Human dihydroorotate dehydrogenase ( hDHODH) catalyzes the rate-limiting step in de novo pyrimidine biosynthesis, the conversion of dihydroorotate to orotate. hDHODH has recently been found to be associated with acute myelogenous leukemia, a disease for which the standard of intensive care has not changed over decades. This work presents a novel class of hDHODH inhibitors, which are based on an unusual carboxylic group bioisostere 2-hydroxypyrazolo[1,5- a]pyridine, that has been designed starting from brequinar, one of the most potent hDHODH inhibitors. A combination of structure-based and ligand-based strategies produced compound 4, which shows brequinar-like hDHODH potency in vitro and is superior in terms of cytotoxicity and immunosuppression. Compound 4 also restores myeloid differentiation in leukemia cell lines at concentrations that are one log digit lower than those achieved in experiments with brequinar. This Article reports the design, synthesis, SAR, X-ray crystallography, biological assays, and physicochemical characterization of the new class of hDHODH inhibitors.

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TDP-43, encoded by TARDBP, is a ubiquitously expressed, primarily nuclear protein. In recent years, TDP-43 has been identified as the major pathological protein in ALS due to its mislocalisation in the cytoplasm of motor neurons of patients with and without TARDBP mutations and expression in forms that do not match its predicted molecular weight. In this study, the TDP-43 profile was investigated using western immunoblot analysis in whole lysates, nuclei and cytoplasm of circulating lymphomonocytes from 16 ALS patients, 4 with (ALS/TDP+) and 12 without (ALS/TDP-) TARDBP mutations in the protein C-terminal domain, and thirteen age-matched, healthy donors (controls). Three disease-unaffected first-degree relatives of an ALS/TDP+ patient were also included: one carried the parent mutation (Rel/TDP+) whereas the other two did not (Rel/TDP-). In all ALS patients, relatives and controls, TDP-43 retained the predicted molecular weight in whole cell lysates and nuclei, but in the cytoplasm its molecular weight was slightly smaller than expected. In quantitative terms, TDP-43 was expressed at approximately the same levels in whole cell lysates of ALS patients, relatives and controls. In contrast, TDP-43 accumulated in the cytoplasm with concomitant nuclear depletion in all ALS/TDP+ patients, in about 50% of ALS/TDP- patients and in the Rel/TDP+ subject compared to the controls. In the remaining ALS/TDP- patients and in the two Rel/TDP- subjects, TDP-43 matched the control levels in both subcellular compartments. Were these findings further confirmed, circulating lymphomonocytes could be informative of TDP-43 mislocalisation in nervous tissue and used as a biomarker for future disease risk.

Niemann-Pick disease (NPD) type A is a neurodegenerative disorder caused by sphingomyelin (SM) accumulation in lysosomes relying on reduced or absent acid sphingomyelinase (ASM) activity. NPD-A patients develop progressive neurodegeneration including cerebral and cerebellar atrophy, relevant Purkinje cell and myelin deficiency with death within 3 years. ASM 'knock-out' (ASMKO) mice, an animal model of NPD-A, develop a phenotype largely mimicking that of NPD-A. The mechanisms underlying myelin formation are poorly documented in ASMKO mice. In this study we determined the content of four myelin-specific proteins, myelin basic protein (MBP), 2¢,3¢-cyclic nucleotide 3¢-phosphodiesterase (CNP), myelin associated glycoprotein (MAG) and proteolipid protein (PLP), and that of myelin-enriched sphingolipids in the brains of ASMKO and wild-type mice in early stages of post-natal (pn) life. Protein and mRNA analysis revealed that in ASMKO mice beginning from 4 post-natal weeks (wk-pn), the expression levels of MAG, CNP, and MBP were below those observed in wild-type mice and the same applied to PLP at 10 wk-pn. Moreover, at 4 wk-pn the expression of SOX10, one of the transcription factors involved in oligodendrocyte development and maintenance was lower in ASMKO mice. Lipid analysis showed that SM and the gangliosides GM3 and GM2 accumulated in the brains of ASMKO mice, as opposed to galactocerebroside and galactosulfocerebroside that, in parallel with the mRNAs of UDP-galactose ceramide galactosyltransferase and galactose-3-O-sulfotransferase 1, the two transferases involved in their synthesis, decreased. Myelin lipid analysis showed a progressive sphingomyelin accumulation in ASMKO mice; noteworthy, of the two sphingomyelin species known to be resolved by TLC, only that with the lower Rf accumulated. The immunohistochemical analysis showed that the reduced expression of myelin specific proteins in ASMKO mice at 10 wk-pn was not restricted to the Purkinje layer of the cerebellar cortex but involved the cerebral cortex as well. In conclusion, reduced oligodendrocyte metabolic activity is likely to be the chief cause of myelin deficiency in ASMKO mice, thus shedding light on the molecular dysfunctions underlying neurodegeneration in NPD-A. Keywords: absent acid sphingomyelinase 'knock-out' mice, brain sphingolipids, myelin proteins, Niemann-Pick disease type A.

The connection with acute myelogenous leukemia (AML) of dihydroorotate dehydrogenase ( h DHODH), a key enzyme in pyrimidine biosynthesis, has attracted significant interest from pharma as a possible AML therapeutic target. We recently discovered compound 1 , a potent h DHODH inhibitor (IC 50 = 1.2 nM), able to induce myeloid differentiation in AML cell lines (THP1) in the low nM range (EC 50 = 32.8 nM) superior to brequinar’s phase I/II clinical trial (EC 50 = 265 nM). Herein, we investigate the 1 drug-like properties observing good metabolic stability and no toxic profile when administered at doses of 10 and 25 mg/kg every 3 days for 5 weeks (Balb/c mice). Moreover, in order to identify a backup compound, we investigate the SAR of this class of compounds. Inside the series, 17 is characterized by higher potency in inducing myeloid differentiation (EC 50 = 17.3 nM), strong proapoptotic properties (EC 50 = 20.2 nM), and low cytotoxicity toward non-AML cells (EC 30 (Jurkat) > 100 μM).