RESUMO
We here describe the structure-based design of small molecule inhibitors of the type IV secretion system of Helicobacter pylori. The secretion system is encoded by theâ¯cagâ¯pathogenicity island, and we chose Cagα, a hexameric ATPase and member of the family of VirB11-like proteins, as target for inhibitor design. We first solved the crystal structure of Cagα in a complex with the previously identified small molecule inhibitor 1G2. The molecule binds at the interface between two Cagα subunits and mutagenesis of the binding site identified Cagα residues F39 and R73 as critical for 1G2 binding. Based on the inhibitor binding site we synthesized 98 small molecule derivates of 1G2 to improve binding of the inhibitor. We used the production of interleukin-8 of gastric cancer cells during H. pylori infection to screen the potency of inhibitors and we identified five molecules (1G2_1313, 1G2_1338, 1G2_2886, 1G2_2889, and 1G2_2902) that have similar or higher potency than 1G2. Differential scanning fluorimetry suggested that these five molecules bind Cagα, and enzyme assays demonstrated that some are more potent ATPase inhibitors than 1G2. Finally, scanning electron microscopy revealed that 1G2 and its derivatives inhibit the assembly of T4SS-determined extracellular pili suggesting a mechanism for their anti-virulence effect.
Assuntos
Adenosina Trifosfatases , Proteínas de Bactérias , Helicobacter pylori , Helicobacter pylori/enzimologia , Humanos , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/química , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/química , Sistemas de Secreção Tipo IV/metabolismo , Sistemas de Secreção Tipo IV/química , Sistemas de Secreção Tipo IV/antagonistas & inibidores , Desenho de Fármacos , Infecções por Helicobacter/tratamento farmacológico , Infecções por Helicobacter/microbiologia , Cristalografia por Raios X , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Modelos Moleculares , Sítios de Ligação , Relação Estrutura-Atividade , Linhagem Celular Tumoral , Interleucina-8/metabolismoRESUMO
DNA-encoded libraries (DELs) can be considered as one of the most powerful tools for the discovery of small molecules of biological interest. However, the ability to access large DELs is contingent upon having chemical transformations that work in aqueous phase and generate minimal DNA alterations and the availability of building blocks compatible with on-DNA chemistry. In addition, accessing scaffolds of interest to medicinal chemists can be challenging in a DEL setting because of inherent limitations of DNA-supported chemistry. In this context, a squaramide formation reaction was developed by using a two-step process. The mild and high-yielding reaction tolerates a wide array of functional groups and was shown to be safe for DNA, thereby making this methodology ideal for DELs.
Assuntos
DNA , Bibliotecas de Moléculas Pequenas , DNA/química , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/síntese química , Biblioteca Gênica , Ésteres/química , Quinina/análogos & derivadosRESUMO
MOTIVATION: The efficacy of a chemical compound is often tested through dose-response experiments from which efficacy metrics, such as the IC50, can be derived. The Marquardt-Levenberg algorithm (non-linear regression) is commonly used to compute estimations for these metrics. The analysis are however limited and can lead to biased conclusions. The approach does not evaluate the certainty (or uncertainty) of the estimates nor does it allow for the statistical comparison of two datasets. To compensate for these shortcomings, intuition plays an important role in the interpretation of results and the formulations of conclusions. We here propose a Bayesian inference methodology for the analysis and comparison of dose-response experiments. RESULTS: Our results well demonstrate the informativeness gain of our Bayesian approach in comparison to the commonly used Marquardt-Levenberg algorithm. It is capable to characterize the noise of dataset while inferring probable values distributions for the efficacy metrics. It can also evaluate the difference between the metrics of two datasets and compute the probability that one value is greater than the other. The conclusions that can be drawn from such analyzes are more precise. AVAILABILITY AND IMPLEMENTATION: We implemented a simple web interface that allows the users to analyze a single dose-response dataset, as well as to statistically compare the metrics of two datasets.
Assuntos
Algoritmos , Descoberta de Drogas , Teorema de Bayes , Modelos Lineares , ProbabilidadeRESUMO
BACKGROUND: Early diagnostic orientation for differentiating pneumonia from pneumonitis at the early stage after aspiration would be valuable to avoid unnecessary antibiotic therapy. We assessed the accuracy of procalcitonin (PCT) in diagnosing aspiration pneumonia (AP) in intensive care unit (ICU) patients requiring mechanical ventilation after out-of-hospital coma. METHODS: Prospective observational 2-year cohort study in a medical-surgical ICU. PCT, C-reactive protein (CRP) and white blood cell count (WBC) were measured at admission (H0) and 6 h (H), H12, H24, H48, H96, and H120 after inclusion. Lower respiratory tract microbiological investigations performed routinely in patients with aspiration syndrome were the reference standard for diagnosing AP. Performance of PCT, CRP, and WBC up to H48 in diagnosing AP was compared based on the areas under the ROC curves (AUC) and likelihood ratios (LR+ and LR-) computed for the best cutoff values. RESULTS: Of 103 patients with coma, 45 (44%) had AP. Repeated PCT assays demonstrated a significant increase in patients with AP versus without AP from H0 to H120. Among the three biomarkers, PCT showed the earliest change. ROC-AUC values were poor for all three biomarkers. Best ROC-AUC values for diagnosing AP were for CRP at H24 [0.73 (95%CI 0.61-0.84)] and PCT at H48 [0.73 (95%CI 0.61-0.84)]. LR+ was best for PCT at H24 (3.5) and LR- for CRP and WBC at H24 (0.4 and 0.4, respectively). CONCLUSIONS: Early and repeated assays of PCT, CRP, and WBC demonstrated significant increases in all three biomarkers in patients with versus without AP. All three biomarkers had poor diagnostic performance for ruling out AP. Whereas PCT had the fastest kinetics, PCT assays within 48 h after ICU admission do not help to diagnose AP in ICU patients with coma.
Assuntos
Coma/terapia , Cuidados Críticos/normas , Técnicas de Diagnóstico Neurológico/normas , Pneumonia Aspirativa/sangue , Pneumonia Aspirativa/diagnóstico , Pró-Calcitonina/sangue , Respiração Artificial/efeitos adversos , Adulto , Biomarcadores/sangue , Coma/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pneumonia Aspirativa/etiologia , Estudos Prospectivos , Sensibilidade e EspecificidadeRESUMO
In this study, we analyzed RNA-sequencing data of 14 samples characterized by biallelic CEBPA (CEBPA(bi)) mutations included in the Leucegene collection of 415 primary acute myeloid leukemia (AML) specimens, and describe for the first time high frequency recurrent mutations in the granulocyte colony-stimulating factor receptor gene CSF3R, which signals through JAK-STAT proteins. Chemical interrogation of these primary human specimens revealed a uniform and specific sensitivity to all JAK inhibitors tested irrespective of their CSF3R mutation status, indicating a general sensitization of JAK-STAT signaling in this leukemia subset. Altogether, these results identified the co-occurrence of mutations in CSF3R and CEBPA in a well-defined AML subset, which uniformly responds to JAK inhibitors and paves the way to personalized clinical trials for this disease.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/genética , Resistencia a Medicamentos Antineoplásicos/genética , Janus Quinases/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Inibidores de Proteínas Quinases/uso terapêutico , Receptores de Fator Estimulador de Colônias/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Análise Mutacional de DNA/métodos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Perfilação da Expressão Gênica , Frequência do Gene , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Leucemia Mieloide Aguda/classificação , Leucemia Mieloide Aguda/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Medicina de Precisão , Transcriptoma , Células Tumorais Cultivadas , Adulto JovemRESUMO
Leukemic stem cells (LSCs) are considered a major cause of relapse in acute myeloid leukemia (AML). Defining pathways that control LSC self-renewal is crucial for a better understanding of underlying mechanisms and for the development of targeted therapies. However, currently available culture conditions do not prevent spontaneous differentiation of LSCs, which greatly limits the feasibility of cell-based assays. To overcome these constraints we conducted a high-throughput chemical screen and identified small molecules that inhibit differentiation and support LSC activity in vitro. Similar to reports with cord blood stem cells, several of these compounds suppressed the aryl-hydrocarbon receptor (AhR) pathway, which we show to be inactive in vivo and rapidly activated ex vivo in AML cells. We also identified a compound, UM729, that collaborates with AhR suppressors in preventing AML cell differentiation. Together, these findings provide newly defined culture conditions for improved ex vivo culture of primary human AML cells.
Assuntos
Adenina/análogos & derivados , Técnicas de Cultura de Células/métodos , Indóis/farmacologia , Leucemia/metabolismo , Células-Tronco Neoplásicas/fisiologia , Pirimidinas/farmacologia , Adenina/farmacologia , Meios de Cultura Livres de Soro , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Leucemia Mieloide Aguda , Estrutura MolecularRESUMO
Weak protein interactions between ubiquitin and the ubiquitin-proteasome system (UPS) enzymes that mediate its covalent attachment to substrates serve to position ubiquitin for optimal catalytic transfer. We show that a small-molecule inhibitor of the E2 ubiquitin-conjugating enzyme Cdc34A, called CC0651, acts by trapping a weak interaction between ubiquitin and the E2 donor ubiquitin-binding site. A structure of the ternary CC0651-Cdc34A-ubiquitin complex reveals that the inhibitor engages a composite binding pocket formed from Cdc34A and ubiquitin. CC0651 also suppresses the spontaneous hydrolysis rate of the Cdc34A-ubiquitin thioester without decreasing the interaction between Cdc34A and the RING domain subunit of the E3 enzyme. Stabilization of the numerous other weak interactions between ubiquitin and UPS enzymes by small molecules may be a feasible strategy to selectively inhibit different UPS activities.
Assuntos
Aminoácidos/química , Compostos de Bifenilo/química , Enzimas de Conjugação de Ubiquitina/antagonistas & inibidores , Ubiquitina/química , Aminoácidos/farmacologia , Sítios de Ligação , Compostos de Bifenilo/farmacologia , Complexos de Coordenação/química , Cristalografia por Raios X , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Concentração Inibidora 50 , Modelos Moleculares , Ligação Proteica , Estabilidade Proteica/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
The UDP glucuronosyltransferases (UGT) deactivate many therapeutics via glucuronidation while being required for clearance of normal metabolites and xenobiotics. There are 19 UGT enzymes categorized into UGT1A and UGT2B families based on sequence conservation. This presents a challenge in terms of targeting specific UGTs to overcome drug resistance without eliciting overt toxicity. Here, we identified for the first time that UGT1A4 is highly elevated in acute myeloid leukemia (AML) patients and its reduction corresponded to objective clinical responses. To develop inhibitors to UGT1A4, we leveraged previous NMR-based fragment screening data against the C-terminal domain of UGT1A (UGT1A-C). NMR and medicinal chemistry strategies identified novel chemical matter based on fragment compounds with the capacity to bind â¼20 fold more tightly to UGT1A-C (Kd â¼ 600 µM vs â¼30 µM). Some compounds differentially inhibited UGT1A4 versus UGT1A1 enzyme activity and restored drug sensitivity in resistant human cancer cells. NMR-based NOE experiments revealed these novel compounds recognised a region distal to the catalytic site suggestive of allosteric regulation. This binding region is poorly conserved between UGT1A and UGT2B C-terminal sequences, which otherwise exhibit high similarity. Consistently, these compounds did not bind to the C-terminal domain of UGT2B7 nor a triple mutant of UGT1A-C replaced with UGT2B7 residues in this region. Overall, we discovered a site on UGTs that can be leveraged to differentially target UGT1As and UGT2Bs, identified UGT1A4 as a therapeutic target, and found new chemical matter that binds the UGT1A C-terminus, inhibits glucuronidation and restores drug sensitivity.
Assuntos
Descoberta de Drogas , Resistencia a Medicamentos Antineoplásicos , Inibidores Enzimáticos , Glucuronosiltransferase , Humanos , Domínio Catalítico , Química Farmacêutica , Glucuronosiltransferase/antagonistas & inibidores , Difosfato de Uridina , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Espectroscopia de Ressonância Magnética/métodosRESUMO
In a phenotypical screen of 56 acute myeloid leukemia (AML) patient samples and using a library of 10,000 compounds, we identified a hit with increased sensitivity toward SF3B1-mutated and adverse risk AMLs. Through structure-activity relationship studies, this hit was optimized into a potent, specific, and nongenotoxic molecule called UM4118. We demonstrated that UM4118 acts as a copper ionophore that initiates a mitochondrial-based noncanonical form of cell death known as cuproptosis. CRISPR-Cas9 loss-of-function screen further revealed that iron-sulfur cluster (ISC) deficiency enhances copper-mediated cell death. Specifically, we found that loss of the mitochondrial ISC transporter ABCB7 is synthetic lethal to UM4118. ABCB7 is misspliced and down-regulated in SF3B1-mutated leukemia, creating a vulnerability to copper ionophores. Accordingly, ABCB7 overexpression partially rescued SF3B1-mutated cells to copper overload. Together, our work provides mechanistic insights that link ISC deficiency to cuproptosis, as exemplified by the high sensitivity of SF3B1-mutated AMLs. We thus propose SF3B1 mutations as a biomarker for future copper ionophore-based therapies.
Assuntos
Cobre , Leucemia Mieloide Aguda , Humanos , Cobre/metabolismo , Fatores de Processamento de RNA/genética , Mutação , Leucemia Mieloide Aguda/genética , Ionóforos/farmacologia , Fosfoproteínas/metabolismoRESUMO
p21Cip1 (p21) is a universal cyclin-dependent kinase (CDK) inhibitor that halts cell proliferation and tumor growth by multiple mechanisms. The expression of p21 is often downregulated in cancer cells as a result of the loss of function of transcriptional activators, such as p53, or the increased degradation rate of the protein. To identify small molecules that block the ubiquitin-mediated degradation of p21 as a future avenue for cancer drug discovery, we have screened a compound library using a cell-based reporter assay of p21 degradation. This led to the identification of a benzodiazepine series of molecules that induce the accumulation of p21 in cells. Using a chemical proteomic strategy, we identified the ubiquitin-conjugating enzyme UBCH10 as a cellular target of this benzodiazepine series. We show that an optimized benzodiazepine analogue inhibits UBCH10 ubiquitin-conjugating activity and substrate proteolysis by the anaphase-promoting complex.
Assuntos
Benzodiazepinas , Enzimas de Conjugação de Ubiquitina , Enzimas de Conjugação de Ubiquitina/química , Benzodiazepinas/farmacologia , Proteômica , Ubiquitina/metabolismo , Núcleo Celular/metabolismoRESUMO
High-mobility group AT-hook 2 (HMGA2) is a nonhistone chromatin-binding protein that is normally expressed in stem cells of various tissues and aberrantly detected in several tumor types. We recently observed that one-fourth of human acute myeloid leukemia (AML) specimens express HMGA2, which associates with a very poor prognosis. We present results indicating that HMGA2+ AMLs share a distinct transcriptional signature representing an immature phenotype. Using single-cell analyses, we showed that HMGA2 is expressed in CD34+ subsets of stem cells and early progenitors, whether normal or derived from AML specimens. Of interest, we found that one of the strongest gene expression signatures associated with HMGA2 in AML is the upregulation of G2/M checkpoint genes. Whole-genome CRISPR/Cas9 screening in HMGA2 overexpressing cells further revealed a synthetic lethal interaction with several G2/M checkpoint genes. Accordingly, small molecules that target G2/M proteins were preferentially active in vitro and in vivo on HMGA2+ AML specimens. Together, our findings suggest that HMGA2 is a key functional determinant in AML and is associated with stem cell features, G2/M status, and related drug sensitivity.
Assuntos
Leucemia Mieloide Aguda , Antígenos CD34 , Pontos de Checagem do Ciclo Celular , Humanos , Leucemia Mieloide Aguda/patologia , Regulação para CimaRESUMO
Cholesterol homeostasis has been proposed as one mechanism contributing to chemoresistance in AML and hence, inclusion of statins in therapeutic regimens as part of clinical trials in AML has shown encouraging results. Chemical screening of primary human AML specimens by our group led to the identification of lipophilic statins as potent inhibitors of AMLs from a wide range of cytogenetic groups. Genetic screening to identify modulators of the statin response uncovered the role of protein geranylgeranylation and of RAB proteins, coordinating various aspect of vesicular trafficking, in mediating the effects of statins on AML cell viability. We further show that statins can inhibit vesicle-mediated transport in primary human specimens, and that statins sensitive samples show expression signatures reminiscent of enhanced vesicular trafficking. Overall, this study sheds light into the mechanism of action of statins in AML and identifies a novel vulnerability for cytogenetically diverse AML.
Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases , Leucemia Mieloide Aguda , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genéticaRESUMO
Protease-activated receptor 4 (PAR4) is a G-protein coupled receptor that is expressed on human platelets and activated by the coagulation enzyme thrombin. PAR4 plays a key role in blood coagulation, and its importance in pathological thrombosis has been increasingly recognized in recent years. Herein, we describe the optimization of a series of imidazothiadiazole PAR4 antagonists to a first-in-class clinical candidate, BMS-986120 (43), and a backup clinical candidate, BMS-986141 (49). Both compounds demonstrated excellent antithrombotic efficacy and minimal bleeding time prolongation in monkey models relative to the clinically important antiplatelet agent clopidogrel and provide a potential opportunity to improve the standard of care in the treatment of arterial thrombosis.
Assuntos
Agregação Plaquetária , Trombose , Benzofuranos , Plaquetas , Humanos , Imidazóis , Morfolinas , Receptor PAR-1 , Receptores de Trombina , Tiazóis , Trombina , Trombose/tratamento farmacológicoRESUMO
T-cell acute lymphoblastic leukemia (T-ALL) is commonly driven by activating mutations in NOTCH1 that facilitate glutamine oxidation. Here we identify oxidative phosphorylation (OxPhos) as a critical pathway for leukemia cell survival and demonstrate a direct relationship between NOTCH1, elevated OxPhos gene expression, and acquired chemoresistance in pre-leukemic and leukemic models. Disrupting OxPhos with IACS-010759, an inhibitor of mitochondrial complex I, causes potent growth inhibition through induction of metabolic shut-down and redox imbalance in NOTCH1-mutated and less so in NOTCH1-wt T-ALL cells. Mechanistically, inhibition of OxPhos induces a metabolic reprogramming into glutaminolysis. We show that pharmacological blockade of OxPhos combined with inducible knock-down of glutaminase, the key glutamine enzyme, confers synthetic lethality in mice harboring NOTCH1-mutated T-ALL. We leverage on this synthetic lethal interaction to demonstrate that IACS-010759 in combination with chemotherapy containing L-asparaginase, an enzyme that uncovers the glutamine dependency of leukemic cells, causes reduced glutaminolysis and profound tumor reduction in pre-clinical models of human T-ALL. In summary, this metabolic dependency of T-ALL on OxPhos provides a rational therapeutic target.
Assuntos
Leucemia-Linfoma Linfoblástico de Células T Precursoras , Animais , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Glutamina/metabolismo , Camundongos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Receptor Notch1/metabolismo , Linfócitos T/metabolismoRESUMO
Pharmacological control of the ubiquitin-proteasome system (UPS) is of intense interest in drug discovery. Here, we report the development of chemical inhibitors of the ubiquitin-conjugating (E2) enzyme CDC34A (also known as UBE2R1), which donates activated ubiquitin to the cullin-RING ligase (CRL) family of ubiquitin ligase (E3) enzymes. A FRET-based interaction assay was used to screen for novel compounds that stabilize the noncovalent complex between CDC34A and ubiquitin, and thereby inhibit the CDC34A catalytic cycle. An isonipecotamide hit compound was elaborated into analogs with ~1000-fold increased potency in stabilizing the CDC34A-ubiquitin complex. These analogs specifically inhibited CDC34A-dependent ubiquitination in vitro and stabilized an E2~ubiquitin thioester reaction intermediate in cells. The x-ray crystal structure of a CDC34A-ubiquitin-inhibitor complex uncovered the basis for analog structure-activity relationships. The development of chemical stabilizers of the CDC34A-ubiquitin complex illustrates a general strategy for de novo discovery of molecular glue compounds that stabilize weak protein interactions.
RESUMO
Human hematopoietic stem cells (HSCs) exhibit attrition of their self-renewal capacity when cultured ex vivo, a process that is partially reversed upon treatment with epigenetic modifiers, most notably inhibitors of histone deacetylases (HDACs) or lysine-specific demethylase LSD1. A recent study showed that the human HSC self-renewal agonist UM171 modulates the CoREST complex, leading to LSD1 degradation, whose inhibition mimics the activity of UM171. The mechanism underlying the UM171-mediated loss of CoREST function remains undetermined. We now report that UM171 potentiates the activity of a CULLIN3-E3 ubiquitin ligase (CRL3) complex whose target specificity is dictated by the poorly characterized Kelch/BTB domain protein KBTBD4. CRL3KBTBD4 targets components of the LSD1/RCOR1 corepressor complex for proteasomal degradation, hence re-establishing H3K4me2 and H3K27ac epigenetic marks, which are rapidly decreased upon ex vivo culture of human HSCs.
Assuntos
Proteínas Correpressoras , Epigênese Genética , Células-Tronco Hematopoéticas , Proteínas Correpressoras/genética , Proteínas Correpressoras/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Histona Desacetilases/metabolismo , HumanosRESUMO
Internalization and intracellular trafficking of G protein-coupled receptors (GPCRs) play pivotal roles in cell responsiveness. Dysregulation in receptor trafficking can lead to aberrant signaling and cell behavior. Here, using an endosomal BRET-based assay in a high-throughput screen with the prototypical GPCR angiotensin II type 1 receptor (AT1R), we sought to identify receptor trafficking inhibitors from a library of ~115,000 small molecules. We identified a novel dual Ras and ARF6 inhibitor, which we named Rasarfin, that blocks agonist-mediated internalization of AT1R and other GPCRs. Rasarfin also potently inhibits agonist-induced ERK1/2 signaling by GPCRs, and MAPK and Akt signaling by EGFR, as well as prevents cancer cell proliferation. In silico modeling and in vitro studies reveal a unique binding modality of Rasarfin within the SOS-binding domain of Ras. Our findings unveil a class of dual small G protein inhibitors for receptor trafficking and signaling, useful for the inhibition of oncogenic cellular responses.
Assuntos
Fatores de Ribosilação do ADP/antagonistas & inibidores , Endocitose/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Proteínas ras/antagonistas & inibidores , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/metabolismo , Sítios de Ligação , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Descoberta de Drogas , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Células HEK293 , Humanos , Simulação de Dinâmica Molecular , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas ras/química , Proteínas ras/metabolismoRESUMO
Trimethoprim (TMP) is widely used to treat infections in humans and in livestock, accelerating the incidence of TMP resistance. The emergent and largely untracked type II dihydrofolate reductases (DfrBs) are intrinsically TMP-resistant plasmid-borne Dfrs that are structurally and evolutionarily unrelated to chromosomal Dfrs. We report kinetic characterization of the known DfrB family members. Their kinetic constants are conserved and all are poorly inhibited by TMP, consistent with TMP resistance. We investigate their inhibition with known and novel bisubstrate inhibitors of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK). Importantly, all are inhibited by the HPPK inhibitors, making these molecules dual-target inhibitors of two folate pathway enzymes that are strictly microbial.
RESUMO
Acute myeloid leukemias (AML) with mutations in the NPM1 gene (NPM1c+) represent a large AML subgroup with varying response to conventional treatment, highlighting the need to develop targeted therapeutic strategies for this disease. We screened a library of clinical drugs on a cohort of primary human AML specimens and identified the BCL2 inhibitor ABT-199 as a selective agent against NPM1c+ AML. Mutational analysis of ABT-199-sensitive and -resistant specimens identified mutations in NPM1, RAD21, and IDH1/IDH2 as predictors of ABT-199 sensitivity. Comparative transcriptome analysis further uncovered BCL2A1 as a potential mediator of ABT-199 resistance in AML. In line with our observation that RAD21 mutation confers sensitivity to ABT-199, we provide functional evidence that reducing RAD21 levels can sensitize AML cells to BCL2 inhibition. Moreover, we demonstrate that ABT-199 is able to produce selective anti-AML activity in vivo toward AML with mutations associated with compound sensitivity in PDX models. Overall, this study delineates the contribution of several genetic events to the response to ABT-199 and provides a rationale for the development of targeted therapies for NPM1c+ AML.
Assuntos
Antineoplásicos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Leucemia Mieloide Aguda/genética , Antígenos de Histocompatibilidade Menor/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Sulfonamidas/farmacologia , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Mutação , Proteínas Nucleares/genética , Nucleofosmina , Células Tumorais CultivadasRESUMO
BACKGROUND: Benefits of cord blood transplantation include low rates of relapse and chronic graft-versus-host disease (GVHD). However, the use of cord blood is rapidly declining because of the high incidence of infections, severe acute GVHD, and transplant-related mortality. UM171, a haematopoietic stem cell self-renewal agonist, has been shown to expand cord blood stem cells and enhance multilineage blood cell reconstitution in mice. We aimed to investigate the safety and feasibility of single UM171-expanded cord blood transplantation in patients with haematological malignancies who do not have a suitable HLA-matched donor. METHODS: This single-arm, open-label, phase 1-2 safety and feasibility study was done at two hospitals in Canada. The study had two parts. In part 1, patients received two cord blood units (one expanded with UM171 and one unmanipulated cord blood) until UM171-expanded cord blood demonstrated engraftment. Once engraftment was documented we initiated part 2, reported here, in which patients received a single UM171-expanded cord blood unit with a dose de-escalation design to determine the minimal cord blood unit cell dose that achieved prompt engraftment. Eligible patients were aged 3-64 years, weighed 12 kg or more, had a haematological malignancy with an indication for allogeneic hematopoietic stem cell transplant and did not have a suitable HLA-matched donor, and a had a Karnofsky performance status score of 70% or more. Five clinical sites were planned to participate in the study; however, only two study sites opened, both of which only treated adult patients, thus no paediatric patients (aged <18 years) were recruited. Patients aged younger than 50 years without comorbidities received a myeloablative conditioning regimen (cyclophosphamide 120 mg/kg, fludarabine 75 mg/m2, and 12 Gy total body irradiation) and patients aged older than 50 years and those with comorbidities received a less myeloablative conditioning regimen (cyclophosphamide 50 mg/kg, thiotepa 10 mg/kg, fludarabine 150 mg/m2, and 4 Gy total body irradiation). Patients were infused with the 7-day UM171-expanded CD34-positive cells and the lymphocyte-containing CD34-negative fraction. The primary endpoints were feasibility of UM171 expansion, safety of the transplant, kinetics of hematopoietic reconstitution (time to neutrophil and platelet engraftment) of UM171-expanded cord blood, and minimal pre-expansion cord blood unit cell dose that achieved prompt engraftment. We analysed feasibility in all enrolled patients and all other primary outcomes were analysed per protocol, in all patients who received single UM171-expanded cord blood transplantation. This trial has been completed and was registered with ClinicalTrials.gov, NCT02668315. FINDINGS: Between Feb 17, 2016, and Nov 11, 2018, we enrolled 27 patients, four of whom received two cord blood units for safety purposes in part 1 of the study. 23 patients were subsequently enrolled in part 2 to receive a single UM171-expanded cord blood transplant and 22 patients received a single UM171-expanded cord blood transplantation. At data cutoff (Dec 31, 2018), median follow-up was 18 months (IQR 12-22). The minimal cord blood unit cell dose at thaw that achieved prompt engraftment as a single cord transplant after UM171 expansion was 0·52â×â105 CD34-positive cells. We successfully expanded 26 (96%) of 27 cord blood units with UM171. Among the 22 patients who received single UM171-expanded cord blood transplantation, median time to engraftment of 100 neutrophils per µL was 9·5 days (IQR 8-12), median time to engraftment of 500 neutrophils per µL was 18 days (12·5-20·0), and no graft failure occurred. Median time to platelet recovery was 42 days (IQR 35-47). The most common non-haematological adverse events were grade 3 febrile neutropenia (16 [73%] of 22 patients) and bacteraemia (nine [41%]). No unexpected adverse events were observed. One (5%) of 22 patients died due to treatment-related diffuse alveolar haemorrhage. INTERPRETATION: Our preliminary findings suggest that UM171 cord blood stem cell expansion is feasible, safe, and allows for the use of small single cords without compromising engraftment. UM171-expanded cord blood might have the potential to overcome the disadvantages of other cord blood transplants while maintaining the benefits of low risk of chronic GVHD and relapse, and warrants further investigation in randomised trials. FUNDING: Canadian Institutes of Health Research, Canadian Cancer Society and Stem Cell Network.