Comparative small molecule screening of primary human acute leukemias, engineered human leukemia and leukemia cell lines.

Targeted therapeutics for high-risk cancers remain an unmet medical need. Here we report the results of a large-scale screen of over 11,000 molecules for their ability to inhibit the survival and growth in vitro of human leukemic cells from multiple sources including patient samples, de novo generated human leukemia models, and established human leukemic cell lines. The responses of cells from de novo models were most similar to those of patient samples, both of which showed striking differences from the cell-line responses.

A Novel Confocal Scanning Protein-Protein Interaction Assay (PPI-CONA) Reveals Exceptional Selectivity and Specificity of CC0651, a Small Molecule Binding Enhancer of the Weak Interaction between the E2 Ubiquitin-Conjugating Enzyme CDC34A and Ubiquitin.

Protein-protein interactions (PPIs) are some of the most challenging target classes in drug discovery. Highly sensitive detection techniques are required for the identification of chemical modulators of PPIs. Here, we introduce PPI confocal nanoscanning (PPI-CONA), a miniaturized, microbead based high-resolution fluorescence imaging assay.

Squaramide Formation for DNA-Encoded Library Synthesis.

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.

mutations provide genetic vulnerability to copper ionophores in human acute myeloid leukemia.

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 -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.

Structure-based design of small molecule inhibitors of the cagT4SS ATPase Cagα of .

We here describe the structure-based design of small molecule inhibitors of the type IV secretion system of . The secretion system is encoded by the  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.

Medicinal Chemistry and NMR Driven Discovery of Novel UDP-glucuronosyltransferase 1A Inhibitors That Overcome Therapeutic Resistance in Cells.

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.

Discovery of Benzodiazepine-Based Inhibitors of the E2 Enzyme UBCH10 from a Cell-Based p21 Degradation Screen.

p21 (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.

HMGA2 expression defines a subset of human AML with immature transcriptional signature and vulnerability to G2/M inhibition.

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.

Discovery of Two Novel Antiplatelet Clinical Candidates (BMS-986120 and BMS-986141) That Antagonize Protease-Activated Receptor 4.

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 (), and a backup clinical candidate, BMS-986141 ().

Inhibition of mitochondrial complex I reverses NOTCH1-driven metabolic reprogramming in T-cell acute lymphoblastic leukemia.

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.

Vesicular trafficking is a key determinant of the statin response in acute myeloid leukemia.

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.

Identification and optimization of molecular glue compounds that inhibit a noncovalent E2 enzyme-ubiquitin complex.

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.

Discovery of a dual Ras and ARF6 inhibitor from a GPCR endocytosis screen.

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.

UM171 Preserves Epigenetic Marks that Are Reduced in Ex Vivo Culture of Human HSCs via Potentiation of the CLR3-KBTBD4 Complex.

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.

Dual-Target Inhibitors of the Folate Pathway Inhibit Intrinsically Trimethoprim-Resistant DfrB Dihydrofolate Reductases.

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.

Hematopoietic stem cell transplantation using single UM171-expanded cord blood: a single-arm, phase 1-2 safety and feasibility study.

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.

Enhancing the drug discovery process: Bayesian inference for the analysis and comparison of dose-response experiments.

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.

Structure-Based Design of Dimeric Bisbenzimidazole Inhibitors to an Emergent Trimethoprim-Resistant Type II Dihydrofolate Reductase Guides the Design of Monomeric Analogues.

The worldwide use of the broad-spectrum antimicrobial trimethoprim (TMP) has induced the rise of TMP-resistant microorganisms. In addition to resistance-causing mutations of the microbial chromosomal dihydrofolate reductase (Dfr), the evolutionarily and structurally unrelated type II Dfrs (DfrBs) have been identified in TMP-resistant microorganisms. DfrBs are intrinsically TMP-resistant and allow bacterial proliferation when the microbial chromosomal Dfr is TMP-inhibited, making these enzymes important targets for inhibitor development.

Genetic characterization of ABT-199 sensitivity in human AML.

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.

Mubritinib Targets the Electron Transport Chain Complex I and Reveals the Landscape of OXPHOS Dependency in Acute Myeloid Leukemia.

To identify therapeutic targets in acute myeloid leukemia (AML), we chemically interrogated 200 sequenced primary specimens. Mubritinib, a known ERBB2 inhibitor, elicited strong anti-leukemic effects in vitro and in vivo. In the context of AML, mubritinib functions through ubiquinone-dependent inhibition of electron transport chain (ETC) complex I activity.

Discovery of Potent Protease-Activated Receptor 4 Antagonists with in Vivo Antithrombotic Efficacy.

In an effort to identify novel antithrombotics, we have investigated protease-activated receptor 4 (PAR4) antagonism by developing and evaluating a tool compound, , in a monkey thrombosis model. Beginning with a high-throughput screening hit, we identified an imidazothiadiazole-based PAR4 antagonist chemotype. Detailed structure-activity relationship studies enabled optimization to a potent, selective, and orally bioavailable PAR4 antagonist, .

Identification of Allosteric Inhibitors against Active Caspase-6.

Caspase-6 is a cysteine protease that plays essential roles in programmed cell death, axonal degeneration, and development. The excess neuronal activity of Caspase-6 is associated with Alzheimer disease neuropathology and age-dependent cognitive impairment. Caspase-6 inhibition is a promising strategy to stop early stage neurodegenerative events, yet finding potent and selective Caspase-6 inhibitors has been a challenging task due to the overlapping structural and functional similarities between caspase family members.

Mild and Diazo-Free Synthesis of Trifluoromethyl-Cyclopropanes Using Sulfonium Ylides.

The synthesis of several 1,1-disubstituted trifluoromethyl-cyclopropanes (TFCPs), known as tert-butyl bioisosteres, has been achieved from the reaction between trifluoromethylalkenes and unstabilized sulfonium ylides in yields of ≤97%. This method offers practical access to this cyclopropyl moiety of pharmacological interest, employing a commercially available reagent at low temperatures. The synthesis of cyclopropanes bearing other electron-withdrawing groups as well as trisubstituted TFCPs was also accomplished.

Complex karyotype AML displays G2/M signature and hypersensitivity to PLK1 inhibition.

Patients diagnosed with acute myeloid leukemia with complex karyotype (CK AML) have an adverse prognosis using current therapies, especially when accompanied by alterations. We hereby report the RNA-sequencing analysis of the 68 CK AML samples included in the Leucegene 415 patient cohort. We confirm the frequent occurrence of alterations in this subgroup and further characterize the allele expression profile and transcript alterations of this gene.

Diagnostic Accuracy of Procalcitonin for Early Aspiration Pneumonia in Critically Ill Patients with Coma: A Prospective Study.

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.