Cancer-Stem-Cell Phenotype-Guided Discovery of a Microbiota-Inspired Synthetic Compound Targeting NPM1 for Leukemia

Abstract

The human microbiota plays an important role in human health and disease, through the secretion of metabolites that regulate key biological functions. We propose that microbiota metabolites represent an unexplored chemical space of small drug-like molecules in the search of new hits for drug discovery. Here, we describe the generation of a set of complex chemotypes inspired on selected microbiota metabolites, which have been synthesized using asymmetric organocatalytic reactions. Following a primary screening in CSC models, we identified the novel compound UCM-13369 (4b) whose cytotoxicity was mediated by NPM1. This protein is one of the most frequent mutations of AML, and NPM1-mutated AML is recognized by the WHO as a distinct hematopoietic malignancy. UCM-13369 inhibits NPM1 expression, downregulates the pathway associated with mutant NPM1 C+, and specifically recognizes the C-end DNA-binding domain of NPM1 C+, avoiding the nucleus-cytoplasm translocation involved in the AML tumorological process. The new NPM1 inhibitor triggers apoptosis in AML cell lines and primary cells from AML patients and reduces tumor infiltration in a mouse model of AML with NPM1 C+ mutation. The disclosed phenotype-guided discovery of UCM-13369, a novel small molecule inspired on microbiota metabolites, confirms that CSC death induced by NPM1 inhibition represents a promising therapeutic opportunity for NPM1-mutated AML, a high-mortality disease.

Figure 1

Design of new chemotypes inspired on human microbiota metabolites. (A) Selected small-molecule metabolites produced by the microbiota. (B) Synthetic approach via asymmetric aminocatalysis to generate microbiota-inspired molecules 1–13. (C) Overview of the synthetic strategy based on a reaction between an α,β-unsaturated aldehyde and a nitro compound or a second aldehyde. PrSc: privileged scaffold. Untapered lines represent the relative stereochemistry; a and b are used to designate the two enantiomers of each compound.

Figure 2

(A) Synthesis of compounds 1–3 containing 2-piperidinone or piperidine scaffolds. Reagents and conditions: (a) (i) 10 mol % chiral cat., 20 mol % benzoic acid, DCM, 0 °C to rt, 5 h; (ii) PDC, rt, on, 45–58%; (b) TFA, DCM, rt, 45 min-4 h, 73–93%; (c) (i) NiCl₂·6H₂O, NaBH₄, MeOH, 0 °C, 1 h; (ii) Boc₂O, rt, on, quantitative; (d) (i) NaH, DMF, 0 °C, 1 h; (ii) (bromomethyl)cyclopropane, NaI, rt, on, 36–42%; (e) LiAlH₄, THF, rt, on, 77–85%; (f) TFA, rt, 2 h, 84–87%; (g) NiCl₂·6H₂O, NaBH₄, MeOH, 0 °C to rt, on, 53–60%; (h) (i) cyclopropranecarbaldehyde, MeOH, rt, 4 h; (ii) NaBH₄, rt, 2 h, 63–64%. (B) Synthesis of compound 4 containing a tetrahydrocarbazole scaffold. Reagents and conditions: (a) 20 mol % chiral cat., 20 mol % benzoic acid, toluene, MW, 110 °C, 5.5 h, 78%; (b) (i) (cyclopropylmethyl)amine, MeOH/DCM, rt, 3 h; (ii) NaBH₄, rt, 2 h, 83–86%; (c) Zn, AcOH/MeOH, 2 h, quantitative; (d) HCl, MeOH, rt, 4 h, 40–50%. (C) Synthesis of compounds 5–12 containing chromane and tetrahydrobenzo[c]chromene scaffolds. Reagents and conditions: (a) 20 mol % chiral cat., 20 mol % AcOH, CHCl₃, rt, on, 21–26% for 17, 22–31% for 19, 52–61% for 20; (b) 20 mol % chiral cat., 20 mol % AcOH, CHCl₃, rt, 1 h then 24 h, 43–48%; (c) (i) (cyclopropylmethyl)amine, MeOH/DCM, rt, 3 h; (ii) NaBH₄, rt, 2 h, 17%-quantitative; (d) Zn, AcOH/MeOH, rt, 2 h, 41–93%; (e) (cyclopropylmethyl)amine, DCM, rt, 3 h, quantitative; (f) NaBH₄, MeOH, rt, 3 h, 30–33%; (g) HSiCl₃, DCM, DMF, rt, on, 80–89%; (h) NaNO₂, AcOH, MeOH/H₂O, rt, on, 30–40%; (i) NaClO₂, KH₂PO₄, 2-methylbut-2-ene, t-BuOH/THF, 30 °C, on, 79%-quantitative; (j) (i) (COCl)₂ (2 M in DCM), cat. DMF, DCM, rt, 1 h; (ii) (cyclopropylmethyl)amine, 0 °C to rt, 3–18 h, 55%-quantitative; (k) LiAlH₄, THF, reflux, 24 h, 20–22%. (D) Synthesis of compound 13 containing a dihydropyrido[2,3-b]pyrazine scaffold. Reagents and conditions: (a) 20 mol % chiral cat., 20% mol AcOH, CHCl₃, rt, 7 days, 40–45%; (b) CH₃I, Cs₂CO₃, DMF, rt, 1 h, 95%-quantitative; (c) (i) (cyclopropylmethyl)amine, MeOH/DCM, rt, 3 h; (ii) NaBH₄, 0 °C to rt, 1–2 h, 49–50%.

Figure 3

Compound UCM-13369 targets NPM1 protein, binding mainly through the C-end domain of the C+ mutant form. (A) Dose–response curves of UCM-13369 in AML cell lines MOLM13 (NPM1 WT) and OCI-AML3 (NPM1 C+). (B) Confocal microscopy images with staining of NPM1 (Alexa Fluor-488, green), UCM-13369-Cy5 (red), and nuclei (DAPI, blue) in OCI-AML3 cells. (C,D) 1D ¹H NMR spectra of the C-end domain of NPM1 WT (C) and C+ (D). (E) ITC thermograms and binding isotherms of the interaction of UCM-13369 with the WT (blue) and C+ mutant (red) forms of NPM1. ITC-derived thermodynamic parameters of UCM-13369–NPM1 interaction. Gibbs free energy (ΔG), enthalpy (ΔH), entropic term (-TΔS), equilibrium dissociation constant (K_D), and binding stoichiometry (n). (F) Visualization of UCM-13369 binding interfaces to NPM1 C-end domain. The centroids of UCM-13369 in the four lowest-energy structures of each 10 clusters are plotted as balls colored according to the HADDOCK score, as indicated in the color legend. The structure of UCM-13369 of the top-ranked structure of each cluster is represented in the insets (i-v).

Figure 4

UCM-13369 inhibits gene and mutant NPM1 C+ protein expression and restores nucleolar localization. (A) Schematic representation of the molecular signaling pathways of NPM1 mutation in AML. (B) Dot plot showing decreased NPM1 expression with UCM-13369 treatment (10 μM) in OCI-AML3 cells. (C) Western blot showing decreased NPM1 and c-Myc expression and increased FBXW7 expression with UCM-13369 treatment (10 μM) in OCI-AML3 cells at 24 and 48 h. Densitometry values for NPM1: reduction of 50% and 70%, respectively. (D) Top: Confocal microscopy images with NPM1 (Alexa Fluor-647, red) and nuclei (DAPI, blue) staining in MOLM13, OCI-AML3, and UCM-13369-treated OCI-AML3 cells (10 μM). Bottom: Violin plot of NPM1 nucleus/cytoplasm ratio in MOLM13 and OCI-AML3 cell lines, and in untreated and UCM-13369-treated OCI-AML3 cells.

Figure 5

UCM-13369 has efficacy against AML cell lines and primary cells. (A) Dose–response curve of UCM-13369 in colony-forming unit (CFU) assays performed with primary HSCs bone marrow mononuclear fraction from healthy donors and AML patients. (B) Left: Dose–response curve of UCM-13369 in short-term cultures of HSCs performed with primary HSCs from bone marrow mononuclear fraction from healthy donors and AML patients. Right: Dose–response curve of the same assay with samples from AML patients with NPM1 WT vs NPM1 C+. (C) Dot plot of a representative sample from flow cytometry analysis using CD34-PE/Annexin V-FITC staining of short-term cultures of primary HSCs from AML patients performed with primary HSCs from bone marrow mononuclear fraction with and without treatment.

Figure 6

UCM-13369 shows efficacy against AML cells in vivo. (A) Values of UCM-13369 concentration in vivo at different time points after injection (25 mg/kg) analyzed by HPLC-MS. (B) Bioluminescence images acquired using an in vivo imaging system of NSG mice after injection of OCI-AML3 cell line with and without UCM-13369 treatment (50 mg/kg). (C) Microscopy analysis of the OCI-AML3 infiltration by H&E staining slides of bone marrow samples from NSG mice with paired time points after engraftment with and without UCM-13369 treatment (50 mg/kg).