RESUMEN
The structure-based design of small-molecule inhibitors targeting protein-protein interactions (PPIs) remains a huge challenge as the drug must bind typically wide and shallow protein sites. A PPI target of high interest for hematological cancer therapy is myeloid cell leukemia 1 (Mcl-1), a prosurvival guardian protein from the Bcl-2 family. Despite being previously considered undruggable, seven small-molecule Mcl-1 inhibitors have recently entered clinical trials. Here, we report the crystal structure of the clinical-stage inhibitor AMG-176 bound to Mcl-1 and analyze its interaction along with clinical inhibitors AZD5991 and S64315. Our X-ray data reveal high plasticity of Mcl-1 and a remarkable ligand-induced pocket deepening. Nuclear Magnetic Resonance (NMR)-based free ligand conformer analysis demonstrates that such unprecedented induced fit is uniquely achieved by designing highly rigid inhibitors, preorganized in their bioactive conformation. By elucidating key chemistry design principles, this work provides a roadmap for targeting the largely untapped PPI class more successfully.
Asunto(s)
Apoptosis , Naftalenos , Modelos Moleculares , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , LigandosRESUMEN
PARP-1 is a key early responder to DNA damage in eukaryotic cells. An allosteric mechanism links initial sensing of DNA single-strand breaks by PARP-1's F1 and F2 domains via a process of further domain assembly to activation of the catalytic domain (CAT); synthesis and attachment of poly(ADP-ribose) (PAR) chains to protein sidechains then signals for assembly of DNA repair components. A key component in transmission of the allosteric signal is the HD subdomain of CAT, which alone bridges between the assembled DNA-binding domains and the active site in the ART subdomain of CAT. Here we present a study of isolated CAT domain from human PARP-1, using NMR-based dynamics experiments to analyse WT apo-protein as well as a set of inhibitor complexes (with veliparib, olaparib, talazoparib and EB-47) and point mutants (L713F, L765A and L765F), together with new crystal structures of the free CAT domain and inhibitor complexes. Variations in both dynamics and structures amongst these species point to a model for full-length PARP-1 activation where first DNA binding and then substrate interaction successively destabilise the folded structure of the HD subdomain to the point where its steric blockade of the active site is released and PAR synthesis can proceed.
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Poli(ADP-Ribosa) Polimerasa-1/química , Regulación Alostérica , Amidas/química , Dominio Catalítico , Cristalografía por Rayos X , Daño del ADN , Activación Enzimática , Modelos Moleculares , Mutación , Poli(ADP-Ribosa) Polimerasa-1/antagonistas & inhibidores , Poli(ADP-Ribosa) Polimerasa-1/genética , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas/química , Dominios ProteicosRESUMEN
The receptor tyrosine kinase, MERTK, plays an essential role in homeostasis of the retina via efferocytosis of shed outer nuclear segments of photoreceptors. The Royal College of Surgeons rat model of retinal degeneration has been linked to loss-of-function of MERTK, and together with the MERTK knock-out mouse, phenocopy retinitis pigmentosa in humans with MERTK mutations. Given recent efforts and interest in MERTK as a potential immuno-oncology target, development of a strategy to assess ocular safety at an early pre-clinical stage is critical. We have applied a state-of-the-art, multi-modal imaging platform to assess the in vivo effects of pharmacological inhibition of MERTK in mice. This involved the application of mass spectrometry imaging (MSI) to characterize the ocular spatial distribution of our highly selective MERTK inhibitor; AZ14145845, together with histopathology and transmission electron microscopy to characterize pathological and ultra-structural change in response to MERTK inhibition. In addition, we assessed the utility of a human retinal in vitro cell model to identify perturbation of phagocytosis post MERTK inhibition. We identified high localized total compound concentrations in the retinal pigment epithelium (RPE) and retinal lesions following 28 days of treatment with AZ14145845. These lesions were present in 4 of 8 treated animals, and were characterized by a thinning of the outer nuclear layer, loss of photoreceptors (PR) and accumulation of photoreceptor outer segments at the interface of the RPE and PRs. Furthermore, the lesions were very similar to that shown in the RCS rat and MERTK knock-out mouse, suggesting a MERTK-induced mechanism of PR cell death. This was further supported by the observation of reduced phagocytosis in the human retinal cell model following treatment with AZ14145845. Our study provides a viable, translational strategy to investigate the pre-clinical toxicity of MERTK inhibitors but is equally transferrable to novel chemotypes.
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Fagocitosis/efectos de los fármacos , Células Fotorreceptoras de Vertebrados/efectos de los fármacos , Tirosina Quinasa c-Mer/antagonistas & inhibidores , Animales , Línea Celular , Femenino , Humanos , Masculino , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Imagen Multimodal , Células Fotorreceptoras de Vertebrados/patología , Ratas , Ratas Long-Evans , Ratas Wistar , Degeneración Retiniana/inducido químicamente , Epitelio Pigmentado de la Retina/metabolismo , Distribución Tisular , Tirosina Quinasa c-Mer/genéticaRESUMEN
Free Energy Perturbation (FEP) calculations can provide high-confidence predictions of the interaction strength between a ligand and its protein target. We sought to explore a series of triazolopyrimidines which bind to the EED subunit of the PRC2 complex as potential anticancer therapeutics, using FEP calculations to inform compound design. Combining FEP predictions with a late-stage functionalisation (LSF) inspired synthetic approach allowed us to rapidly evaluate structural modifications in a previously unexplored region of the EED binding site. This approach generated a series of novel triazolopyrimidine EED ligands with improved physicochemical properties and which inhibit PRC2 methyltransferase activity in a cancer-relevant G401 cell line.
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Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Complejo Represivo Polycomb 2/antagonistas & inhibidores , Purinas/farmacología , Termodinámica , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Ligandos , Microsomas Hepáticos/química , Microsomas Hepáticos/metabolismo , Estructura Molecular , Complejo Represivo Polycomb 2/metabolismo , Purinas/síntesis química , Purinas/química , Teoría Cuántica , Relación Estructura-ActividadRESUMEN
The activation loop (A-loop) plays a key role in regulating the catalytic activity of protein kinases. Phosphorylation in this region enhances the phosphoryl transfer rate of the kinase domain and increases its affinity for ATP. Furthermore, the A-loop possesses autoinhibitory functions in some kinases, where it collapses onto the protein surface and blocks substrate binding when unphosphorylated. Due to its flexible nature, the A-loop is usually disordered and untraceable in kinase domain crystal structures. The resulting lack of structural information is regrettable as it impedes the design of drug A-loop contacts, which have proven favourable in multiple cases. Here, we characterize the binding with A-loop engagement between type 1.5 kinase inhibitor 'example 172' (EX172) and Mer tyrosine kinase (MerTK). With the help of crystal structures and binding kinetics, we portray how the recruitment of the A-loop elicits a two-step binding mechanism which results in a drug-target complex characterized by high affinity and long residence time. In addition, the type 1.5 compound possesses excellent kinome selectivity and a remarkable preference for the phosphorylated over the dephosphorylated form of MerTK. We discuss these unique characteristics in the context of known type 1 and type 2 inhibitors and highlight opportunities for future kinase inhibitor design.
Asunto(s)
Adenosina Trifosfato/química , Inhibidores de Proteínas Quinasas/química , Tirosina Quinasa c-Mer/antagonistas & inhibidores , Tirosina Quinasa c-Mer/química , Humanos , Estructura Secundaria de ProteínaRESUMEN
Targeted covalent inhibition of disease-associated proteins has become a powerful methodology in the field of drug discovery, leading to the approval of new therapeutics. Nevertheless, current approaches are often limited owing to their reliance on a cysteine residue to generate the covalent linkage. Here we used aryl boronic acid carbonyl warheads to covalently target a noncatalytic lysine side chain, and generated to our knowledge the first reversible covalent inhibitors for Mcl-1, a protein-protein interaction (PPI) target that has proven difficult to inhibit via traditional medicinal chemistry strategies. These covalent binders exhibited improved potency in comparison to noncovalent congeners, as demonstrated in biochemical and cell-based assays. We identified Lys234 as the residue involved in covalent modification, via point mutation. The covalent binders discovered in this study will serve as useful starting points for the development of Mcl-1 therapeutics and probes to interrogate Mcl-1-dependent biological phenomena.
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Ácidos Borónicos/química , Ácidos Borónicos/farmacología , Lisina/química , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Ácidos Borónicos/síntesis química , Relación Dosis-Respuesta a Droga , Humanos , Concentración de Iones de Hidrógeno , Lisina/metabolismo , Estructura Molecular , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/química , Relación Estructura-ActividadRESUMEN
The duration of action of adenosine A2A receptor (A2A) agonists is critical for their clinical efficacy, and we sought to better understand how this can be optimized. The in vitro temporal response profiles of a panel of A2A agonists were studied using cAMP assays in recombinantly (CHO) and endogenously (SH-SY5Y) expressing cells. Some agonists (e.g., 3cd; UK-432,097) but not others (e.g., 3ac; CGS-21680) demonstrated sustained wash-resistant agonism, where residual receptor activation continued after washout. The ability of an antagonist to reverse pre-established agonist responses was used as a surrogate read-out for agonist dissociation kinetics, and together with radioligand binding studies suggested a role for slow off-rate in driving sustained effects. One compound, 3ch, showed particularly marked sustained effects, with a reversal t1/2 > 6 hours and close to maximal effects that remained for at least 5 hours after washing. Based on the structure-activity relationship of these compounds, we suggest that lipophilic N6 and bulky C2 substituents can promote stable and long-lived binding events leading to sustained agonist responses, although a high compound logD is not necessary. This provides new insight into the binding interactions of these ligands and we anticipate that this information could facilitate the rational design of novel long-acting A2A agonists with improved clinical efficacy.
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Agonistas del Receptor de Adenosina A2/química , Agonistas del Receptor de Adenosina A2/farmacología , Animales , Técnicas Biosensibles , Células CHO , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Cricetinae , Cricetulus , Células HEK293 , Humanos , Cinética , Ensayo de Unión Radioligante , Receptor de Adenosina A2A/metabolismo , Relación Estructura-Actividad , Factores de TiempoRESUMEN
The development of orally bioavailable PROTACs presents a significant challenge due to the inflated physicochemical properties of such heterobifunctional molecules. Molecules occupying this "beyond rule of five" space often demonstrate limited oral bioavailability due to the compounding effects of elevated molecular weight and hydrogen bond donor count (among other properties), but it is possible to achieve sufficient oral bioavailability through physicochemical optimization. Herein, we disclose the design and evaluation of a low hydrogen bond donor count (≤1 HBD) fragment screening set to aid hit generation of PROTACs intended for an oral route of delivery. We demonstrate that application of this library can enhance fragment screens against PROTAC proteins of interest and ubiquitin ligases, yielding fragment hits containing ≤1 HBD suitable for optimizing toward orally bioavailable PROTACs.
Asunto(s)
Proteínas , Quimera Dirigida a la Proteólisis , Enlace de Hidrógeno , Proteínas/metabolismo , Disponibilidad Biológica , Administración Oral , Proteolisis , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Surface plasmon resonance (SPR) is a widely used method to study ligand-protein interactions. The throughput and sensitivity of SPR has made it an important technology for measuring low-affinity, ultralow weight fragments (<200 Da) in the early stages of drug discovery. However, the biochemistry of membrane proteins, such as G-protein-coupled receptors (GPCRs), makes their SPR fragment screening particularly challenging, especially for native/wild-type, nonthermostabilized mutant receptors. In this study, we demonstrate the use of SPR-based biosensors to study the entire human family of adenosine receptors and present biologically active novel binders with a range of selectivity to human adenosine 2a receptor (hA2AR) from an ultralow weight fragment library and the public GlaxoSmithKline (GSK) kinase library. Thus, we demonstrate the ability of SPR to screen ultra-low-affinity fragments and identify biologically meaningful chemical equity and that SPR campaigns are highly effective "chemical filters" for screening small building block fragments that can be used to enable drug discovery programs.
RESUMEN
Fragment based drug discovery is a critical part of the lead generation toolbox and relies heavily on a readily available, high quality fragment library. Over years of use, the AstraZeneca fragment set had become partially depleted and instances of compound deterioration had been found. It was recognised that a redevelopment was required. This provided an opportunity to evolve our screening sets strategy, whilst ensuring that the quality of the fragment set met the robust requirements of fragment screening campaigns. In this communication we share the strategy employed, in particular highlighting two aspects of our approach that we believe others in the community would benefit from, namely that; (i) fragments were selected with input from Medicinal Chemists at an early stage, and (ii) the library was arranged in a layered format to ensure maximum flexibility on a per target basis.
RESUMEN
A key activity in small-molecule drug discovery is the characterization of compound-target interactions. Surface plasmon resonance (SPR) is a flexible technique for this purpose, with a wide affinity range (micromoles to picomoles), low protein requirements, and the ability to characterize the kinetics of compound binding. However, a key requirement of SPR is the immobilization of the target protein to the surface of the sensor chip. The most commonly used immobilization techniques (covalent immobilization, streptavidin-biotin) are irreversible in nature, which can afford excellent baseline stability but impose limitations throughput for slowly dissociating compounds or unstable targets. Reversible immobilization (e.g., His-tag-Ni-NTA) is possible but typically precludes accurate quantification of slow dissociation kinetics due to baseline drift.Here we present our investigation of three immobilization strategies (dual-His-tagged target protein, His-tagged streptavidin, and switchavidin) that combine the robustness of irreversible immobilization with the flexibility of reversible immobilization. Each has its own advantages and limitations, and while a universal immobilization procedure remains to be found, these strategies add to the immobilization toolbox that enables previously out-of-scope applications. Such applications are highlighted in two examples that greatly increased throughput for the kinetic characterization of potent kinase inhibitors and kinetic profiling of covalent inhibitors.
Asunto(s)
Técnicas Biosensibles/métodos , Descubrimiento de Drogas/métodos , Resonancia por Plasmón de Superficie/métodos , Humanos , Cinética , Bibliotecas de Moléculas PequeñasRESUMEN
Mer is a member of the TAM (Tyro3, Axl, Mer) kinase family that has been associated with cancer progression, metastasis, and drug resistance. Their essential function in immune homeostasis has prompted an interest in their role as modulators of antitumor immune response in the tumor microenvironment. Here we illustrate the outcomes of an extensive lead-generation campaign for identification of Mer inhibitors, focusing on the results from concurrent, orthogonal high-throughput screening approaches. Data mining, HT (high-throughput), and DECL (DNA-encoded chemical library) screens offered means to evaluate large numbers of compounds. We discuss campaign strategy and screening outcomes, and exemplify series resulting from prioritization of hits that were identified. Concurrent execution of HT and DECL screening successfully yielded a large number of potent, selective, and novel starting points, covering a range of selectivity profiles across the TAM family members and modes of kinase binding, and offered excellent start points for lead development.
Asunto(s)
Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Tirosina Quinasa c-Mer/antagonistas & inhibidores , Animales , Cristalografía por Rayos X , Minería de Datos , Descubrimiento de Drogas , Humanos , Modelos Moleculares , Tirosina Quinasa c-Mer/química , Tirosina Quinasa c-Mer/metabolismoRESUMEN
Inhibition of Mer and Axl kinases has been implicated as a potential way to improve the efficacy of current immuno-oncology therapeutics by restoring the innate immune response in the tumor microenvironment. Highly selective dual Mer/Axl kinase inhibitors are required to validate this hypothesis. Starting from hits from a DNA-encoded library screen, we optimized an imidazo[1,2-a]pyridine series using structure-based compound design to improve potency and reduce lipophilicity, resulting in a highly selective in vivo probe compound 32. We demonstrated dose-dependent in vivo efficacy and target engagement in Mer- and Axl-dependent efficacy models using two structurally differentiated and selective dual Mer/Axl inhibitors. Additionally, in vivo efficacy was observed in a preclinical MC38 immuno-oncology model in combination with anti-PD1 antibodies and ionizing radiation.
Asunto(s)
Antineoplásicos/uso terapéutico , Imidazoles/uso terapéutico , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Piridinas/uso terapéutico , Animales , Antineoplásicos/síntesis química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Imidazoles/síntesis química , Masculino , Ratones Endogámicos C57BL , Ratones Desnudos , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Proteínas Proto-Oncogénicas/metabolismo , Piridinas/síntesis química , Proteínas Tirosina Quinasas Receptoras/metabolismo , Relación Estructura-Actividad , Tirosina Quinasa c-Mer/metabolismo , Tirosina Quinasa del Receptor AxlRESUMEN
Multidrug transporters can confer drug resistance on cells by extruding structurally unrelated compounds from the cellular interior. In transport assays, Hoechst 33342 (referred to as Hoechst) is a commonly used substrate, the fluorescence of which changes in the transport process. With three basic nitrogen atoms that can be protonated, Hoechst can exist as cationic and neutral species that have different fluorescence emissions and different abilities to diffuse across cell envelopes and interact with lipids and intracellular nucleic acids. Due to this complexity, the mechanism of Hoechst transport by multidrug transporters is poorly characterised. We investigated Hoechst transport by the bacterial major facilitator superfamily multidrug-proton antiporter LmrP in Lactococcus lactis and developed a novel assay for the direct quantitation of cell-associated Hoechst. We observe that changes in Hoechst fluorescence in cells do not always correlate with changes in the amount of Hoechst. Our data indicate that chemical proton gradient-dependent efflux by LmrP in cells converts populations of highly fluorescent, membrane-intercalated Hoechst in the alkaline interior into populations of less fluorescent, cell surface-bound Hoechst in the acidic exterior. Our methods and findings are directly relevant for the transport of many amphiphilic antibiotics, antineoplastic agents and cytotoxic compounds that are differentially protonated within the physiological pH range.
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Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Resistencia a Múltiples Medicamentos , Lactococcus lactis/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Protones , Proteínas Bacterianas/genética , Transporte Biológico , Membrana Celular/efectos de los fármacos , Lactococcus lactis/efectos de los fármacos , Lactococcus lactis/crecimiento & desarrollo , Proteínas de Transporte de Membrana/genética , Mutagénesis Sitio-Dirigida , MutaciónRESUMEN
Apoptosis is a crucial process by which multicellular organisms control tissue growth, removal and inflammation. Disruption of the normal apoptotic function is often observed in cancer, where cell death is avoided by the overexpression of anti-apoptotic proteins of the Bcl-2 (B-cell lymphoma 2) family, including Mcl-1 (myeloid cell leukaemia 1). This makes Mcl-1 a potential target for drug therapy, through which normal apoptosis may be restored by inhibiting the protective function of Mcl-1. Here, the discovery and biophysical properties of an anti-Mcl-1 antibody fragment are described and the utility of both the scFv and Fab are demonstrated in generating an Mcl-1 crystal system amenable to iterative structure-guided drug design.
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Descubrimiento de Drogas , Fragmentos Fab de Inmunoglobulinas/química , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/química , Anticuerpos de Cadena Única/química , Animales , Apoptosis , Células CHO , Clonación Molecular , Cricetulus , Escherichia coli/genética , HumanosRESUMEN
Enhancer of zeste homologue 2 (EZH2), the catalytic subunit of polycomb repressive complex 2 (PRC2), regulates chromatin state and gene expression by methylating histone H3 lysine 27. EZH2 is overexpressed or mutated in various hematological malignancies and solid cancers. Our previous efforts to identify inhibitors of PRC2 methyltransferase activity by high-throughput screening (HTS) resulted in large numbers of false positives and thus a significant hit deconvolution challenge. More recently, others have reported compounds that bind to another PRC2 core subunit, EED, and allosterically inhibit EZH2 activity. This mechanism is particularly appealing as it appears to retain potency in cell lines that have acquired resistance to orthosteric EZH2 inhibition. By designing a fluorescence polarization probe based on the reported EED binding compounds, we were able to quickly and cleanly re-triage our previously challenging HTS hit list and identify novel allosteric PRC2 inhibitors.
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Benzofuranos/química , Inhibidores Enzimáticos/química , Complejo Represivo Polycomb 2/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/química , Regulación Alostérica/efectos de los fármacos , Benzofuranos/metabolismo , Carbocianinas/química , Línea Celular Tumoral , Inhibidores Enzimáticos/metabolismo , Colorantes Fluorescentes/química , Ensayos Analíticos de Alto Rendimiento , Humanos , Ligandos , Complejo Represivo Polycomb 2/aislamiento & purificación , Complejo Represivo Polycomb 2/metabolismo , Unión Proteica , Bibliotecas de Moléculas Pequeñas/metabolismoRESUMEN
Mcl-1 is a member of the Bcl-2 family of proteins that promotes cell survival by preventing induction of apoptosis in many cancers. High expression of Mcl-1 causes tumorigenesis and resistance to anticancer therapies highlighting the potential of Mcl-1 inhibitors as anticancer drugs. Here, we describe AZD5991, a rationally designed macrocyclic molecule with high selectivity and affinity for Mcl-1 currently in clinical development. Our studies demonstrate that AZD5991 binds directly to Mcl-1 and induces rapid apoptosis in cancer cells, most notably myeloma and acute myeloid leukemia, by activating the Bak-dependent mitochondrial apoptotic pathway. AZD5991 shows potent antitumor activity in vivo with complete tumor regression in several models of multiple myeloma and acute myeloid leukemia after a single tolerated dose as monotherapy or in combination with bortezomib or venetoclax. Based on these promising data, a Phase I clinical trial has been launched for evaluation of AZD5991 in patients with hematological malignancies (NCT03218683).
Asunto(s)
Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Leucemia Mieloide Aguda/tratamiento farmacológico , Mieloma Múltiple/tratamiento farmacológico , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Animales , Bortezomib/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Línea Celular Tumoral , Cristalografía por Rayos X , Humanos , Leucemia Mieloide Aguda/patología , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Mieloma Múltiple/patología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Ratas , Ratas Desnudas , Sulfonamidas/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
B-cell lymphoma 6 (BCL6) inhibition is a promising mechanism for treating hematological cancers but high quality chemical probes are necessary to evaluate its therapeutic potential. Here we report potent BCL6 inhibitors that demonstrate cellular target engagement and exhibit exquisite selectivity for BCL6 based on mass spectrometry analyses following chemical proteomic pull down. Importantly, a proteolysis-targeting chimera (PROTAC) was also developed and shown to significantly degrade BCL6 in a number of diffuse large B-cell lymphoma (DLBCL) cell lines, but neither BCL6 inhibition nor degradation selectively induced marked phenotypic response. To investigate, we monitored PROTAC directed BCL6 degradation in DLBCL OCI-Ly1 cells by immunofluorescence and discovered a residual BCL6 population. Analysis of subcellular fractions also showed incomplete BCL6 degradation in all fractions despite having measurable PROTAC concentrations, together providing a rationale for the weak antiproliferative response seen with both BCL6 inhibitor and degrader. In summary, we have developed potent and selective BCL6 inhibitors and a BCL6 PROTAC that effectively degraded BCL6, but both modalities failed to induce a significant phenotypic response in DLBCL despite achieving cellular concentrations.
Asunto(s)
Antineoplásicos/farmacología , Proteínas Proto-Oncogénicas c-bcl-6/antagonistas & inhibidores , Quinolonas/farmacología , Talidomida/análogos & derivados , Talidomida/farmacología , Proteínas Adaptadoras Transductoras de Señales , Antineoplásicos/síntesis química , Antineoplásicos/metabolismo , Línea Celular Tumoral , Células HEK293 , Humanos , Ligandos , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Péptido Hidrolasas/metabolismo , Unión Proteica , Proteolisis , Proteínas Proto-Oncogénicas c-bcl-6/química , Proteínas Proto-Oncogénicas c-bcl-6/metabolismo , Quinolonas/síntesis química , Quinolonas/metabolismo , Talidomida/síntesis química , Talidomida/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Surface plasmon resonance (SPR) is a powerful method for obtaining detailed molecular interaction parameters. Modern instrumentation with its increased throughput has enabled routine screening by SPR in hit-to-lead and lead optimization programs, and SPR has become a mainstream drug discovery technology. However, the processing and reporting of SPR data in drug discovery are typically performed manually, which is both time-consuming and tedious. Here, we present the workflow concept, design and experiences with a software module relying on a single, browser-based software platform for the processing, analysis, and reporting of SPR data. The efficiency of this concept lies in the immediate availability of end results: data are processed and analyzed upon loading the raw data file, allowing the user to immediately quality control the results. Once completed, the user can automatically report those results to data repositories for corporate access and quickly generate printed reports or documents. The software module has resulted in a very efficient and effective workflow through saved time and improved quality control. We discuss these benefits and show how this process defines a new benchmark in the drug discovery industry for the handling, interpretation, visualization, and sharing of SPR data.
Asunto(s)
Técnicas Biosensibles/métodos , Análisis de Datos , Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos/tendencias , Diseño de Fármacos , Humanos , Investigación Farmacéutica , Programas Informáticos , Resonancia por Plasmón de Superficie , Flujo de TrabajoRESUMEN
Inhibition of the protein-protein interaction between B-cell lymphoma 6 (BCL6) and corepressors has been implicated as a therapeutic target in diffuse large B-cell lymphoma (DLBCL) cancers and profiling of potent and selective BCL6 inhibitors are critical to test this hypothesis. We identified a pyrazolo[1,5-a]pyrimidine series of BCL6 binders from a fragment screen in parallel with a virtual screen. Using structure-based drug design, binding affinity was increased 100000-fold. This involved displacing crystallographic water, forming new ligand-protein interactions and a macrocyclization to favor the bioactive conformation of the ligands. Optimization for slow off-rate constant kinetics was conducted as well as improving selectivity against an off-target kinase, CK2. Potency in a cellular BCL6 assay was further optimized to afford highly selective probe molecules. Only weak antiproliferative effects were observed across a number of DLBCL lines and a multiple myeloma cell line without a clear relationship to BCL6 potency. As a result, we conclude that the BCL6 hypothesis in DLBCL cancer remains unproven.