCL-1 co-dependent SCLC cells, but not normal cells. Mechanistically, the combination caused BCL-XL degradation and suppression of MCL-1 expression, and thus disrupted MCL-1 interaction with BIM leading to an enhanced apoptotic induction. In vivo, the DT2216 + AZD8055 combination significantly inhibited the growth of cell line-derived and patient-derived xenografts and reduced tumor burden accompanied by increased survival in a genetically engineered mouse model of SCLC without causing appreciable thrombocytopenia or other normal tissue injuries. Thus, these preclinical findings lay a strong foundation for future clinical studies to test DT2216 + mTOR inhibitor combinations in a subset of SCLC patients whose tumors are co-driven by BCL-XL and MCL-1.

8.
JCI Insight ; 8(10)2023 04 25.
Artículo en Inglés | MEDLINE | ID: mdl-37097751

RESUMEN

Although thymidylate synthase (TYMS) inhibitors have served as components of chemotherapy regimens, the currently available inhibitors induce TYMS overexpression or alter folate transport/metabolism feedback pathways that tumor cells exploit for drug resistance, limiting overall benefit. Here we report a small molecule TYMS inhibitor that i) exhibited enhanced antitumor activity as compared with current fluoropyrimidines and antifolates without inducing TYMS overexpression, ii) is structurally distinct from classical antifolates, iii) extended survival in both pancreatic xenograft tumor models and an hTS/Ink4a/Arf null genetically engineered mouse tumor model, and iv) is well tolerated with equal efficacy using either intraperitoneal or oral administration. Mechanistically, we verify the compound is a multifunctional nonclassical antifolate, and using a series of analogs, we identify structural features allowing direct TYMS inhibition while maintaining the ability to inhibit dihydrofolate reductase. Collectively, this work identifies nonclassical antifolate inhibitors that optimize inhibition of thymidylate biosynthesis with a favorable safety profile, highlighting the potential for enhanced cancer therapy.


Asunto(s)
Antagonistas del Ácido Fólico , Ratones , Animales , Humanos , Antagonistas del Ácido Fólico/farmacología , Antagonistas del Ácido Fólico/uso terapéutico , Antagonistas del Ácido Fólico/química , Inhibidores Enzimáticos/farmacología , Resistencia a Medicamentos , Timidilato Sintasa
9.
medRxiv ; 2023 Mar 17.
Artículo en Inglés | MEDLINE | ID: mdl-36993772

RESUMEN

Messenger RNA (mRNA) has emerged as a remarkable tool for COVID-19 prevention but its use for induction of therapeutic cancer immunotherapy remains limited by poor antigenicity and a regulatory tumor microenvironment (TME). Herein, we develop a facile approach for substantially enhancing immunogenicity of tumor-derived mRNA in lipid-particle (LP) delivery systems. By using mRNA as a molecular bridge with ultrapure liposomes and foregoing helper lipids, we promote the formation of 'onion-like' multi-lamellar RNA-LP aggregates (LPA). Intravenous administration of RNA-LPAs mimics infectious emboli and elicits massive DC/T cell mobilization into lymphoid tissues provoking cancer immunogenicity and mediating rejection of both early and late-stage murine tumor models. Unlike current mRNA vaccine designs that rely on payload packaging into nanoparticle cores for toll-like receptor engagement, RNA-LPAs stimulate intracellular pathogen recognition receptors (RIG-I) and reprogram the TME thus enabling therapeutic T cell activity. RNA-LPAs were safe in acute/chronic murine GLP toxicology studies and immunologically active in client-owned canines with terminal gliomas. In an early phase first-in-human trial for patients with glioblastoma, we show that RNA-LPAs encoding for tumor-associated antigens elicit rapid induction of pro-inflammatory cytokines, mobilization/activation of monocytes and lymphocytes, and expansion of antigen-specific T cell immunity. These data support the use of RNA-LPAs as novel tools to elicit and sustain immune responses against poorly immunogenic tumors.

10.
Biochemistry ; 51(11): 2331-47, 2012 Mar 20.
Artículo en Inglés | MEDLINE | ID: mdl-22339203

RESUMEN

A series of neomycin dimers have been synthesized using "click chemistry" with varying functionality and length in the linker region to target the human immunodeficiency virus type 1 (HIV-1) TAR RNA region of the HIV virus. The TAR (Trans-Activation Responsive) RNA region, a 59 bp stem-loop structure located at the 5'-end of all nascent viral transcripts, interacts with its target, a key regulatory protein, Tat, and necessitates the replication of HIV-1. Neomycin, an aminosugar, has been shown to exhibit multiple binding sites on TAR RNA. This observation prompted us to design and synthesize a library of triazole-linked neomycin dimers using click chemistry. The binding between neomycin dimers and TAR RNA was characterized using spectroscopic techniques, including FID (fluorescent intercalator displacement), a FRET (fluorescence resonance energy transfer) competitive assay, circular dichroism (CD), and UV thermal denaturation. UV thermal denaturation studies demonstrate that binding of neomycin dimers increases the melting temperature (T(m)) of the HIV TAR RNA up to 10 °C. Ethidium bromide displacement (FID) and a FRET competition assay revealed nanomolar binding affinity between neomycin dimers and HIV TAR RNA, while in case of neomycin, only weak binding was detected. More importantly, most of the dimers exhibited lower IC(50) values toward HIV TAR RNA, when compared to the fluorescent Tat peptide, and show increased selectivity over mutant TAR RNA. Cytopathic effects investigated using MT-2 cells indicate a number of the dimers with high affinity toward TAR show promising anti-HIV activity.


Asunto(s)
Duplicado del Terminal Largo de VIH , VIH-1/química , VIH-1/genética , ARN Viral/química , Sitios de Unión , Transferencia Resonante de Energía de Fluorescencia , VIH-1/metabolismo , Humanos , Neomicina/química , Neomicina/metabolismo , Conformación de Ácido Nucleico
11.
Cureus ; 12(7): e8982, 2020 Jul 03.
Artículo en Inglés | MEDLINE | ID: mdl-32775064

RESUMEN

Introduction Patellofemoral pain syndrome (PFPS) is one of the leading causes of anterior knee pain treated by orthopedists and physical therapists. This syndrome predominantly affects young, active individuals, and remains a challenging syndrome to manage due to the lack of quantitative diagnostic criteria to monitor during treatment. The etiology of this syndrome is believed to be multifactorial, with the gait and movement patterns of a patient potentially contributing to pain due to increased stress on the knee. In this study, we investigated the gait of participants with PFPS using the GaitRite system (CIR Systems Inc., Clifton, NJ) before and after the application of Kinesio Tape in order to assess the impact of Kinesio Tape on cadence, stance time, and pain. Methods A convenience sample of 10 participants were recruited for this study, with five participants without PFPS serving as controls, and five with PFPS in the Kinesio Tape group. Participants in the Kinesio Tape groups served as their own internal control, ambulating both before and after taping. All participants ambulated across the GaitRite carpet three times and completed a visual analogue scale pain score for each trip. Results The results of our study found there to be no significant difference in the cadence for gait between the participants without PFPS and participants with PFPS (105.2 seconds vs. 105.1 seconds, p = 0.272), or in the stance time between the control and PFPS group (1.43 seconds vs. 1.44, p = 0.907). Similarly, no significant difference was found in participants with PFPS before and after Kinesio Taping in the cadence and stance times (105.1 seconds vs. 107.4 seconds, p =0.288, and 1.44 vs. 1.40, p = 0.272). There was a significant difference in pain in PFPS participants before and after taping, with a 112.5% reduction in pain reported after taping (3.4 vs. 1.6, p < 0.05). Discussion and conclusion This study is one of the first studies to utilize the GaitRite system in order to analyze the impact of Kinesio Tape on gait in participants with PFPS. While our study failed to demonstrate a significant difference in the gait of participants with PFPS in comparison to those without PFPS, we did demonstrate a significant reduction in pain after the application of Kinesio Tape. These results suggest other variables addressed by the Kinesio Tape may be causing the pain associated with PFPS.

12.
J Clin Invest ; 129(6): 2279-2292, 2019 04 29.
Artículo en Inglés | MEDLINE | ID: mdl-31033480

RESUMEN

Oncolytic virotherapy has been proposed as an ablative and immunostimulatory treatment strategy for solid tumors that are resistant to immunotherapy alone; however, there is a need to optimize host immune activation using preclinical immunocompetent models in previously untested common adult tumors. We studied a modified oncolytic myxoma virus (MYXV) that shows high efficiency for tumor-specific cytotoxicity in small-cell lung cancer (SCLC), a neuroendocrine carcinoma with high mortality and modest response rates to immune checkpoint inhibitors. Using an immunocompetent SCLC mouse model, we demonstrated the safety of intrapulmonary MYXV delivery with efficient tumor-specific viral replication and cytotoxicity associated with induction of immune cell infiltration. We observed increased SCLC survival following intrapulmonary MYXV that was enhanced by combined low-dose cisplatin. We also tested intratumoral MYXV delivery and observed immune cell infiltration associated with tumor necrosis and growth inhibition in syngeneic murine allograft tumors. Freshly collected primary human SCLC tumor cells were permissive to MYXV and intratumoral delivery into patient-derived xenografts resulted in extensive tumor necrosis. We confirmed MYXV cytotoxicity in classic and variant SCLC subtypes as well as cisplatin-resistant cells. Data from 26 SCLC human patients showed negligible immune cell infiltration, supporting testing MYXV as an ablative and immune-enhancing therapy.


Asunto(s)
Cisplatino/farmacología , Neoplasias Pulmonares/terapia , Myxoma virus , Viroterapia Oncolítica , Virus Oncolíticos , Carcinoma Pulmonar de Células Pequeñas/terapia , Animales , Línea Celular Tumoral , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/patología , Ratones , Ratones Noqueados , Carcinoma Pulmonar de Células Pequeñas/genética , Carcinoma Pulmonar de Células Pequeñas/inmunología , Carcinoma Pulmonar de Células Pequeñas/patología , Ensayos Antitumor por Modelo de Xenoinjerto
13.
Medchemcomm ; 5(8): 1235-1246, 2014 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-27076899

RESUMEN

Neomycin dimers synthesized using "click chemistry" with varying functionality and length in the linker region have been shown to be effective in targeting the HIV-1 TAR RNA region of the HIV virus. TAR (Transactivation Response) RNA region, a 59 base pair stem loop structure located at the 5'-end of all nascent viral transcripts interacts with its target, a key regulatory protein, Tat, and necessitates the replication of HIV-1 virus. Ethidium bromide displacement and FRET competition assays have revealed nanomolar binding affinity between neomycin dimers and wildtype TAR RNA while in case of neomycin, only a weak binding was detected. Here, NMR and FID-based comparisons reveal an extended binding interface for neomycin dimers involving the upper stem of the TAR RNA thereby offering an explanation for increased affinities. To further explore the potential of these modified aminosugars we have extended binding studies to include four TAR RNA mutants that display conformational differences with minimal sequence variation. The differences in binding between neomycin and neomycin dimers is characterized with TAR RNA mutants that include mutations to the bulge region, hairpin region, and both the bulge and hairpin regions. Our results demonstrate the effect of these mutations on neomycin binding and our results show that linker functionalities between dimeric units of neomycin can distinguish between the conformational differences of mutant TAR RNA structures.

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