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The pharmacological management of influenza virus (IV) infections still poses a series of challenges due to the limited anti-IV drug arsenal. Therefore, the development of new anti-influenza agents effective against antigenically different IVs is therefore an urgent priority. To meet this need, host-targeting antivirals (HTAs) can be evaluated as an alternative or complementary approach to current direct-acting agents (DAAs) for the therapy of IV infections. As a contribution to this antiviral strategy, in this study, we characterized the anti-IV activity of MEDS433, a novel small molecule inhibitor of the human dihydroorotate dehydrogenase (

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).

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.

Although coronaviruses (CoVs) have long been predicted to cause zoonotic diseases and pandemics with high probability, the lack of effective anti-pan-CoVs drugs rapidly usable against the emerging SARS-CoV-2 actually prevented a promptly therapeutic intervention for COVID-19. Development of host-targeting antivirals could be an alternative strategy for the control of emerging CoVs infections, as they could be quickly repositioned from one pandemic event to another. To contribute to these pandemic preparedness efforts, here we report on the broad-spectrum CoVs antiviral activity of MEDS433, a new inhibitor of the human dihydroorotate dehydrogenase (hDHODH), a key cellular enzyme of the de novo pyrimidine biosynthesis pathway. MEDS433 inhibited the in vitro replication of hCoV-OC43 and hCoV-229E, as well as of SARS-CoV-2, at low nanomolar range. Notably, the anti-SARS-CoV-2 activity of MEDS433 against SARS-CoV-2 was also observed in kidney organoids generated from human embryonic stem cells. Then, the antiviral activity of MEDS433 was reversed by the addition of exogenous uridine or the product of hDHODH, the orotate, thus confirming hDHODH as the specific target of MEDS433 in hCoVs-infected cells. Taken together, these findings suggest MEDS433 as a potential candidate to develop novel drugs for COVID-19, as well as broad-spectrum antiviral agents exploitable for future CoVs threats.

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.

[Image: see text] 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.

Tetrahydroisoquinoline (THIQ) is a key structural component in many biologically active molecules including natural products and synthetic pharmaceuticals. Here, we report on the use of transition-metal mediated [2 + 2 + 2] cyclotrimerisation of alkynes to generate tricyclic THIQs with potential to selectively inhibit AKR1C3.

The aldo-keto reductase 1C3 (AKR1C3) enzyme is considered an attractive target in Castration Resistant Prostate Cancer (CRPC) because of its role in the biosynthesis of androgens. Flufenamic acid, a non-selective AKR1C3 inhibitor, has previously been subjected to bioisosteric modulation to give rise to a series of compounds with the hydroxytriazole core. In this work, the hit compound of the previous series has been modulated further, and new, more potent, and selective derivatives have been obtained. The poor solubility of the most active compound (cpd 5) has been improved by substituting the triazole core with an isoxazole heteronucleous, with similar enzymatic activity being retained. Potent AKR1C3 inhibition is translated into antiproliferative effects against the 22RV1 CRPC cellular model, and the in-silico design, synthesis and biological activity of new compounds are described herein. Compounds have also been assayed in combination with two approved antitumor drugs, abiraterone and enzalutamide.

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).

During the optimization process that leads to drug candidates, bioisosterism is often used to achieve improved potency/selectivity, optimal ADME-T profiles as well as reach intellectual property (IP). Hydroxyazoles, due to their acidity are considered isosteres of the carboxylic acid group, offering an efficient tool for designing active compounds with added IP value. Recently, we and other groups have explored the chemical space of these systems, shaping their applications in the framework of hit-to-lead optimization processes. This chapter covers the chemo-physical properties, synthetic methodologies, as well as the application in drug design, of monocyclic hydroxyazoles.

Oropharynx squamous cell carcinoma (OPSCC) is a subtype of head and neck squamous cell carcinoma (HNSCC) arising from the base of the tongue, lingual tonsils, tonsils, oropharynx or pharynx. The majority of HPV-positive OPSCCs has a good prognosis, but a fraction of them has a poor prognosis, similar to HPV-negative OPSCCs. An in-depth understanding of the molecular mechanisms underlying OPSCC is mandatory for the identification of novel prognostic biomarkers and/or novel therapeutic targets. 14 HPV-positive and 15 HPV-negative OPSCCs with 5-year follow-up information were subjected to gene expression profiling and, subsequently, compared to three extensive published OPSCC cohorts to define robust biomarkers for HPV-negative lesions. Validation of Aldo-keto-reductases 1C3 (AKR1C3) by qRT-PCR was carried out on an independent cohort (n = 111) of OPSCC cases. In addition, OPSCC cell lines Fadu and Cal-27 were treated with Cisplatin and/or specific AKR1C3 inhibitors to assess their (combined) therapeutic effects. Gene set enrichment analysis (GSEA) on the four datasets revealed that the genes down-regulated in HPV-negative samples were mainly involved in immune system, whereas those up-regulated mainly in glutathione derivative biosynthetic and xenobiotic metabolic processes. A panel of 30 robust HPV-associated transcripts was identified, with AKR1C3 as top-overexpressed transcript in HPV-negative samples. AKR1C3 expression in 111 independent OPSCC cases positively correlated with a worse survival, both in the entire cohort and in HPV-positive samples. Pretreatment with a selective AKR1C3 inhibitor potentiated the effect of Cisplatin in OPSCC cells exhibiting higher basal AKR1C3 expression levels. We identified AKR1C3 as a potential prognostic biomarker in OPSCC and as a potential drug target whose inhibition can potentiate the effect of Cisplatin.

Dihydroorotate Dehydrogenase (DHODH) is a key enzyme of the de novo pyrimidine biosynthesis, whose inhibition can induce differentiation and apoptosis in acute myeloid leukemia (AML). DHODH inhibitors had shown promising in vitro and in vivo activity on solid tumors, but their effectiveness was not confirmed in clinical trials, probably because cancer cells exploited the pyrimidine salvage pathway to survive. Here, we investigated the antileukemic activity of MEDS433, the DHODH inhibitor developed by our group, against AML. Learning from previous failures, we mimicked human conditions (performing experiments in the presence of physiological uridine plasma levels) and looked for synergic combinations to boost apoptosis, including classical antileukemic drugs and dipyridamole, a blocker of the pyrimidine salvage pathway. MEDS433 induced apoptosis in multiple AML cell lines, not only as a consequence of differentiation, but also directly. Its combination with antileukemic agents further increased the apoptotic rate, but when experiments were performed in the presence of physiological uridine concentrations, results were less impressive. Conversely, the combination of MEDS433 with dipyridamole induced metabolic lethality and differentiation in all AML cell lines, this extraordinary synergism was confirmed on AML primary cells with different genetic backgrounds and was unaffected by physiological uridine concentrations, predicting in human activity.

Identification and development of effective drugs active against SARS-CoV-2 are urgently needed. Here, we report on the anti-SARS-CoV-2 activity of MEDS433, a novel inhibitor of human dihydroorotate dehydrogenase (hDHODH), a key cellular enzyme of thede novopyrimidines biosynthesis. MEDS433 inhibitsin vitrovirus replication in the low nanomolar range, and through a mechanism that stems from its ability to block hDHODH activity. MEDS433 thus represents an attractive candidate to develop novel anti-SARS-CoV-2 agents.

Emergence of drug resistance and adverse effects often affect the efficacy of nucleoside analogues in the therapy of Herpes simplex type 1 (HSV-1) and type 2 (HSV-2) infections. Host-targeting antivirals could therefore be considered as an alternative or complementary strategy in the management of HSV infections. To contribute to this advancement, here we report on the ability of a new generation inhibitor of a key cellular enzyme of de novo pyrimidine biosynthesis, the dihydroorotate dehydrogenase (DHODH), to inhibit HSV-1 and HSV-2 in vitro replication, with a potency comparable to that of the reference drug acyclovir. Analysis of the HSV replication cycle in MEDS433-treated cells revealed that it prevented the accumulation of viral genomes and reduced late gene expression, thus suggesting an impairment at a stage prior to viral DNA replication consistent with the ability of MEDS433 to inhibit DHODH activity. In fact, the anti-HSV activity of MEDS433 was abrogated by the addition of exogenous uridine or of the product of DHODH, the orotate, thus confirming DHODH as the MEDS433 specific target in HSV-infected cells. A combination of MEDS433 with dipyridamole (DPY), an inhibitor of the pyrimidine salvage pathway, was then observed to be effective in inhibiting HSV replication even in the presence of exogenous uridine, thus mimicking in vivo conditions. Finally, when combined with acyclovir and DPY in checkerboard experiments, MEDS433 exhibited highly synergistic antiviral activity. Taken together, these findings suggest that MEDS433 is a promising candidate as either single agent or in combination regimens with existing direct-acting anti-HSV drugs to develop new strategies for treatment of HSV infections.

BackgroundDihydroorotate Dehydrogenase (DHODH) is a key enzyme of the de novo pyrimidine biosynthesis, whose inhibition was recently found to induce differentiation and apoptosis in acute myeloid leukemia (AML). DHODH inhibitors were previously investigated in solid tumors, where they showed promising antiproliferative activity, both in vitro and in vivo. However, their effectiveness was not confirmed in clinical trials, probably due to the pyrimidine salvage pathway that cancer cells could exploit to survive. In this study we investigated the pro-apoptotic activity of MEDS433, the DHODH inhibitor developed by our group, against AML. Learning from previous failures, we challenged our model mimicking in vivo conditions, and looked for synergic combination to boost apoptosis.MethodsWe evaluated the apoptotic rate of multiple AML cell lines and AML primary cells treated with MEDS433 or other DHODH inhibitors, alone and in combination with classical antileukemic drugs or with dipyridamole, a blocker of the pyrimidine salvage pathway. Experiments were also performed mimicking in vivo conditions, i.e., in the presence of physiological uridine plasma levels (5 μM).ResultsMEDS433 showed a strong apoptotic effect against multiple AML cell lines, which was at least partially independent from the differentiation process. Its combination with classical antileukemic agents resulted in a further increase of the apoptotic rate. However, when MEDS433 was tested in the presence of 5 μM uridine and/or in primary AML cells, results were less impressive. On the contrary, the combination of MEDS433 with dipyridamole resulted in an outstanding synergistic effect, with a dramatic increase of the apoptotic rate both in AML cell lines and AML primary cells, which was unaffected by physiological uridine concentrations. Preliminary analyses show that the toxicity of this treatment should be limited to proliferating cells.ConclusionsThe combination of a DHODH inhibitor and dipyridamole is characterized by differentiating and pro-apoptotic features and induces metabolic lethality on a wide variety of AMLs with different genetic backgrounds.

The correct application of bio(iso)steric replacement, a potent tool for the design of optimized compounds, requires the continuous development of new isosters able to respond to specific target requirements. Among carboxylic acid isosters, as the hydroxylated pentatomic heterocyclic systems, the hydroxy-1,2,3-triazole represents one of the most versatile but less investigated. With the purpose to enlarge its bioisosteric application, we report the results of a study devoted to obtain potential biomimetics of the γ-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the central nervous system (CNS). A series of N1- and N2- functionalized 4-hydroxy-1,2,3-triazole analogues of the previous reported GABAAR ligands, including muscimol, 4-PIOL, and 4-PHP has been synthesized and characterized pharmacologically. Furthermore, this study led to development of straightforward chemical strategies directed to decorate the hydroxytriazole core scaffold, opening for further elaborative studies based on this system. The unsubstituted N1- and N2-piperidin-4-yl-4-hydroxy-1,2,3-triazole analogues (3a, 4a) of 4-PIOL and 4-PHP showed weak affinity (high to medium micromolar range), whereas substituting the 5-position of the triazole core with a 2-naphthylmethyl or 3,3-diphenylpropyl led to binding affinities in the low micromolar range. Based on electrostatic analysis and docking studies using a α1β2γ2 GABAAR homology model we were able to rationalize the observed divergence in SAR for the series of N1- and N2- piperidin-4-yl-4-hydroxy-1,2,3-triazole analogues, offering more detailed insight into the orthosteric GABAAR binding site.

The aldo-keto reductase 1C3 (AKR1C3) isoform plays a vital role in the biosynthesis of androgens and is considered an attractive target in prostate cancer (PCa). No AKR1C3-targeted agent has to date been approved for clinical use. Flufenamic acid and indomethacine are non-steroidal anti-inflammatory drugs known to inhibit AKR1C3 in a non-selective manner as COX off-target effects are also observed. Recently, we employed a scaffold hopping approach to design a new class of potent and selective AKR1C3 inhibitors based on a N-substituted hydroxylated triazole pharmacophore. Following a similar strategy, we designed a new series focused around an acidic hydroxybenzoisoxazole moiety, which was rationalised to mimic the benzoic acid role in the flufenamic scaffold. Through iterative rounds of drug design, synthesis and biological evaluation, several compounds were discovered to target AKR1C3 in a selective manner. The most promising compound of series (6) was found to be highly selective (up to 450-fold) for AKR1C3 over the 1C2 isoform with minimal COX1 and COX2 off-target effects. Other inhibitors were obtained modulating the best example of hydroxylated triazoles we previously presented. In cell-based assays, the most promising compounds of both series reduced the cell proliferation, prostate specific antigen (PSA) and testosterone production in AKR1C3-expressing 22RV1 prostate cancer cells and showed synergistic effect when assayed in combination with abiraterone and enzalutamide. Structure determination of AKR1C3 co-crystallized with one representative compound from each of the two series clearly identified both compounds in the androstenedione binding site, hence supporting the biochemical data.

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.

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.

The NF-κB signaling pathway is a validated oncological target. Here, we applied scaffold hopping to IMD-0354, a presumed IKKβ 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 IKKβ enzyme assays.

Given the heterogeneity within the γ-aminobutyric acid (GABA) receptor and transporter families, a detailed insight into the pharmacology is still relatively sparse. To enable studies of the physiological roles governed by specific receptor and transporter subtypes, a series of GABA analogues comprising five-membered nitrogen- and sulfur-containing heterocycles as amine bioisosteres were synthesized and pharmacologically characterized at native and selected recombinant GABAA receptors and GABA transporters. The dihydrothiazole and imidazoline analogues, 5-7, displayed moderate GAT activities and GABAA receptor binding affinities in the mid-nanomolar range ( Ki, 90-450 nM). Moreover, they exhibited full and equipotent agonist activity compared to GABA at GABAA-αβγ receptors but somewhat lower potency as partial agonists at the GABAA-ρ1 receptor. Stereoselectivity was observed for compounds 4 and 7 for the GABAA-αβγ receptors but not the GABAA-ρ1 receptor. This study illustrates how subtle differences in these novel amino GABA bioisosteres result in diverse pharmacological profiles in terms of selectivity and efficacy.

Pyrimidines are essential for the cell survival and proliferation of living parasitic organisms, such as Helicobacter pylori, Plasmodium falciparum and Schistosoma mansoni, that are able to impact upon human health. Pyrimidine building blocks, in human cells, are synthesised via both de novo biosynthesis and salvage pathways, the latter of which is an effective way of recycling pre-existing nucleotides. As many parasitic organisms lack pyrimidine salvage pathways for pyrimidine nucleotides, blocking de novo biosynthesis is seen as an effective therapeutic means to selectively target the parasite without effecting the human host. Dihydroorotate dehydrogenase (DHODH), which is involved in the de novo biosynthesis of pyrimidines, is a validated target for anti-infective drug research. Recent advances in the DHODH microorganism field are discussed herein, as is the potential for the development of DHODH-targeted therapeutics.

In acute myeloid leukemia (AML), blasts lose their ability to differentiate into mature cells and to undergo apoptosis. Accordingly, a proapoptotic and differentiating therapy (arsenic and retinoic acid) has dramatically improved survival in acute promyelocytic leukemia; however, a similar combination therapy is not available for other AML subtypes. In 2016, inhibition of dihydroorotate dehydrogenase (DHODH), a key enzyme of the pyrimidine biosynthesis, was found to induce differentiation in several AML models; for in vivo studies, brequinar (BRQ) was utilized. Starting from BRQ and applying a scaffold-hopping replacement, we have recently developed a new DHODH inhibitor, Meds433, which could induce differentiation at a 1-log lower concentration compared to BRQ (Sainas, J Med Chem 2018). Here we characterize Meds433 with in vitro and in vivo experiments, showing that it has a significant pro-apoptotic effect in several AML cell lines, which is at least partially independent from the differentiating effect. Analyzing the kinetic of differentiation induced by Meds433 on U937 and THP1 cell lines, and comparing the data with the number of viable cells, we noticed that cells started to die before the differentiation effect could be significant. Hence, we decided to investigate the proapoptotic effect of Meds433, treating several AML (U937, THP1, OCI-AML3, NB4) and non-AML (CEM, P3j, peripheral blood mononuclear cells-PBMC) cell lines with Meds433, and analyzing the expression of Annexin V and propidium in flow cytometry. Experiments demonstrated that Meds433 had a significant pro-apoptotic effect on several AML cell lines (Fig.1), but not on non-AML cell lines. The apoptotic rate increased with the time of exposure (3 vs 6 days), allowing to obtain a good apoptotic rate also in OCI-AML3, the most resistant cell line in our hands. As for the differentiation experiments, Meds433 could induce apoptosis at a 1-log inferior concentration compared to BRQ. More interestingly, in NB4 cells, a strong apoptotic effect was not associated with any differentiating feature, indicating that DHODH inhibition can induce apoptosis directly. As NB4 is a promyelocytic cell line, we also compared the effects of ATRA (all-trans retinoic acid), Meds433 and their combination. ATRA alone was found to induce strong differentiation and mild apoptosis on NB4 cells, while Meds433 alone could induce exclusively apoptosis; finally, the combination of ATRA and Meds433 increased both the rate of differentiating and apoptotic cells compared to ATRA only. We next tried to further characterize the apoptotic effect. When experiments were performed in the presence of uridine, a downstream product of DHODH in the pyrimidine biosynthesis, the apoptotic effect was totally abrogated. This phenomenon was already observed in the differentiation experiments (Sainas, J Med Chem 2018), and it confirms that both differentiation and apoptosis are indeed caused by the pyrimidine depletion rather than off-target mechanisms. Moreover, when experiments were performed in hypoxic conditions, the rate of apoptosis did not change, suggesting that Meds433 could work in the bone marrow hypoxic niche of (leukemic) stem cells. Further analyzing the effect of Meds433 on non-AML cells, we evaluated the maturation of T-lymphocytes, from naïve to TEMRA (T effector memory RA), finding no influence at all. Finally, preliminary results from in vivo experiments show that i) Meds433 is not toxic on Balb/c mice after 5 weeks of intraperitoneal administration; ii) the half-life is limited to 4-6 hours and iii) Meds433 has a good antileukemic activity (approximately 50% reduction of the tumor volume compared with control, after an 18-day treatment of THP1-xenograft in NSG mice). In conclusion, our work demonstrates that: i) DHODH inhibition can induce apoptosis in AML cells both directly and as a result of differentiation, partially depending on the cell line; ii) Meds433 is a novel, promising antileukemic drug, with limited toxicity, which could be active on a wide variety of AML. Since continuous exposure to the drug is fundamental in the pyrimidine starvation strategy, we are currently optimizing Meds433 pharmacokinetic profile in order to maximize the in vivo antileukemic activity. Disclosures Saglio: BMS: Consultancy; Novartis: Consultancy; Ariad: Consultancy; Incyte: Consultancy; Pfizer: Consultancy; Jansen: Consultancy; Celgene: Consultancy.

Context The inhibition of Dihydroorotate Dehydrogenase (DHODH) was recently found to induce differentiation and apoptosis in acute myeloid leukemia (AML). Multiple clinical trials testing DHODH inhibitors are currently ongoing, but the clinical effectiveness of these compounds is expected to be lower than in vitro due to: i) the heterogeneity of the AML genetic landscape and the well-known ability of leukemic cells to undergo clonal escape when treated with single agents, and ii) the pyrimidine salvage pathway, which could mitigate the pyrimidine starvation induced by DHODH inhibitors. Objective To increase the apoptotic rate induced by DHODH inhibitors in AML cells by testing multiple associations both with classical chemotherapeutical agents and targeted compounds, leveraging the synthetic lethality concept. Design MEDS433, the DHODH inhibitor developed by our group, was tested on several AML cell lines (THP1, MV4-11, NB4, U937, OCI-AML3) and primary cells, alone and in combination with cytarabine, anthracyclines, and decitabine. Moreover, in order to block the pyrimidine salvage pathway, MEDS433 was also combined with dipyridamole, a potent inhibitor of hENT1 and hENT2 nucleotide/nucleoside transport channels. Results The association of MEDS433 with cytarabine, idarubicin, and decitabine resulted in an additive effect, with a significant increase of the apoptotic rate in all tested AML cell lines. Moreover, the combination of MEDS433 with dipyridamole resulted in a synergistic effect, with an apoptotic rate ranging between 69% and 95% after just 3 days of treatment at the lowest effective MEDS433 concentration (0.1 μM), depending on the cell line. The combination of MEDS433, dipyridamole, and chemotherapy resulted in the massive death of all AML cells (>90%). The best associations were evaluated on primary cells, confirming optimal results. Conclusions The addition of DHODH inhibitors to the (3+7) regimen or to hypomethylating agents could significantly increase the performances of these classical treatments. Moreover, a deep pyrimidine starvation obtained through the inhibition of both DHODH and the pyrimidine salvage pathway, could further increase the antileukemic activity. Ongoing in vivo experiments will be determinant to verify this hypothesis and to assess the toxicity on normal cells. This work was supported by AIRC (IG-23344).

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.

RATIONALE The gas-phase fragmentation chemistry of multifunctional cations is highly influenced by the site of protonation. Possible relationships between protonation site and fragmentation processes were studied using 4-aminoalkyl-3-hydroxyfurazans. For these heterocyclic amines, the starting points for competing fragmentation pathways varied with protonation at multiple sites in two tautomers. METHODS Mass spectra were acquired using electrospray ionization (positive mode) coupled to triple quadrupole and ion trap mass spectrometers; precursor-product ion relationships were studied by collision-induced dissociation. Quantum mechanical computations were performed at the MP2/6-311++G(2d,p)//ωB97X-D/6-311+G(d) level of theory. RESULTS Prominent successive losses of NO and CO and competing losses of CH2 =NH or NH3 were observed as fragmentation processes. The lowest barrier computed for the initial step in a fragmentation pathway was associated with the [M + H]+ ion protonated at N5 in the heterocyclic ring, whereas an alternative ring cleavage leading to complementary product ions was initiated by protonation of the ring at N2. Side-chain protonation led to loss of NH3 without cleavage of the 3-hydroxyfurazan ring. CONCLUSIONS The product ions obtained by the competing fragmentation processes varied with the site of protonation. Interestingly, the most abundant product ions observed at low collision energies were formed by cleavage of protonated molecules possessing more internal energy than other isomers.

Onchocerciasis, also called river blindness, is a neglected tropical disease which is in desperate need of a therapeutic revolution. This debilitating disease, endemic in 31 countries in sub-Saharan Africa, Yemen, and Latin America, is a leading cause of blindness in the developing world. The infection is caused by the filarial parasitic nematode Onchocerca volvulus that is transmitted to humans by the black fly Simulium spp. Its pathology, whose symptoms are onchodermatitis, musculoskeletal pain, and various stages of blindness, is a result of the death of the microfilariae in the skin and eyes. This review covers the drug design and early detection fields of this pathology and pays particular attention to the period after the introduction of ivermectin, which is the only drug available for mass treatment. The emergence of ivermectin resistance justifies the crucial need to identify new drug targets and agents that can effectively treat onchocerciasis.

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.

The NF-κB signaling pathway is a validated oncological target. Here, we applied scaffold hopping to IMD-0354, a presumed IKKβ inhibitor, and identified 4-hydroxy

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.

Bioisosterism and scaffold hopping are two widely used approaches in medicinal chemistry for the purpose of lead optimization. The study highlights the physicochemical properties of the 4-hydroxy-1,2,3-triazole scaffold, a less investigated heterocyclic system. Synthetic strategies to obtain different N-substituted 4-hydroxy-1,2,3-triazole isomers are presented, and their role as possible isosteres of the carboxylic acid is discussed. The aim is to use this system to modulate the acidic moieties present in lead compounds and, at the same time, to regiodirect substituents in set directions, through targeted substitution on the three nitrogenatoms of the triazole ring. Through this approach, compounds having enhanced binding affinity, will be sought. Two examples of bioisosteric applications of this moiety are presented. In the first example, a classical bioisosteric approach mimicking the distal (S)-glutamic acid carboxyl group using the 4-hydroxy-1,2,3-triazole moiety is applied, to obtain two promising glutamate analogs. In the second example, a scaffold hopping approach is applied, replacing the phenolic moiety present in MDG-1-33A, a potent inhibitor of Onchocerca volvulus chitinase, with the 4-hydroxy-1,2,3-triazole scaffold. The 4-hydroxy-1,2,3-triazole system appears to be useful and versatile in drug design.

The synthesis and study of the antioxidant and vasodilator properties of a new class of phenols able to release nitric oxide are described. The products were designed through a symbiotic approach using selected phenols and selected nitrooxy and furoxan NO-donors as reference models. The antioxidant activities of the hybrid products were assessed by detecting the 2-thiobarbituric acid reactive substances (TBARS) produced in the ferrous salt/ascorbate-induced autoxidation of lipids present in microsomial membranes of rat hepatocytes. The vasodilator activity was assessed on rat aortic strips precontracted with phenylephrine. Some of the products (13, 35, 37, 60-62, 64) behave principally as vasodilators and others as antioxidants (24, 32, 72), and the two properties are relatively balanced in 19, 41, and 68. Further in vivo studies should clarify whether some of these products may become preclinical candidates for the treatment of cardiovascular disease underpinned by atheroma.

A series of furazan and furoxan sulfonamides were prepared and studied for their ability to inhibit human carbonic anhydrase (CA, EC 4.2.1.1) isoforms hCA I, hCA II, hCA IX, and hCA XII. The simple methyl substituted products 3–5 were potent inhibitors. Differing structural modifications of these leads had differing effects on potency and selectivity. In particular, products in which the sulfonamide group is separated from the hetero ring by a phenylene bridge retained high potency only on the hCA XII isoform. The sulfonamides 3–5 exerted intraocular pressure (IOP) lowering effects in vivo in hypertensive rabbits more efficiently than dorzolamide. Some other products (39–42), although less effective in vitro hCA II/XII inhibitors, also effectively lowered IOP in two different animal models of glaucoma.