RESUMEN
In this study, we present a modular synthesis and evaluation of two prostate-specific membrane antigen (PSMA) targeted small molecule drug conjugates (SMDCs) incorporating the potent chemotherapeutic agent monomethyl auristatin E (MMAE). These SMDCs are distinguished by their cleavable linker modules: one utilizing the widely known valine-citrulline linker, susceptible to cleavage by cathepsin B, and the other featuring a novel acid-labile phosphoramidate-based (PhosAm) linker. Both SMDCs maintained nanomolar affinity to PSMA. Furthermore, we confirmed the selective release of the payload and observed chemotherapeutic efficacy specifically within PSMA-positive prostate cancer cells, while maintaining cell viability in PSMA-negative cells. These findings not only validate the efficacy of our approach but also highlight the potential of the innovative pH-responsive PhosAm linker. This study contributes significantly to the field and also paves the way for future advancements in targeted cancer therapy.
Asunto(s)
Antineoplásicos , Inmunoconjugados , Neoplasias de la Próstata , Humanos , Masculino , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Citrulina , Sistemas de Liberación de Medicamentos , Inmunoconjugados/uso terapéutico , Valina , Neoplasias de la Próstata/tratamiento farmacológicoRESUMEN
We developed a model small-molecule drug conjugate (SMDC) that employed doxorubicin as a representative chemotherapeutic targeted to the cell membrane biomarker PSMA (prostate-specific membrane antigen) expressed on prostate cancer cells. The strategy capitalized on the clatherin-mediated internalization of PSMA to facilitate the selective uptake and release of doxorubicin in the target cells. The SMDC was prepared and assessed for binding kinetics, plasma stability, cell toxicity, and specificity towards PSMA expressing prostate cancer cell lines. We observed high affinity of the SMDC for PSMA (IC50 5 nM) with irreversible binding, as well as specific effectiveness against PSMA(+) cells. These findings validated the strategy for a small molecule-based approach in targeted cancer therapy.
Asunto(s)
Antígenos de Superficie , Doxorrubicina , Glutamato Carboxipeptidasa II , Neoplasias de la Próstata , Humanos , Masculino , Antígenos de Superficie/metabolismo , Línea Celular Tumoral , Doxorrubicina/farmacología , Doxorrubicina/uso terapéutico , Sistemas de Liberación de Medicamentos , Glutamato Carboxipeptidasa II/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismoRESUMEN
Herein, we report the modular synthesis and evaluation of a prostate-specific membrane antigen (PSMA) targeted small molecule drug conjugate (SMDC) carrying the chemotherapeutic agent, SN38. Due to the fluorogenic properties of SN38, payload release kinetics from the platform was observed in buffers representing the pH conditions of systemic circulation and cellular internalization. It was found that this platform is stable with minimal payload release at physiological pH with most rapid payload release observed at pH values representing the endosome complex. We confirmed selective payload release and chemotherapeutic efficacy for PSMA(+) prostate cancer cells over PSMA(-) cells. These results demonstrate that chemotherapeutic agents with limited solubility can be conjugated to a water-soluble targeting and linker platform without attenuating efficacy.
Asunto(s)
Glutamato Carboxipeptidasa II , Neoplasias de la Próstata , Masculino , Humanos , Línea Celular Tumoral , Glutamato Carboxipeptidasa II/química , Antígenos de Superficie/química , Neoplasias de la Próstata/tratamiento farmacológicoRESUMEN
Since their first discovery in the 1960s by Alec Bangham, liposomes have been shown to be effective drug delivery systems for treating various cancers. Several liposome-based formulations received approval by the U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA), with many others in clinical trials. Liposomes have several advantages, including improved pharmacokinetic properties of the encapsulated drug, reduced systemic toxicity, extended circulation time, and targeted disposition in tumor sites due to the enhanced permeability and retention (EPR) mechanism. However, it is worth noting that despite their efficacy in treating various cancers, liposomes still have some potential toxicity and lack specific targeting and disposition. This explains, in part, why their translation into the clinic has progressed only incrementally, which poses the need for more research to focus on addressing such translational limitations. This review summarizes the main properties of liposomes, their current status in cancer therapy, and their limitations and challenges to achieving maximal therapeutic efficacy.
Asunto(s)
Antineoplásicos , Neoplasias , Humanos , Liposomas/uso terapéutico , Sistemas de Liberación de Medicamentos , Neoplasias/tratamiento farmacológico , Neoplasias/patologíaRESUMEN
Histone arginine methylation is a key post-translational modification that mediates epigenetic events that activate or repress gene transcription. Protein arginine methyltransferases (PRMTs) are the driving force for the process of arginine methylation, and the core histone proteins have been shown to be substrates for most PRMT family members. However, previous reports of the enzymatic activities of PRMTs on histones in the context of nucleosomes seem contradictory. Moreover, what governs nucleosomal substrate recognition of different PRMT members is not understood. We sought to address this key biological question by examining how different macromolecular contexts where the core histones reside may regulate arginine methylation catalyzed by individual PRMT members (i.e., PRMT1, PRMT3, PRMT4, PRMT5, PRMT6, PRMT7, and PRMT8). Our results demonstrated that the substrate context exhibits a huge impact on the histone arginine methylation activity of PRMTs. Although all the tested PRMTs methylate multiple free histones individually, they show a preference for one particular histone substrate in the context of the histone octamer. We found that PRMT1, PRMT3, PRMT5, PRMT6, PRMT7, and PRMT8 preferentially methylate histone H4, whereas PRMT4/coactivator-associated arginine methyltransferase 1 prefers histone H3. Importantly, neither reconstituted nor cell-extracted mononucleosomes could be methylated by any PRMTs tested. Structural analysis suggested that the electrostatic interaction may play a mechanistic role in priming the substrates for methylation by PRMT enzymes. Taken together, this work expands our knowledge on the molecular mechanisms of PRMT substrate recognition and has important implications for understanding cellular dynamics and kinetics of histone arginine methylation in regulating gene transcription and other chromatin-templated processes.
Asunto(s)
Histonas/química , Complejos Multiproteicos/química , Proteína-Arginina N-Metiltransferasas/química , Arginina/química , Arginina/genética , Arginina/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Estructura Cuaternaria de Proteína , Proteína-Arginina N-Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/metabolismo , Especificidad por SustratoRESUMEN
Protein post-translational modifications (PTMs) in eukaryotic cells play important roles in the regulation of functionalities of the proteome and in the tempo-spatial control of cellular processes. Most PTMs enact their regulatory functions by affecting the biochemical properties of substrate proteins such as altering structural conformation, protein-protein interaction, and protein-nucleic acid interaction. Amid various PTMs, arginine methylation is widespread in all eukaryotic organisms, from yeasts to humans. Arginine methylation in many situations can drastically or subtly affect the interactions of substrate proteins with their partnering proteins or nucleic acids, thus impacting major cellular programs. Recently, arginine methylation has become an important regulator of the formation of membrane-less organelles inside cells, a phenomenon of liquid-liquid phase separation (LLPS), through altering π-cation interactions. Another unique feature of arginine methylation lies in its impact on cellular physiology through its downstream amino acid product, asymmetric dimethylarginine (ADMA). Accumulation of ADMA in cells and in the circulating bloodstream is connected with endothelial dysfunction and a variety of syndromes of cardiovascular diseases. Herein, we review the current knowledge and understanding of protein arginine methylation in regards to its canonical function in direct protein regulation, as well as the biological axis of protein arginine methylation and ADMA biology.
Asunto(s)
Arginina/análogos & derivados , Arginina/metabolismo , Proteínas/metabolismo , Animales , Arginina/química , Humanos , Metaboloma , Metilación , Proteína-Arginina N-Metiltransferasas/metabolismoRESUMEN
Histone methylation plays an important regulatory role in chromatin restructuring and RNA transcription. Arginine methylation that is enzymatically catalyzed by the family of protein arginine methyltransferases (PRMTs) can either activate or repress gene expression depending on cellular contexts. Given the strong correlation of PRMTs with pathophysiology, great interest is seen in understanding molecular mechanisms of PRMTs in diseases and in developing potent PRMT inhibitors. Herein, we reviewed key research advances in the study of biochemical mechanisms of PRMT catalysis and their relevance to cell biology. We highlighted how a random binary, ordered ternary kinetic model for PRMT1 catalysis reconciles the literature reports and endorses a distributive mechanism that the enzyme active site utilizes for multiple turnovers of arginine methylation. We discussed the impacts of histone arginine methylation and its biochemical interplays with other key epigenetic marks. Challenges in developing small-molecule PRMT inhibitors were also discussed.
Asunto(s)
Arginina/metabolismo , Histonas/metabolismo , Animales , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Histonas/antagonistas & inhibidores , Humanos , Metilación , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Proteína-Arginina N-Metiltransferasas/metabolismo , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismoRESUMEN
Although EGFR is a highly sought-after drug target, inhibitor resistance remains a challenge. As an alternative strategy for kinase inhibition, we sought to explore whether allosteric activation mechanisms could effectively be disrupted. The kinase domain of EGFR forms an atypical asymmetric dimer via head-to-tail interactions and serves as a requisite for kinase activation. The kinase dimer interface is primarily formed by the H-helix derived from one kinase monomer and the small lobe of the second monomer. We hypothesized that a peptide designed to resemble the binding surface of the H-helix may serve as an effective disruptor of EGFR dimerization and activation. A library of constrained peptides was designed to mimic the H-helix of the kinase domain and interface side chains were optimized using molecular modeling. Peptides were constrained using peptide "stapling" to structurally reinforce an alpha-helical conformation. Peptide stapling was demonstrated to notably enhance cell permeation of an H-helix derived peptide termed EHBI2. Using cell-based assays, EHBI2 was further shown to significantly reduce EGFR activity as measured by EGFR phosphorylation and phosphorylation of the downstream signaling substrate Akt. To our knowledge, this is the first H-helix-based compound targeting the asymmetric interface of the kinase domain that can successfully inhibit EGFR activation and signaling. This study presents a novel, alternative targeting site for allosteric inhibition of EGFR.
Asunto(s)
Receptores ErbB/metabolismo , Péptidos/química , Inhibidores de Proteínas Quinasas/química , Regulación Alostérica , Línea Celular Tumoral , Dimerización , Receptores ErbB/química , Humanos , Microscopía Fluorescente , Péptidos/síntesis química , Péptidos/metabolismo , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/metabolismo , Estructura Secundaria de ProteínaRESUMEN
Chemical modifications of the DNA and nucleosomal histones tightly control the gene transcription program in eukaryotic cells. The "histone code" hypothesis proposes that the frequency, combination, and location of post-translational modifications (PTMs) of the core histones compose a complex network of epigenetic regulation. Currently, there are at least 23 different types and >450 histone PTMs that have been discovered, and the PTMs of lysine and arginine residues account for a crucial part of the histone code. Although significant progress has been achieved in recent years, the molecular basis for the histone code is far from being fully understood. In this study, we investigated how naturally occurring N-terminal acetylation and PTMs of histone H4 lysine-5 (H4K5) affect arginine-3 methylation catalyzed by both type I and type II PRMTs at the biochemical level. Our studies found that acylations of H4K5 resulted in decreased levels of arginine methylation by PRMT1, PRMT3, and PRMT8. In contrast, PRMT5 exhibits an increased rate of arginine methylation upon H4K5 acetylation, propionylation, and crotonylation, but not upon H4K5 methylation, butyrylation, or 2-hydroxyisobutyrylation. Methylation of H4K5 did not affect arginine methylation by PRMT1 or PRMT5. There was a small increase in the rate of arginine methylation by PRMT8. Strikingly, a marked increase in the rate of arginine methylation was observed for PRMT3. Finally, N-terminal acetylation reduced the rate of arginine methylation by PRMT3 but had little influence on PRMT1, -5, and -8 activity. These results together highlight the underlying mechanistic differences in substrate recognition among different PRMTs and pave the way for the elucidation of the complex interplay of histone modifications.
Asunto(s)
Arginina/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Acetilación , Acilación , Secuencia de Aminoácidos , Arginina/química , Histonas/química , Humanos , Lisina/química , Proteínas de la Membrana/metabolismo , Metilación , Procesamiento Proteico-Postraduccional , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Represoras/metabolismoRESUMEN
Prostate cancer (PCa) is the second most common cause of cancer death among American men after lung cancer. Unfortunately, current therapies do not provide effective treatments for patients with advanced, metastatic, or hormone refractory disease. Therefore, we seek to generate therapeutic agents for a novel PCa treatment strategy by delivering a suicide enzyme (yCDtriple) to a cell membrane bound biomarker found on PCa cells (prostate-specific membrane antigen (PSMA)). This approach has resulted in a new PCa treatment strategy reported here as inhibitor-directed enzyme prodrug therapy (IDEPT). The therapeutic agents described were generated using a click chemistry reaction between the unnatural amino acid (p-azidophenylalanine (pAzF)) incorporated into yCDtriple and the dibenzylcyclooctyne moiety of our PSMA targeting agent (DBCO-PEG4-AH2-TG97). After characterization of the therapeutic agents, we demonstrate significant PCa cell killing of PSMA-positive cells. Importantly, we demonstrate that this click chemistry approach can be used to efficiently couple a therapeutic protein to a targeting agent and may be applicable to the ablation of other types of cancers and/or malignancies.
Asunto(s)
Antígenos de Superficie/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacología , Azidas/química , Azidas/farmacología , Glutamato Carboxipeptidasa II/metabolismo , Fenilalanina/análogos & derivados , Neoplasias de la Próstata/tratamiento farmacológico , Antineoplásicos/administración & dosificación , Antineoplásicos/síntesis química , Azidas/administración & dosificación , Azidas/síntesis química , Línea Celular Tumoral , Química Clic , Sistemas de Liberación de Medicamentos , Humanos , Masculino , Fenilalanina/administración & dosificación , Fenilalanina/síntesis química , Fenilalanina/química , Fenilalanina/farmacología , Profármacos/administración & dosificación , Profármacos/síntesis química , Profármacos/química , Profármacos/farmacología , Próstata/efectos de los fármacos , Próstata/metabolismo , Próstata/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patologíaRESUMEN
BACKGROUND: The enzyme-biomarker prostate-specific membrane antigen (PSMA) is an active target for imaging and therapeutic applications for prostate cancer. The internalization of PSMA has been shown to vary with inhibitors' mode of binding: irreversible, slowly reversible, and reversible. METHODS: In the present study, PSMA-targeted clickable derivatives of an irreversible phosphoramidate inhibitor DBCO-PEG(4) -CTT-54 (IC(50) = 1.0 nM) and a slowly reversible phosphate inhibitor, DBCO-PEG(4) -CTT-54.2 (IC(50) = 6.6 nM) were clicked to (99m) Tc(CO)(3) -DPA-azide to assemble a PSMA-targeted SPECT agent. The selectivity, percent uptake, and internalization of these PSMA-targeted SPECT agents were evaluated in PSMA-positive and PSMA-negative cells. RESULTS: In vitro studies demonstrated that PSMA-targeted SPECT agents exhibited selective cellular uptake in the PSMA-positive LNCaP cells compared to PSMA-negative PC3 cells. More importantly, it was found that (99m) Tc(CO)(3) -DPA-DBCO-PEG(4) -CTT-54 based on an irreversible PSMA inhibitor core, exhibited greater uptake and internalization than (99m) Tc(CO)(3) -DPA-DBCO-PEG(4) -CTT-54.2 constructed from a slowly reversible PSMA inhibitor core. CONCLUSIONS: We have demonstrated that a PSMA-targeted SPECT agent can be assembled efficiently using copper-less click chemistry. In addition, we demonstrated that mode of binding has an effect on internalization and percent uptake of PSMA-targeted SPECT agents; with the irreversible targeting agent demonstrating superior uptake and internalization in PSMA+ cells. The approach demonstrated in this work now supports a modular approach for the assembly of PSMA-targeted imaging and therapeutic agents.
Asunto(s)
Antígenos de Superficie/metabolismo , Sistemas de Liberación de Medicamentos/métodos , Glutamato Carboxipeptidasa II/metabolismo , Neoplasias de la Próstata/metabolismo , Tomografía Computarizada de Emisión de Fotón Único , Línea Celular Tumoral , Supervivencia Celular/fisiología , Humanos , Masculino , Neoplasias de la Próstata/diagnóstico , Neoplasias de la Próstata/patología , Unión Proteica/fisiología , Radioisótopos/metabolismoRESUMEN
Histone arginine methylation is a prevalent posttranslational modification (PTM) in eukaryotic cells and contributes to the histone codes for epigenetic regulation of gene transcription. In this study, we determined how local changes on adjacent residues in the histone H4 substrate regulate arginine asymmetric dimethylation and symmetric dimethylation catalysed by the major protein arginine methyltransferase (PRMT) enzymes PRMT1 and PRMT5, respectively. We found that phosphorylation at histone H4 Ser-1 site (H4S1) was inhibitory to activities of PRMT1 and PRMT5 in both monomethylating and dimethylating H4R3. Also, a positively charged H4K5 was important for PRMT1 catalysis because acetylation of H4K5 or the loss of the H4K5 ε-amine had a similar effect in reducing the catalytic efficiency of asymmetric dimethylation of H4R3. An opposite effect was observed in that acetylation of H4K5 or the loss of the H4K5 ε-amine enhanced PRMT5-mediated symmetric dimethylation of H4R3. Furthermore, we observed that N-terminal acetylation of H4 modestly decreased asymmetric dimethylation of H4R3 by PRMT1 and symmetric dimethylation of H4R3 by PRMT5. This work highlights the significance of local chemical changes in the substrate to regulating PRMT activity and unravels the pattern complexities and subtleties of histone codes.
Asunto(s)
Arginina , Epigénesis Genética , Arginina/química , Arginina/genética , Arginina/metabolismo , Metilación de ADN , Histonas/metabolismo , Proteína-Arginina N-Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/metabolismoRESUMEN
Aberrant expression of protein arginine methyltransferases (PRMTs) has been implicated in a number of cancers, making PRMTs potential therapeutic targets. But it remains not well understood how PRMTs impact specific oncogenic pathways. We previously identified PRMTs as important regulators of cell growth in neuroblastoma, a deadly childhood tumor of the sympathetic nervous system. Here, we demonstrate a critical role for PRMT1 in neuroblastoma cell survival. PRMT1 depletion decreased the ability of murine neuroblastoma sphere cells to grow and form spheres, and suppressed proliferation and induced apoptosis of human neuroblastoma cells. Mechanistic studies reveal the prosurvival factor, activating transcription factor 5 (ATF5) as a downstream effector of PRMT1-mediated survival signaling. Furthermore, a diamidine class of PRMT1 inhibitors exhibited anti-neuroblastoma efficacy both in vitro and in vivo. Importantly, overexpression of ATF5 rescued cell apoptosis triggered by PRMT1 inhibition genetically or pharmacologically. Taken together, our findings shed new insights into PRMT1 signaling pathway, and provide evidence for PRMT1 as an actionable therapeutic target in neuroblastoma.
RESUMEN
Kinases are amongst the largest families in the human proteome and serve as critical mediators of a myriad of cell signaling pathways. Since altered kinase activity is implicated in a variety of pathological diseases, kinases have become a prominent class of proteins for targeted inhibition. Although numerous small molecule and antibody-based inhibitors have already received clinical approval, several challenges may still exist with these strategies including resistance, target selection, inhibitor potency and in vivo activity profiles. Constrained peptide inhibitors have emerged as an alternative strategy for kinase inhibition. Distinct from small molecule inhibitors, peptides can provide a large binding surface area that allows them to bind shallow protein surfaces rather than defined pockets within the target protein structure. By including chemical constraints within the peptide sequence, additional benefits can be bestowed onto the peptide scaffold such as improved target affinity and target selectivity, cell permeability and proteolytic resistance. In this review, we highlight examples of diverse chemistries that are being employed to constrain kinase-targeting peptide scaffolds and highlight their application to modulate kinase signaling as well as their potential clinical implications.
Asunto(s)
Péptidos/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/efectos de los fármacos , Animales , Sitios de Unión , Diseño de Fármacos , Humanos , Terapia Molecular Dirigida , Unión Proteica , Proteínas Quinasas/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Glutamate carboxypeptidase II (GCPII) is a membrane-bound cell surface peptidase. There is significant interest in the inhibition of GCPII as a means of neuroprotection, while GCPII inhibition as a method to treat prostate cancer remains a topic of further investigation. The key zinc-binding functional group of the well-characterized classes of GCPII inhibitors (phosphonates and phosphoramidates) is tetrahedral and negatively charged at neutral pH, while glutamyl urea class of inhibitors possesses a planar and neutral zinc-binding group. This study explores a new class of GCPII inhibitors, glutamyl sulfamides, which possess a putative net neutral tetrahedral zinc-binding motif. A small library containing six sulfamides was prepared and evaluated for inhibitory potency against purified GCPII in an enzymatic assay. While most inhibitors have potencies in the micromolar range, one showed promising sub-micromolar potency, with the optimal inhibitor in this series being aspartyl-glutamyl sulfamide (2d). Lastly, computational docking was used to develop a tentative binding model on how the most potent inhibitors interact with the ligand-binding site of GCPII.
Asunto(s)
Glutamato Carboxipeptidasa II/antagonistas & inhibidores , Inhibidores de Proteasas/química , Sulfonamidas/química , Sitios de Unión , Diseño de Fármacos , Glutamato Carboxipeptidasa II/metabolismo , Humanos , Simulación del Acoplamiento Molecular , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/metabolismo , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Sulfonamidas/síntesis química , Sulfonamidas/metabolismoRESUMEN
Emergence of androgen-independent cancer cells during androgen deprivation therapy presents a significant challenge to successful treatment outcomes in prostate cancer. Elucidating the role of androgen deprivation in the transition from an androgen-dependent to an androgen-independent state may enable the development of more effective therapeutic strategies against prostate cancer. Herein, we describe an in vitro model for assessing the effects of continuous androgen-deprivation on prostate cancer cells (LNCaP) with respect to the expression of two prostate-specific markers: the androgen receptor (AR) and prostate-specific membrane antigen (PSMA). Compared with androgen-containing normal growth medium, androgen-deprived medium apparently induced the concomitant downregulation of AR and PSMA over time. Decreased protein levels were confirmed by fluorescence imaging, western blotting and enzymatic activity studies. In contrast to the current understanding of AR and PSMA in prostate cancer progression, our data demonstrated that androgen-deprivation induced a decrease in AR and PSMA levels in androgen-sensitive LNCaP cells, which may be associated with the development of more aggressive disease-state following androgen deprivation therapy.