Tuning mTORC1 activity dictates the response of acute myeloid leukemia to LSD1 inhibition.

Lysine specific demethylase-1 (LSD1) has been shown to be critical in acute myeloid leukemia (AML) pathogenesis and this has led to the development of LSD1 inhibitors (LSD1i) which are currently tested in clinical trials. Nonetheless, preclinical studies reported that AML cells frequently exhibit intrinsic resistance to LSD1 inhibition, and the molecular basis for this phenomenon is largely unknown. We explored the potential involvement of mammalian target of rapamycin (mTOR) in mediating the resistance of leukemic cells to LSD1i. Strikingly, unlike sensitive leukemias, mTOR complex 1 (mTORC1) signaling was robustly triggered in resistant leukemias following LSD1 inhibition. Transcriptomic, chromatin immunoprecipitation and functional studies revealed that insulin receptor substrate 1(IRS1)/extracellular-signal regulated kinases (ERK1/2) signaling critically controls LSD1i induced mTORC1 activation. Notably, inhibiting mTOR unlocked the resistance of AML cell lines and primary patient-derived blasts to LSD1i both in vitro and in vivo In conclusion, mTOR activation might act as a novel pro-survival mechanism of intrinsic as well as acquired resistance to LSD1i, and combination regimens co-targeting LSD1/mTOR could represent a rational approach in AML therapy.

Inhibition of the lysine demethylase LSD1 modulates the balance between inflammatory and antiviral responses against coronaviruses.

Innate immune responses to coronavirus infections are highly cell specific. Tissue-resident macrophages, which are infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in patients but are inconsistently infected in vitro, exert critical but conflicting effects by secreting both antiviral type I interferons (IFNs) and tissue-damaging inflammatory cytokines. Steroids, the only class of host-targeting drugs approved for the treatment of coronavirus disease 2019 (COVID-19), indiscriminately suppress both responses, possibly impairing viral clearance. Here, we established in vitro cell culture systems that enabled us to separately investigate the cell-intrinsic and cell-extrinsic proinflammatory and antiviral activities of mouse macrophages infected with the prototypical murine coronavirus MHV-A59. We showed that the nuclear factor κB-dependent inflammatory response to viral infection was selectively inhibited by loss of the lysine demethylase LSD1, which was previously implicated in innate immune responses to cancer, with negligible effects on the antiviral IFN response. LSD1 ablation also enhanced an IFN-independent antiviral response, blocking viral egress through the lysosomal pathway. The macrophage-intrinsic antiviral and anti-inflammatory activity of Lsd1 inhibition was confirmed in vitro and in a humanized mouse model of SARS-CoV-2 infection. These results suggest that LSD1 controls innate immune responses against coronaviruses at multiple levels and provide a mechanistic rationale for potentially repurposing LSD1 inhibitors for COVID-19 treatment.

Measurement of S-methylcysteine and S-methyl-mercapturic acid in human urine by alkyl-chloroformate extractive derivatization and isotope-dilution gas chromatography-mass spectrometry.

S-methylcysteine (SMC) is a minor amino acid naturally excreted in human urine, a protective agent against oxidative stress and a biotransformation product of the fumigant biocide methyl bromide and of nicotine. A metabolic source of SMC is catabolism of the repair catalytic protein MGMT (EC 2.1.1.37), which specifically removes the methyl group from the modified DNA nucleotide O-6-methyl-guanine to revert the normal GC base pairing. To assess the value of SMC and of S-methylmercapturic acid (SMMA) as candidate biomarkers of proliferative phenomena, a sensitive analytical method by GC-MS was applied in a pilot study of healthy subjects to assess their urinary elimination and the intra- and inter-individual variability. Extractive alkylation with butylchloroformate-n-butanol-pyridine (Husek technique) was employed for sample derivatization and isotope dilution GC-MS with S-[CD(3) ]-SMC and -SMMA was applied for specific and sensitive detection. To resolve the target analytes from the main coeluting interferents in the derivatized urine extract a medium-polarity stationary phase was employed. SMMA was not detected in the morning urine of three healthy fertile-age women followed for one month above the minimum detectable level of approx. 500 µg/L while SMC concentrations were in the 0.02-0.7 µg/mL range (n = 61) with large inter-day and inter-individual variations. In a young healthy male urine samples taken throughout a few days yielded concentrations in the same 90-810 µg/L range (n = 11). These preliminary results points at SMC as a candidate biomarker for the study of methylation turnover in several biochemical processes.

Enhanced activity or resistance of adenosine derivatives towards adenosine deaminase-catalyzed deamination: Influence of ribose modifications.

The effect of the presence of the 1'-C-methyl group and 2',3'-O-substitution in the adenosine structure on ADA activity has been investigated by modeling studies. Results show that the 2'- and 3'-O- substituents are harbored in a quite large cavity of intermediate polarity, whereas the 1'-C-substituent clashes against Ala180 distorting the architecture of the catalytic centre. Globally, the study emphasizes the ability of ADA to transform a large set of 2',3'-O-substituted adenosine analogues as well as the opportunity to design 1'-C-substituted adenosine derivatives resistant to ADA-catalyzed deamination.

Novel potent inhibitors of the histone demethylase KDM1A (LSD1), orally active in a murine promyelocitic leukemia model.

BACKGROUND: Histone lysine demethylases (KDMs) are well-recognized targets in oncology drug discovery. They function at the post-translation level controlling chromatin conformation and gene transcription. KDM1A is a flavin adenine dinucleotide-dependent amine oxidase, overexpressed in several tumor types, including acute myeloid leukemia, neuroblastoma and non-small-cell lung cancer. Among the many known monoamine oxidase inhibitors screened for KDM1A inhibition, tranylcypromine emerged as a moderately active hit, which irreversibly binds to the flavin adenine dinucleotide cofactor. MATERIAL & METHODS: The KDM1A inhibitors 5a-w were synthesized and tested in vitro and in vivo. The biochemical potency was determined, modulation of target in cells was demonstrated on KDM1A-dependent genes and the anti-clonogenic activity was performed in murine acute promyelocytic Leukemia (APL) blasts. An in vivo efficacy experiment was conducted using an established murine promyelocytic leukemia model. RESULTS: We report a new series of tranylcypromine derivatives substituted on the cyclopropyl moiety, endowed with high potency in both biochemical and cellular assays. CONCLUSION: The most interesting derivative (5a) significantly improved survival rate after oral administration in a murine model of promyelocitic leukemia.

Thieno[3,2-b]pyrrole-5-carboxamides as New Reversible Inhibitors of Histone Lysine Demethylase KDM1A/LSD1. Part 2: Structure-Based Drug Design and Structure-Activity Relationship.

The balance of methylation levels at histone H3 lysine 4 (H3K4) is regulated by KDM1A (LSD1). KDM1A is overexpressed in several tumor types, thus representing an emerging target for the development of novel cancer therapeutics. We have previously described ( Part 1, DOI 10.1021.acs.jmedchem.6b01018 ) the identification of thieno[3,2-b]pyrrole-5-carboxamides as novel reversible inhibitors of KDM1A, whose preliminary exploration resulted in compound 2 with biochemical IC50 = 160 nM. We now report the structure-guided optimization of this chemical series based on multiple ligand/KDM1A-CoRest cocrystal structures, which led to several extremely potent inhibitors. In particular, compounds 46, 49, and 50 showed single-digit nanomolar IC50 values for in vitro inhibition of KDM1A, with high selectivity in secondary assays. In THP-1 cells, these compounds transcriptionally affected the expression of genes regulated by KDM1A such as CD14, CD11b, and CD86. Moreover, 49 and 50 showed a remarkable anticlonogenic cell growth effect on MLL-AF9 human leukemia cells.

Thieno[3,2-b]pyrrole-5-carboxamides as New Reversible Inhibitors of Histone Lysine Demethylase KDM1A/LSD1. Part 1: High-Throughput Screening and Preliminary Exploration.

Lysine specific demethylase 1 KDM1A (LSD1) regulates histone methylation and it is increasingly recognized as a potential therapeutic target in oncology. We report on a high-throughput screening campaign performed on KDM1A/CoREST, using a time-resolved fluorescence resonance energy transfer (TR-FRET) technology, to identify reversible inhibitors. The screening led to 115 hits for which we determined biochemical IC50, thus identifying four chemical series. After data analysis, we have prioritized the chemical series of N-phenyl-4H-thieno[3, 2-b]pyrrole-5-carboxamide for which we obtained X-ray structures of the most potent hit (compound 19, IC50 = 2.9 µM) in complex with the enzyme. Initial expansion of this chemical class, both modifying core structure and decorating benzamide moiety, was directed toward the definition of the moieties responsible for the interaction with the enzyme. Preliminary optimization led to compound 90, which inhibited the enzyme with a submicromolar IC50 (0.162 µM), capable of inhibiting the target in cells.

Discovery of a Novel Inhibitor of Histone Lysine-Specific Demethylase 1A (KDM1A/LSD1) as Orally Active Antitumor Agent.

We report the stereoselective synthesis and biological activity of a novel series of tranylcypromine (TCPA) derivatives (14a-k, 15, 16), potent inhibitors of KDM1A. The new compounds strongly inhibit the clonogenic potential of acute leukemia cell lines. In particular three molecules (14d, 14e, and 14g) showing selectivity versus MAO A and remarkably inhibiting colony formation in THP-1 human leukemia cells, were assessed in mouse for their preliminary pharmacokinetic. 14d and 14e were further tested in vivo in a murine acute promyelocytic leukemia model, resulting 14d the most effective. Its two enantiomers were synthesized: the (1S,2R) enantiomer 15 showed higher activity than its (1R,2S) analogue 16, in both biochemical and cellular assays. Compound 15 exhibited in vivo efficacy after oral administration, determining a 62% increased survival in mouse leukemia model with evidence of KDM1A inhibition. The biological profile of compound 15 supports its further investigation as a cancer therapeutic.

Further insights into the SAR of α-substituted cyclopropylamine derivatives as inhibitors of histone demethylase KDM1A.

Epigenetics alterations including histone methylation and acetylation, and DNA methylation, are thought to play important roles in the onset and progression of cancer in numerous tumour cell lines. Lysine-specific demethylase 1 (LSD1 or KDM1A) is highly expressed in different cancer types and inhibiting KDM1A activity seems to have high therapeutic potential in cancer treatment. In the recent years, several inhibitors of KDM1A have been prepared and disclosed. The majority of these derivatives were designed based on the structure of tranylcypromine, as the cyclopropane core is responsible for the covalent interaction between the inhibitor and the catalytic domain of KDM proteins. In this study, we have further extended the SAR regarding compounds 1a-e, which were recently found to inhibit KDM1A with good activity. The decoration of the phenyl ring at the ß-position of the cyclopropane ring with small functional groups, mostly halogenated, and in particular at the meta position, led to a significant improvement of the inhibitory activity against KDM1A, as exemplified by compound 44a, which has a potency in the low nanomolar range (31 nM).

Synthesis, biological activity and mechanistic insights of 1-substituted cyclopropylamine derivatives: a novel class of irreversible inhibitors of histone demethylase KDM1A.

Histone demethylase KDM1A (also known as LSD1) has become an attractive therapeutic target for the treatment of cancer as well as other disorders such as viral infections. We report on the synthesis of compounds derived from the expansion of tranylcypromine as a chemical scaffold for the design of novel demethylase inhibitors. These compounds, which are substituted on the cyclopropyl core moiety, were evaluated for their ability to inhibit KDM1A in vitro as well as to function in cells by modulating the expression of Gfi-1b, a well recognized KDM1A target gene. The molecules were all found to covalently inhibit KDM1A and to become increasingly selective against human monoamine oxidases MAO A and MAO B through the introduction of bulkier substituents on the cyclopropylamine ring. Structural and biochemical analysis of selected trans isomers showed that the two stereoisomers are endowed with similar inhibitory activities against KDM1A, but form different covalent adducts with the FAD co-enzyme.

Synthesis of enantiopure Delta2-isoxazoline derivatives and evaluation of their affinity and efficacy profiles at human beta-adrenergic receptor subtypes.

The new enantiomerically pure 3-substituted-Delta(2)-isoxazolin-5-yl-ethanolamines (+)-6a/(-)-6b, (-)-6a/(+)-6b, and (+)-7a/(-)-7b, prepared via a 1,3-dipolar cycloaddition-based approach, were tested for their affinity at human beta(1)-, beta(2)-, and beta(3)-adrenergic receptor (beta-AR) subtypes stably expressed in CHO cells. The corresponding 3-isopropenyl derivatives (+)-5a/(-)-5b, (-)-5a/(+)-5b, and some isoxazole analogs were also tested. The binding affinities at the beta-ARs of the isoxazolinyl amino alcohols were significantly lower than those of the corresponding isoxazole derivatives. A stereochemical effect was observed, since the process of molecular recognition is predominantly controlled by the (S)-configuration of the stereogenic center located at the 5 position of the heterocycle rather than by that of the stereocenter carrying the secondary alcohol group. On the contrary, the stereochemical features marginally affected the efficacy response; as a matter of fact, functional tests carried out on Delta(2)-isoxazoline derivatives provided with a detectable binding affinity showed the overall profile of neutral antagonists at all three beta-AR subtypes.