Exploring the Potential of Sulfur Moieties in Compounds Inhibiting Steroidogenesis.

This study reports on the synthesis and evaluation of novel compounds replacing the nitrogen-containing heterocyclic ring on the chemical backbone structure of cytochrome P450 17α-hydroxylase/12,20-lyase (CYP17A1) inhibitors with a phenyl bearing a sulfur-based substituent. Initial screening revealed compounds with marked inhibition of CYP17A1 activity. The selectivity of compounds was thereafter determined against cytochrome P450 21-hydroxylase, cytochrome P450 3A4, and cytochrome P450 oxidoreductase. Additionally, the compounds showed weak inhibitory activity against aldo-keto reductase 1C3 (AKR1C3). The compounds' impact on steroid hormone levels was also assessed, with some notable modulatory effects observed. This work paves the way for developing more potent dual inhibitors specifically targeting CYP17A1 and AKR1C3.

Targeting Acute Myelogenous Leukemia Using Potent Human Dihydroorotate Dehydrogenase Inhibitors Based on the 2-Hydroxypyrazolo[1,5-a]pyridine Scaffold: SAR of the Aryloxyaryl Moiety.

In recent years, human dihydroorotate dehydrogenase inhibitors have been associated with acute myelogenous leukemia as well as studied as potent host targeting antivirals. Starting from MEDS433 (IC50 1.2 nM), we kept improving the structure-activity relationship of this class of compounds characterized by 2-hydroxypyrazolo[1,5-a]pyridine scaffold. Using an in silico/crystallography supported design, we identified compound 4 (IC50 7.2 nM), characterized by the presence of a decorated aryloxyaryl moiety that replaced the biphenyl scaffold, with potent inhibition and pro-differentiating abilities on AML THP1 cells (EC50 74 nM), superior to those of brequinar (EC50 249 nM) and boosted when in combination with dipyridamole. Finally, compound 4 has an extremely low cytotoxicity on non-AML cells as well as MEDS433; it has shown a significant antileukemic activity in vivo in a xenograft mouse model of AML.

Targeting Acute Myelogenous Leukemia Using Potent Human Dihydroorotate Dehydrogenase Inhibitors Based on the 2-Hydroxypyrazolo[1,5-a]pyridine Scaffold: SAR of the Biphenyl Moiety.

The connection with acute myelogenous leukemia (AML) of dihydroorotate dehydrogenase (hDHODH), 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 hDHODH inhibitor (IC50 = 1.2 nM), able to induce myeloid differentiation in AML cell lines (THP1) in the low nM range (EC50 = 32.8 nM) superior to brequinar's phase I/II clinical trial (EC50 = 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 (EC50 = 17.3 nM), strong proapoptotic properties (EC50 = 20.2 nM), and low cytotoxicity toward non-AML cells (EC30(Jurkat) > 100 µM).

A New NF-κB Inhibitor, MEDS-23, Reduces the Severity of Adverse Post-Ischemic Stroke Outcomes in Rats.

AIM: Nuclear factor kappa B (NF-κB) is known to play an important role in the inflammatory process which takes place after ischemic stroke. The major objective of the present study was to examine the effects of MEDS-23, a potent inhibitor of NF-κB, on clinical outcomes and brain inflammatory markers in post-ischemic stroke rats. MAIN METHODS: Initially, a Toxicity Experiment was performed to determine the appropriate dose of MEDS-23 for use in animals, as MEDS-23 was analyzed in vivo for the first time. We used the middle cerebral artery occlusion (MCAO) model for inducing ischemic stroke in rats. The effects of MEDS-23 (at 10 mg/kg, ip) on post-stroke outcomes (brain inflammation, fever, neurological deficits, mortality, and depression- and anxiety-like behaviours) was tested in several efficacy experiments. KEY FINDINGS: MEDS-23 was found to be safe and significantly reduced the severity of some adverse post-stroke outcomes such as fever and neurological deficits. Moreover, MEDS-23 significantly decreased prostaglandin E2 levels in the hypothalamus and hippocampus of post-stroke rats, but did not prominently alter the levels of interleukin-6 and tumor necrosis factor-α. SIGNIFICANCE: These results suggest that NF-κB inhibition is a potential therapeutic strategy for the treatment of ischemic stroke.

Bioisosteres of Indomethacin as Inhibitors of Aldo-Keto Reductase 1C3.

Aldo-keto reductase 1C3 (AKR1C3) is an attractive target in drug design for its role in resistance to anticancer therapy. Several nonsteroidal anti-inflammatory drugs such as indomethacin are known to inhibit AKR1C3 in a nonselective manner because of COX-off target effects. Here we designed two indomethacin analogues by proposing a bioisosteric connection between the indomethacin carboxylic acid function and either hydroxyfurazan or hydroxy triazole rings. Both compounds were found to target AKR1C3 in a selective manner. In particular, hydroxyfurazan derivative is highly selective for AKR1C3 over the 1C2 isoform (up to 90-times more) and inactive on COX enzymes. High-resolution crystal structure of its complex with AKR1C3 shed light onto the binding mode of the new inhibitors. In cell-based assays (on colorectal and prostate cancer cells), the two indomethacin analogues showed higher potency than indomethacin. Therefore, these two AKR1C3 inhibitors can be used to provide further insight into the role of AKR1C3 in cancer.

Hydroxyazole scaffold-based Plasmodium falciparum dihydroorotate dehydrogenase inhibitors: Synthesis, biological evaluation and X-ray structural studies.

Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) has been clinically validated as a target for antimalarial drug discovery, as a triazolopyrimidine class inhibitor (DSM265) is currently undergoing clinical development. Here, we have identified new hydroxyazole scaffold-based PfDHODH inhibitors belonging to two different chemical series. The first series was designed by a scaffold hopping strategy that exploits the use of hydroxylated azoles. Within this series, the hydroxythiadiazole 3 was identified as the best selective PfDHODH inhibitor (IC50 12.0 µM). The second series was designed by modulating four different positions of the hydroxypyrazole scaffold. In particular, hydroxypyrazoles 7e and 7f were shown to be active in the low µM range (IC50 2.8 and 5.3 µM, respectively). All three compounds, 3, 7e and 7f showed clear selectivity over human DHODH (IC50 > 200 µM), low cytotoxicity, and retained micromolar activity in P. falciparum-infected erythrocytes. The crystallographic structures of PfDHODH in complex with compounds 3 and 7e proved their binding mode, supplying essential data for future optimization of these scaffolds.

N-Acetyl-3-aminopyrazoles block the non-canonical NF-kB cascade by selectively inhibiting NIK.

NF-κB-inducing kinase (NIK), an oncogenic drug target that is associated with various cancers, is a central signalling component of the non-canonical pathway. A blind screening process, which established that amino pyrazole related scaffolds have an effect on IKKbeta, led to a hit-to-lead optimization process that identified the aminopyrazole 3a as a low µM selective NIK inhibitor. Compound 3a effectively inhibited the NIK-dependent activation of the NF-κB pathway in tumour cells, confirming its selective inhibitory profile.

Targeting Myeloid Differentiation Using Potent 2-Hydroxypyrazolo[1,5- a]pyridine Scaffold-Based Human Dihydroorotate Dehydrogenase Inhibitors.

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.

Use of human Dihydroorotate Dehydrogenase (hDHODH) Inhibitors in Autoimmune Diseases and New Perspectives in Cancer Therapy.

BACKGROUND: Human dihydroorotate dehydrogenase (hDHODH, EC 1.3.5.2), a flavindependent mitochondrial enzyme involved in de novo pyrimidine biosynthesis, is a validated therapeutic target for the treatment of autoimmune diseases, such as rheumatoid arthritis and multiple sclerosis. However, human DHODH inhibitors have also been investigated as treatment for cancer, parasite infections (i.e. malaria) and viruses as well as in the agrochemicals industry. OBJECTIVE: An overview of current knowledge of hDHODH inhibitors and their potential uses in diseases where hDHODH is involved. METHOD: This review focuses on recent advances in the development and application of hDHODH inhibitors, specifically covering the patent field, starting from a brief description of enzyme topography and of the strategies usually followed in designing its selective inhibitors. RESULTS: The most important and well-described novelty is the fact that the discovery, in the autumn of 2016, that hDHODH inhibitors are able to induce in vivo myeloid differentiation has led to the possibility of developing novel hDHODH based treatments for Acute Myelogenous Leukemia (AML). CONCLUSION: The review will describe a variety of specific inhibitor classes and conclude on recent and future therapeutic perspectives for this target.

Hydroxytriazole derivatives as potent and selective aldo-keto reductase 1C3 (AKR1C3) inhibitors discovered by bioisosteric scaffold hopping approach.

The aldo-keto reductase 1C3 isoform (AKR1C3) plays a vital role in the biosynthesis of androgens, making this enzyme an attractive target for castration-resistant prostate cancer therapy. Although AKR1C3 is a promising drug target, no AKR1C3-targeted agent has to date been approved for clinical use. Flufenamic acid, a non-steroidal anti-inflammatory drug, is known to potently inhibit AKR1C3 in a non-selective manner as COX off-target effects are also observed. To diminish off-target effects, we have applied a scaffold hopping strategy replacing the benzoic acid moiety of flufenamic acid with an acidic hydroxyazolecarbonylic scaffold. In particular, differently N-substituted hydroxylated triazoles were designed to simultaneously interact with both subpockets 1 and 2 in the active site of AKR1C3, larger for AKR1C3 than other AKR1Cs isoforms. Through computational design and iterative rounds of synthesis and biological evaluation, novel compounds are reported, sharing high selectivity (up to 230-fold) for AKR1C3 over 1C2 isoform and minimal COX1 and COX2 off-target inhibition. A docking study of compound 8, the most interesting compound of the series, suggested that its methoxybenzyl substitution has the ability to fit inside subpocket 2, being involved in π-π staking interaction with Trp227 (partial overlapping) and in a T-shape π-π staking with Trp86. This compound was also shown to diminish testosterone production in the AKR1C3-expressing 22RV1 prostate cancer cell line while synergistic effect was observed when 8 was administered in combination with abiraterone or enzalutamide.

Design, synthesis, biological evaluation and X-ray structural studies of potent human dihydroorotate dehydrogenase inhibitors based on hydroxylated azole scaffolds.

A new generation of potent hDHODH inhibitors designed by a scaffold-hopping replacement of the quinolinecarboxylate moiety of brequinar, one of the most potent known hDHODH inhibitors, is presented here. Their general structure is characterized by a biphenyl moiety joined through an amide bridge with an acidic hydroxyazole scaffold (hydroxylated thiadiazole, pyrazole and triazole). Molecular modelling suggested that these structures should adopt a brequinar-like binding mode involving interactions with subsites 1, 2 and 4 of the hDHODH binding site. Initially, the inhibitory activity of the compounds was studied on recombinant hDHODH. The most potent compound of the series in the enzymatic assays was the thiadiazole analogue 4 (IC50 16 nM). The activity was found to be dependent on the fluoro substitution pattern at the biphenyl moiety as well as on the choice/substitution of the heterocyclic ring. Structure determination of hDHODH co-crystallized with one representative compound from each series (4, 5 and 6) confirmed the brequinar-like binding mode as suggested by modelling. The specificity of the observed effects of the compound series was tested in cell-based assays for antiproliferation activity using Jurkat cells and PHA-stimulated PBMC. These tests were also verified by addition of exogenous uridine to the culture medium. In particular, the triazole analogue 6 (IC50 against hDHODH: 45 nM) exerted potent in vitro antiproliferative and immunosuppressive activity without affecting cell survival.

4-Hydroxy-N-[3,5-bis(trifluoromethyl)phenyl]-1,2,5-thiadiazole-3-carboxamide: a novel inhibitor of the canonical NF-κB cascade.

The NF-κB signaling pathway is a validated oncological target. Here, we applied scaffold hopping to IMD-0354, a presumed IKKß inhibitor, and identified 4-hydroxy-N-[3,5-bis(trifluoromethyl)phenyl]-1,2,5-thiadiazole-3-carboxamide (4) as a nM-inhibitor of the NF-κB pathway. However, both 4 and IMD-0354, being potent inhibitors of the canonical NF-κB pathway, were found to be inactive in human IKKß enzyme assays.

Heterocyclic ring cleavage upon collision-induced dissociation of deprotonated 3-hydroxy-1,2,5-oxadiazoles (3-hydroxyfurazans).

A series of 4-substituted 3-hydroxyfurazans were subjected to electrospray ionization tandem mass spectrometry. At low collision energy, oxyisocyanate ([O=C=N-O](-), m/z 58) was formed as the predominant product ion from each deprotonated 3-hydroxyfurazan, indicating cleavage of the heterocyclic ring. The facile energetics of this characteristic fragmentation process was confirmed by density functional computations.

Discovery and verification of panels of T-lymphocyte proteins as biomarkers of Parkinson's disease.

The diagnosis of Parkinson's disease (PD) is currently based on the clinical evaluation of extrapyramidal signs with a considerable error rate. The identification of specific markers might allow PD diagnosis before the onset of classical motor symptoms. By two-dimensional electrophoresis we identified proteome alterations in T-lymphocytes of 17 control subjects and 15 PD patients. The observed changes were used to build predictive models that were verified by the leave-one-out cross-validation. We further built two functions able to stage the subjects. We chose to verify by Western blotting the identity of spots corresponding to ß-fibrinogen and transaldolase, two recurrent proteins in six out of 20 spots. ß-Fibrinogen levels are lowered in PD patients, whereas a heavy transaldolase set of isoforms was more abundant. Eventually, we identified a list of seven proteins showing different levels in early-onset with respect to late-onset PD patients.

Dopaminergic therapies modulate the T-CELL proteome of patients with Parkinson's disease.

Dopamine receptor agonists and L-dihydroxyphenylalanine (L-DOPA) counteract dopamine loss in the striatum and are therefore used in the treatment of Parkinson's disease (PD). T-Lymphocytes express some features of the dopaminergic system, and their function or activation might be regulated by dopaminergic treatments. Two-dimensional electrophoresis of total protein extract from T-lymphocytes was performed to identify therapy-induced proteome changes in T-cells of 17 patients with PD. Specific protein level alterations were further validated by Western blotting. Of 17 enrolled patients, 11 were treated with different doses of L-DOPA; in this group, we found that the levels of two spots, corresponding to ATP synthase subunit ß and proteasome subunit ß type-2, correlated linearly with the L-DOPA daily dose. Moreover, we identified seven proteins (prolidase, actin-related protein 2, F-actin-capping protein subunit ß, tropomyosin α-3 chain, proteasome activator complex subunit 1, peroxiredoxin 6, and a glyceraldehyde-3-phosphate dehydrogenase isoform) whose levels were significantly different in patients treated with dopamine agonists. These findings demonstrate that dopaminergic stimulation has important effects on T-cell proteome in patients under long-term treatment. Therefore, therapies acting on the dopaminergic system may have additional effects on the immune system.