AAA ATPases as therapeutic targets: Structure, functions, and small-molecule inhibitors

ATPases Associated with Diverse Cellular Activity (AAA ATPase) are essential enzymes found in all organisms. They are involved in various processes such as DNA replication, protein degradation, membrane fusion, microtubule serving, peroxisome biogenesis, signal transduction, and the regulation of gene expression. Due to the importance of AAA ATPases, several researchers identified and developed small-molecule inhibitors against these enzymes. We discuss six AAA ATPases that are potential drug targets and have well-developed inhibitors. We compare available structures that suggest significant differences of the ATP binding pockets among the AAA ATPases with or without ligand. The distances from ADP to the His20 in the His-Ser-His motif and the Arg finger (Arg353 or Arg378) in both RUVBL1/2 complex structures bound with or without ADP have significant differences, suggesting dramatically different interactions of the binding site with ADP. Taken together, the inhibitors of six well-studied AAA ATPases and their structural information suggest further development of specific AAA ATPase inhibitors due to difference in their structures. Future chemical biology coupled with proteomic approaches could be employed to develop variant specific, complex specific, and pathway specific inhibitors or activators for AAA ATPase proteins

A covalent p97/VCP ATPase inhibitor can overcome resistance to CB-5083 and NMS-873 in colorectal cancer cells

Small-molecule inhibitors of p97 are useful tools to study p97 function. Human p97 is an important AAA ATPase due to its diverse cellular functions and implication in mediating the turnover of proteins involved in tumorigenesis and virus infections. Multiple p97 inhibitors identified from previous high-throughput screening studies are thiol-reactive compounds targeting Cys522 in the D2 ATP-binding domain. Thus, these findings suggest a potential strategy to develop covalent p97 inhibitors. We first used purified p97 to assay several known covalent kinase inhibitors to determine if they can inhibit ATPase activity. We evaluated their selectivity using our dual reporter cells that can distinguish p97 dependent and independent degradation. We selected a β-nitrostyrene scaffold to further study the structure-activity relationship. In addition, we used p97 structures to design and synthesize analogues of pyrazolo[3,4-d]pyrimidine (PP). We incorporated electrophiles into a PP-like compound 17 (4-amino-1-tert-butyl-3-phenyl pyrazolo[3,4-d]pyrimidine) to generate eight compounds. A selective compound 18 (N-(1-(tert-butyl)-3-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)acrylamide, PPA) exhibited excellent selectivity in an in vitro ATPase activity assay: IC50 of 0.6 μM, 300 μM, and 100 μM for wild type p97, yeast Cdc48, and N-ethylmaleimide sensitive factor (NSF), respectively. To further examine the importance of Cys522 on the active site pocket during PPA inhibition, C522A and C522T mutants of p97 were purified and shown to increase IC50 values by 100-fold, whereas replacement of Thr532 of yeast Cdc48 with Cysteine decreased the IC50 by 10-fold. The molecular modeling suggested the hydrogen bonds and hydrophobic interactions in addition to the covalent bonding at Cys522 between WT-p97 and PPA. Furthermore, tandem mass spectrometry confirmed formation of a covalent bond between Cys522 and PPA. An anti-proliferation assay indicated that the proliferation of HCT116, HeLa, and RPMI8226 was inhibited by PPA with IC50 of 2.7 μM, 6.1 μM, and 3.4 μM, respectively. In addition, PPA is able to inhibit proliferation of two HCT116 cell lines that are resistant to CB-5083 and NMS-873, respectively. Proteomic analysis of PPA-treated HCT116 revealed Gene Ontology enrichment of known p97 functional pathways such as the protein ubiquitination and the ER to Golgi transport vesicle membrane. In conclusion, we have identified and characterized PPA as a selective covalent p97 inhibitor, which will allow future exploration to improve the potency of p97 inhibitors with different mechanisms of action

Analyzing Resistance to Design Chemical Inhibitors of AAA Proteins

Chemotype-specific resistance is a major factor limiting the efficacy of molecularly targeted therapeutics. Analyses of resistance mechanisms to drugs reveal that mutations conferring resistance often arise in the drug’s binding sites. As selective binding of inhibitors to their protein targets is mediated by specific interactions of the inhibitor with the protein backbone and side chains, mutations that disrupt these interactions can lead to resistance. Identifying resistance-conferring alleles can thus reveal and suggest the biochemical determinants of the drug’s selectivity and potency. In this thesis, I explore how mutations in active sites of proteins can be leveraged to understand inhibitor binding and to design selective chemical inhibitors. In my thesis work I focus on the design of chemical probes for proteins from the AAA (ATPase Associated with diverse cellular Activities) superfamily, for which only a few inhibitors are available. As the number of inhibitor-bound models of AAA proteins is also limited, the key protein-inhibitor interactions needed for design of probes for these proteins are not known. I have developed an approach - Resistance Analysis During Design - that involves testing selected heterocyclic scaffolds against wild-type protein and constructs with engineered mutations that retain enzymatic activity. These analyses, along with computational docking, can guide optimization of inhibitor potency and selectivity. We used this approach to design the pyrazolylpyrrolopyrimidine-based spastazoline - the first potent and selective chemical probe for spastin, a microtubule-severing AAA protein needed for cell division and intracellular vesicle transport. I confirmed the predicted binding mode of spastazoline analogs by X-ray crystallography and used these high-resolution structural models, along with biochemical analyses of spastin mutant alleles, to design an allele-specific inhibitor of spastin. Further, I show that RADD can be used to analyze the binding modes of diaminotriazole-based chemical inhibitors that are chemically unrelated to spastazoline. I also identified a more potent, diaminotriazole-based analog and used our approach to show that it binds spastin’s active site in a different orientation in comparison to the starting compound. The distinct binding modes of these compounds predicted by RADD also match the high-resolution models I generated using X-ray crystallography. Together, these data show how analyses of resistance can be useful at the early stages of the inhibitor design process. In summary, the work in my thesis outlines how mutations in active sites of proteins can be identified and how they could facilitate inhibitor design. I discuss how the binding models of spastin inhibitors I developed can inform on the design of new inhibitors for other AAA proteins and suggest experiments that could be valuable to advance these efforts. I also propose how analyses of resistance to chemical inhibitors could be valuable for designing drugs against which resistance might be less likely to arise. Věnováno babičce Anně Sochnové

Inhibition of VCP preserves retinal structure and function in autosomal dominant retinal degeneration

Due to continuously high production rates of rhodopsin (RHO) and high metabolic activity, photoreceptor neurons are especially vulnerable to defects in proteostasis. A proline to histidine substitution at position 23 (P23H) leads to production of structurally misfolded RHO, causing the most common form of autosomal dominant Retinitis Pigmentosa (adRP) in North America. The AAA-ATPase valosin-containing protein (VCP) extracts misfolded proteins from the ER membrane for cytosolic degradation. Here, we provide the first evidence that inhibition of VCP activity rescues degenerating P23H rod cells and improves their functional properties in P23H transgenic rat and P23H knock-in mouse retinae, both in vitro and in vivo. This improvement correlates with the restoration of the physiological RHO localization to rod outer segments (OS) and properly-assembled OS disks. As a single intravitreal injection suffices to deliver a long-lasting benefit in vivo, we suggest VCP inhibition as a potential therapeutic strategy for adRP patients carrying mutations in the RHO gene

Conserved L464 in p97 D1–D2 linker is critical for p97 cofactor regulated ATPase activity

p97 protein is a highly conserved, abundant, functionally diverse, structurally dynamic homohexameric AAA enzyme-containing N, D1, and D2 domains. A truncated p97 protein containing the N and D1 domains and the D1–D2 linker (ND1L) exhibits 79% of wild-type (WT) ATPase activity whereas the ND1 domain alone without the linker only has 2% of WT activity. To investigate the relationship between the D1–D2 linker and the D1 domain, we produced p97 ND1L mutants and demonstrated that this 22-residue linker region is essential for D1 ATPase activity. The conserved amino acid leucine 464 (L464) is critical for regulating D1 and D2 ATPase activity by p97 cofactors p37, p47, and Npl4–Ufd1 (NU). Changing leucine to alanine, proline, or glutamate increased the maximum rate of ATP turnover (k_(cat)) of p47-regulated ATPase activities for these mutants, but not for WT. p37 and p47 increased the k_(cat) of the proline substituted linker, suggesting that they induced linker conformations facilitating ATP hydrolysis. NU inhibited D1 ATPase activities of WT and mutant ND1L proteins, but activated D2 ATPase activity of full-length p97. To further understand the mutant mechanism, we used single-particle cryo-EM to visualize the full-length p97^(L464P) and revealed the conformational change of the D1–D2 linker, resulting in a movement of the helix-turn-helix motif (543–569). Taken together with the biochemical and structural results we conclude that the linker helps maintain D1 in a competent conformation and relays the communication to/from the N-domain to the D1 and D2 ATPase domains, which are ∼50 Å away

Novel mechanism of inhibition of the proteasome by Bruton’s Tyrosine Kinase inhibitors

Bruton’s tyrosine kinase (BTK) inhibitor Ibrutinib has been shown to synergize in vitro with proteasome inhibitors (PIs) in reducing the viability of cells derived from B-cell malignancies. This synergy was also observed in cells that do not express BTK and in cells that do express BTK, synergy was observed at concentrations much higher than the concentration at which BTK activity was completely inhibited; however, the mechanism responsible for synergy is not known. We report here that an off-target effect of Ibrutinib causes synergy and another BTK inhibitor, CGI-1746, showed even stronger synergy. We reveal through inhibition of the degradation of a model substrate by a purified 26S proteasome as well as inhibition of classical fluorescent proteasome substrates and inhibition of ATP hydrolysis by the 19S subunit of the proteasome, that CGI-1746 allosterically inhibits the proteasome’s ATPase and peptidase activities. We show that the inhibition of the proteasome by CGI-1746 is responsible for the synergy with proteasome inhibitors by comparing the cytotoxic mechanisms governing cell death due to CGI-1746 with FDA-approved proteasome inhibitors. We thereby demonstrate a conceptually novel mode of inhibition that may aid the development of more potent proteasome inhibitors and improve response in solid tumors clinically. We also demonstrate, using CRISPR mutated cell lines, that multi-site inhibition of the proteasome is more efficacious in reducing cancer cell viability compared to single-site inhibition and allows for significant dose reduction which may mitigate off-target adverse events. In addition, through knockdown of small nucleolar RNAs and proteomic analysis, we elucidate a previously unknown non-coding RNA mechanism that regulates proteostasis and modulates the response of cancer cells to proteasome inhibitio

Targeting vulnerabilities through inhibiting the valosin-containing protein (VCP/p97) in ovarian cancer

Ovarian cancer is the fifth leading cause of cancer-related death among women and the deadliest of all gynecological cancers. Treatment failure is a major contributing factor in ovarian cancer-related mortality. Advances in the development of new agents that target critical pathways in cancer may provide better options to overcome treatment failure. One of the molecular mechanisms that can be targeted for ovarian cancer therapy is the protein quality control. Components of the protein quality control, such as heat shock proteins, the ubiquitin-proteasome system, the unfolded protein response and autophagy, regulate protein homeostasis. Protein quality control also provides phenotypic stability by buffering cryptic genetic variations that could potentially reduce the fitness of cancer cells. This concept was best illustrated by studies that genetically or pharmacologically inhibited heat-shock protein 90, and reported variations in the phenotype following the disruption of heat shock protein 90. This dissertation investigates the therapeutic efficacy of targeting valosin-containing protein (VCP), an important component of the protein quality control, in ovarian cancer. VCP or p97, a member of the ATPase Associated with diverse cellular Activities-ATPase (AAA-ATPase) protein family, has been associated with various cellular functions including endoplasmic reticulum associated degradation, the ubiquitin proteasome system, golgi membrane reassembly, autophagy, DNA repair and cell division making it an important regulator of the protein quality control. Recent studies identified VCP and the ubiquitin proteasome system as synthetic lethal targets in ovarian cancer. This dissertation describes the preclinical activity of VCP inhibitors in ovarian cancer. Results presented in this dissertation show that quinazoline-based VCP inhibitors initiate Gap 1 (G1) cell cycle arrest, attenuate cap-dependent protein translation and induce programmed cell death via the intrinsic and the extrinsic modes of apoptosis. Mechanistic studies point to the unresolved unfolded protein response as a mechanism by which VCP inhibitors contribute to cytotoxicity. These results support an emerging concept that the unfolded protein response pathway may be targeted in ovarian cancer as a source of vulnerability. Since prolonged induction of the unfolded protein response results in CCAAT/enhancer binding protein homologous protein (CHOP) mediated cell death, we tested the hypothesis that VCP inhibitors act synergistically with compounds that enhance CHOP expression. Here, we show that VCP inhibitors act synergistically with salubrinal, an inhibitor of growth arrest and DNA-damage-inducible 34 (GADD34), by enhancing CHOP expression in ovarian cancer cell lines. Our results provide a proof-of-concept that VCP inhibitors can be used as a single agent and can be synergized with compounds that enhance CHOP expression to induce cell death. While the synergistic effects observed between VCP inhibitors and GADD34 inhibitor provides an in vitro proof-of-concept, these results have little clinical relevance because salubrinal is not a clinical candidate. Therefore, we identified mifepristone, an FDA approved steroidal progesterone antagonist, to act synergistic with VCP inhibitors across several ovarian cancer cell lines. Combination treatment between clinically achievable doses of mifepristone and oral VCP inhibitor- CB-5083 show an atypical increase in the unfolded protein response, a significant decrease in colony formation as well as an increase in caspase activity. Mechanistic studies upon mifepristone treatment point towards the inhibition of activating transcription factor 6 (ATF6) branch and the activation of heme- regulated inhibitor (HRI) kinase pathway as two plausible mechanisms contributing towards the synergistic effect. Oral VCP inhibitor- CB-5083 is currently in Phase I clinical trials because it shows therapeutic effects in multiple tumor xenograft models; however, the mechanism of resistance to CB-5083 is unknown. Here, we characterize the molecular mechanism of resistance to CB-5083. Using incremental exposure to CB-5083, we established CB-5083- resistant ovarian cancer cells that show 5 to 6-fold resistance in vitro compared to parental cells. Genomic and complementary DNA sequencing of the VCP coding region revealed a pattern of co-selected mutations: (1) missense mutations at codon 470 in one copy resulting in increased ATPase activity; and (2) nonsense or frameshift mutations at codon 606 or codon 616 in another copy causing the loss of allele-specific expression. Unbiased molecular docking studies show codon 470 as a putative binding site for CB-5083. Furthermore, the analysis of somatic mutations in cancer genomes from the cancer genome atlas (TCGA) indicate that codon 616 is frequently mutated in human cancers. Thus, identification of these mutations associated with in vitro resistance to VCP inhibitors may be useful as potential theranostic markers while screening for patients to enroll in clinical trials. VCP has emerged as a viable therapeutic target for several cancer types, and therefore targeting such hyperactive VCP mutants should aid in improving the therapeutic outcome in cancer patients. Overall, this dissertation 1) provides a proof-of-concept that VCP can be targeted in ovarian cancer, 2) proves that VCP inhibitors can act synergistically with other compounds that modulate the protein quality control, 3) identified a clinically relevant drug combination (CB-5083 and mifepristone) that should be investigated further, 4) unearths a novel mechanism of resistance towards the oral VCP inhibitor, CB-5083, in ovarian cancer cells

Estudio de la inmunogenicidad y mecanismos de muerte celular en nuevas terapias antitumorales. Aplicación al mieloma múltiple

En la investigación oncológica, el estudio de los mecanismos moleculares de muerte celular provocados por la quimioterapia es esencial no sólo para llegar a comprender los mecanismos de acción inherentes de estos fármacos, sino también para idear, optimizar y mejorar nuevos enfoques terapéuticos que permitan atajar las tan temidas recaídas. Además, durante los últimos años, la inmunoterapia ha irrumpido en la clínica como un tratamiento que brinda de una cierta esperanza a los pacientes con cáncer. Hoy en día, los tratamientos antitumorales utilizados o en desarrollo son capaces de inducir muerte celular mediante distintos mecanismos como la apoptosis, necroptosis, muerte celular inmunogénica (ICD), catástrofe mitótica, entre otras. Nuestro objetivo durante este trabajo ha sido estudiar los diferentes mecanismos de muerte celular inducidos por fármacos antitumorales actuales o de reciente introducción en la clínica, así como explorar la naturaleza inmunogénica subyacente a dichos tipos de muerte celular.Por un lado, se ha estudiado la contribución de la catástrofe mitótica en la inducción de muerte celular por agentes antimitóticos, así como los mecanismos de muerte accionados cuando dichos fármacos se utilizan en combinación con miméticos de BH3. Los resultados mostraron la existencia de una gran variabilidad inter- e intraindividual en los comportamientos y destinos celulares en respuesta a los distintos compuestos antimitóticos (barasertib, alisertib, vincristina y docetaxel). No obstante, la combinación de los distintos compuestos antimitóticos ensayados con miméticos de BH3 mostró una potenciación de la citotoxicidad en líneas celulares tumorales adherentes, siendo la muerte celular experimentada dependiente de caspasas y de Bax y Bak. En cuanto al papel de la familia de Bcl-2 en la muerte inducida por estos fármacos, los datos parecen indicar que los miembros anti-apoptóticos de esta familia contribuyen de forma cooperativa y acumulativa. Además, a pesar de que se trata un tema bajo debate, los experimentos con microscopía de fluorescencia en time-lapse sugieren que la duración del arresto mitótico influye en el destino ante dicho bloqueo. Cuando se combinó los compuestos antimitóticos con el mimético de BH3, ABT-737, se aceleraba la muerte celular y aumentaba el porcentaje de células que sucumbían durante la mitosis, especialmente en aquellas combinaciones donde el arresto mitótico era más prolongado. En el caso particular del inhibidor de aurora-B (barasertib), la potenciación de la muerte celular cuando se combinaba con ABT-737 sigue un mecanismo molecular diferente. El tratamiento con barasertib indujo marcadores de senescencia en diversas líneas celulares tumorales y la administración posterior de ABT-737 sensibilizaba de forma efectiva a dichas células induciendo una potente respuesta citotóxica. Por otro lado, en este trabajo se ha estudiado la inmunogenicidad y los mecanismos de muerte celular inducidos por la combinación de inhibidores del proteasoma con inhibidores de autofagia o compuestos inductores de estrés en el retículo endoplasmático en diversos modelos de mieloma múltiple. A pesar de que las terapias actuales han conseguido extender la esperanza de vida de esta enfermedad, sigue siendo una neoplasia incurable. En concreto, aunque los inhibidores de proteasoma han demostrado una validada eficacia clínica, la resistencia a estos fármacos sigue siendo recurrente y abarca la mayoría de las recidivas. Esta situación por tanto exige que se diseñen y estudien nuevos esquemas terapéuticos para abordar las recaídas. Así, en un trabajo previo del grupo se demostró la capacidad del inhibidor de autofagia cloroquina para potenciar la muerte celular inducida por carfilzomib. Durante este trabajo, se ha logrado observar también una intensificación similar de la muerte celular en las diferentes líneas celulares humanas y murinas analizadas. Además, la capacidad citotóxica mejorada de esta combinación también se ha constatado en una amplia colección de muestras primarias de médula ósea aisladas de pacientes con mieloma múltiple. De manera similar a lo que ocurre con la cloroquina, también hemos observado que el DBeQ, inhibidor de VCP/p97, también aumenta notablemente la muerte celular inducida por carfilzomib tanto en líneas celulares de MM como en células de mieloma primarias aisladas de la médula ósea de pacientes con esta enfermedad. Además, nuestros resultados indican que las combinaciones de carfilzomib con CLQ o DBeQ no sólo potencian, sino que también aceleran la muerte celular. Sin embargo, el mecanismo de acción por el cual se provoca dicha potenciación no se ha aclarado completamente. Los resultados de este trabajo mostraron que estos tratamientos aumentaban la expresión de varios marcadores de la respuesta a estrés en el retículo. No obstante, dicha respuesta, dependía estrechamente del tiempo y variaba sustancialmente entre las distintas líneas celulares de mieloma. La familia Bcl-2 ocupa una posición trascendental en las respuestas mediadas por estrés en el ER. A este respecto, nuestros datos revelaron una acumulación temprana transitoria de diversos miembros tanto anti- como proapoptóticos de esta familia (Mcl-1, Bim, PUMA y en menor medida NOXA). No obstante, nuestros datos mostraron que la línea celular deficiente para Bim sólo ofrecía una protección parcial contra la muerte celular inducida por los fármacos utilizados. Por lo tanto, su deficiencia podría estar compensada por otros miembros de BH3 capaces de desencadenar la muerte celular tras el tratamiento farmacológico. Por otro lado, aunque todavía no se conoce con exactitud el mecanismo molecular responsable de la muerte celular inducida por estrés en el ER, existen pruebas de la participación tanto de la vía de los receptores mortales como de la vía intrínseca de la apoptosis. Para instigar la muerte celular por la vía canónica, se requiere la permeabilización de la membrana externa mitocondrial (MOMP) inducida por la oligomerización de Bax y Bak. Sin embargo, nuestros datos han demostrado la capacidad de las combinaciones de fármacos utilizadas de inducir la muerte celular en células deficientes en Bax y Bak. Así mismo, la muerte celular inducida por las combinaciones basadas en carfilzomib era dependiente de caspasas en la mayoría de las líneas celulares analizadas, aunque la contribución relativa de cada miembro de esta familia variaba con las diferentes líneas celulares utilizadas. Aunque precisa de un estudio más detallado, los datos obtenidos en este trabajo parecen indicar que la autofagia no juega un papel principal en la potenciación ejercida por la cloroquina sobre la muerte inducida por carfilzomib en nuestros modelos experimentales. En su lugar, los datos apuntan a que la cloroquina pueda ejercer de modulador alostérico sobre el proteasoma potenciando la inhibición de este cuando se utiliza en combinación con carfilzomib. En cuanto al estudio de la capacidad inmunogénica de estos tratamientos, se observó que in vitro, las combinaciones de fármacos eran capaces de inducir la expresión de diversas señales inmunogénicas (expresión de ecto-CRT, Hsp70 y BiP), así como la maduración de células dendríticas cuando estas eran coincubadas con restos apoptóticos de células tratadas con las formulaciones ensayadas. Sin embargo, las respuestas in vivo en los experimentos de vacunación en el modelo ortotópico de mieloma murino MOPC315.BM mostraron que la muerte celular provocada por la combinación de carfilzomib con cloroquina no proporcionaba un efecto protector contra el desarrollo de la enfermedad. Tan solo cuando la combinación de fármacos utilizada para tratar las células de mieloma contenía el inhibidor general de caspasas zVAD-fmk, se consiguió retrasar de forma débil el desarrollo del mieloma. Tal y como apuntan algunos estudios, esto podría indicar que las caspasas juegan un papel en la inmunogenicidad de la muerte celular. A la vista de nuestros datos, la emisión de DAMPs por sí misma podría no ser suficiente para provocar respuestas inmunitarias activas contra el cáncer. De hecho, se considera igualmente decisivo los mecanismos subyacentes involucrados en la recepción, transmisión y respuesta de las células inmunes a estas señales de peligro, así como la propia naturaleza inmunosupresora de esta enfermedad. Una de las razones detrás de la disfunción inmune observada en el mieloma múltiple está mediada por la regulación negativa ejercida por las proteínas inhibidoras del punto de control como el eje PD-1/PD-L1. Sin embargo, ni el tratamiento individual con anticuerpos monoclonales dirigidos contra los inhibidores del punto de control (anti-PD1), ni su combinación con la formulación de vacunación antes mencionada, extendían significativamente la supervivencia de los ratones. Es posible que redes moleculares mas complejas estén involucradas en la generación de una respuesta inmune antitumoral efectiva en la enfermedad de mieloma.Por último, cada vez se está consolidando más la idea de que los DAMPs y los procesos moleculares relacionados con la muerte celular inmunogénica puedan servir como una fuente de biomarcadores pronósticos en pacientes con cáncer. En este trabajo hemos demostrado por primera vez que las células de mieloma en muestras de médula ósea aisladas de pacientes con discrasias de células plasmáticas, muestran niveles elevados de ecto-CRT en su superficie. Además, aunque se observó una gran variabilidad interindividual, nuestros datos sugieren que los niveles de ecto-CRT parecen aumentar con la progresión de la enfermedad. Este hallazgo junto con el hecho de que los pacientes con un perfil citogenético alterado mostraron niveles aumentados de ecto-CRT y que la exposición a la CRT aparentemente no está influenciada por la quimioterapia, puede apuntar a la transformación maligna como el instigador principal de la expresión aumentada de este DAMP. Así mismo el análisis del microambiente en la médula ósea de estos pacientes mostró que los pacientes con niveles altos de ecto-CRT exhibían un perfil de células T alterado, con ratios bajos de células T CD4+/CD8+ consecuencia de una menor frecuencia de linfocitos T CD4+ y un mayor número de células T CD8+. Además, también presentaban un mayor número de células NK, mDC, pDC y Tregs, así como una mayor actividad en el eje PD-1/PD-L1. Así, nuestros datos sugieren que los pacientes con una mayor expresión de este DAMP, poseen rasgos inmunológicos reminiscentes de un microambiente medular óseo asociado a un estado inmunitario debilitado y comprometido, lo que podría traducirse en un mal resultado clínico en el contexto de la enfermedad. De hecho, el grupo de pacientes con un perfil de expresión de ecto-CRT aumentado exhibieron significativamente un menor tiempo medio de progresión de la enfermedad, desarrollaban con mayor frecuencia plasmacitomas extramedulares, habían sido tratados con un mayor número de líneas de tratamiento y albergaban un perfil citogenético de alto riesgo. Todo ello sugiere que la elevada expresión de ecto-CRT en las células de mieloma de estos pacientes está relacionada con una mayor malignidad, un microambiente inmunitario en la médula más deficiente y con un peor pronóstico clínico.<br /

4,6-Diphenyl-pyridines/pyrimidines and pyrazolo[3,4-d]pyrimidines: promising scaffolds as antiviral, anticancer and theranostic agents.

Nitrogen-based heterocyclic molecules received increasing attention in biological and chemical sciences, becoming a significant moiety in drug design. In this thesis, the research work has been focused on the design and the synthesis of novel derivatives based on the 4,6-diphenyl-pyridine/pyrimidine and pyrazolo[3,4-d]pyrimidine scaffolds as key pharmacophores for their antiviral and anticancer activities, respectively. The first part deals with the design and synthesis of a set of small molecules able to interfere with the Influenza (flu) RNA-dependent RNA Polymerase (RdRp) functions, exploiting the protein-protein interactions (PPIs) approach. An introduction of influenza disease and its pathogens is provided, focusing on the replicative mechanism of the influenza viruses and on the structural and functional information of the flu RdRp. The PPIs of the three polymerase subunits PA, PB1 and PB2, are reported and the inhibitors targeting the heterodimer PA-PB1 are introduced. Finally, the rational design and synthesis of new hybrid compounds bearing the 4,6-diphenyl-pyridine/pyrimidine cores are described togheter with the biological evaluation and molecular modeling studies. The second part of the dissertation focuses on the pyrazolo[3,4-d]pyrimidine compounds, on which both a lead optimization study and a theranostic design have been performed. The pyrazolo[3,4-d]pyrimidines have shown a promising activity both in in vitro and in in vivo as protein kinase inhibitors. The lead optimization study has been performed with the aim of obtaining a new class of derivatives as potent Src kinase inhibitors. The rational design, synthesis and biological analysis of the novel compounds are discussed. A further part of the project is dedicated to the design and development of potential theranostic prodrugs of two promising in-house pyrazolo[3,4-d]pyrimidines, SI306 and SI113. These prodrugs have been synthesized as potential agents for the diagnosis and the treatment of glioblastoma multiforme (GBM). The state of the art on theranostic applications of drugs, whose use is continually increasing, is also described

Akt enhances the vulnerability of cancer cells to VCP/p97 inhibition-mediated paraptosis

Valosin-containing protein (VCP)/p97, an AAA+ ATPase critical for maintaining proteostasis, emerges as a promising target for cancer therapy. This study reveals that targeting VCP selectively eliminates breast cancer cells while sparing non-transformed cells by inducing paraptosis, a non-apoptotic cell death mechanism characterized by endoplasmic reticulum and mitochondria dilation. Intriguingly, oncogenic HRas sensitizes non-transformed cells to VCP inhibition-mediated paraptosis. The susceptibility of cancer cells to VCP inhibition is attributed to the non-attenuation and recovery of protein synthesis under proteotoxic stress. Mechanistically, mTORC2/Akt activation and eIF3d-dependent translation contribute to translational rebound and amplification of proteotoxic stress. Furthermore, the ATF4/DDIT4 axis augments VCP inhibition-mediated paraptosis by activating Akt. Given that hyperactive Akt counteracts chemotherapeutic-induced apoptosis, VCP inhibition presents a promising therapeutic avenue to exploit Akt-associated vulnerabilities in cancer cells by triggering paraptosis while safeguarding normal cells