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Human African trypanosomiasis is causing thousands of deaths every year in the rural areas of Africa. In this manuscript we describe the optimization of a family of phtalazinone derivatives. Phosphodiesterases have emerged as attractive molecular targets for a novel treatment for a variety of neglected parasitic diseases. Compound 1 resulted in being a potent TbrPDEB1 inhibitor with interesting activity against T. brucei in a phenotypic screen. Derivative 1 was studied in an acute in vivo mouse disease model but unfortunately showed no efficacy due to low metabolic stability. We report structural modifications to achieve compounds with an improved metabolic stability while maintaining high potency against TbrPDEB1 and T. brucei. Compound 14 presented a good microsomal stability in mouse and human microsomes and provides a good starting point for future efforts.

9 p.-5 fig.-2 tab., We report the evaluation of 265 compounds from a PDE-focused library for their antischistosomal activity, assessed in vitro using Schistosoma mansoni. Of the tested compounds, 171 (64%) displayed selective in vitro activity, with 16 causing worm hypermotility/spastic contractions and 41 inducing various degrees of worm killing at 100 μM, with the surviving worms displaying sluggish movement, worm unpairing and complete absence of eggs. The compounds that did not affect worm viability (n=72) induced a complete cessation of ovipositing. 82% of the compounds had an impact on male worms whereas female worms were barely affected. In vivo evaluation in S. mansoni-infected mice with the in vitro ‘hit’ NPD-0274 at 20 mg/kg/day orally for 5 days resulted in worm burden reductions of 29% and intestinal tissue egg load reduction of 35% at 10 days posttreatment.Combination of praziquantel (PZQ) at 10 mg/kg/day for 5 days with NPD-0274 or NPD-0298 resulted in significantly higher worm killing than PZQ alone, as well as a reduction in intestinal tissue egg load, disappearance of immature eggs and an increase in the number of dead eggs., This work is part of the PDE4NPD consortium supported by Framework Program 7 of the European Commission No: 602666.Funding from RICET/FEDER funds (RD16/0027/0010), and the Ministry of Education, Culture and Sport (MECD) of Spain (Grant FPU15/1465 to V. S.-P.) is also acknowledged. Y. Z. was supported by a grant of the Chinese Science Council; A. R. B. and I. J. P. dE. were supported by the Netherlands Science Foundation.

Increased Gram-negative bacteria resistance to antibiotics is becoming a global problem, and new classes of antibiotics with novel mechanisms of action are required. The caseinolytic protease subunit P (ClpP) is a serine protease conserved among bacteria that is considered as an interesting drug target. ClpP function is involved in protein turnover and homeostasis, stress response, and virulence among other processes. The focus of this study was to identify new inhibitors of Escherichia coli ClpP and to understand their mode of action. A focused library of serine protease inhibitors based on diaryl phosphonate warheads was tested for ClpP inhibition, and a chemical exploration around the hit compounds was conducted. Altogether, 14 new potent inhibitors of E. coli ClpP were identified. Compounds 85 and 92 emerged as most interesting compounds from this study due to their potency and, respectively, to its moderate but consistent antibacterial properties as well as the favorable cytotoxicity profile.

Human African trypanosomiasis is causing thousands of deaths every year in the rural areas of sub-saharan Africa. There is a high unmet medical need since the approved drugs are poorly efficacious, show considerable toxicity and are not easy to administer. This work describes the optimization of the pharmacokinetic properties of a previously published family of triazine lead compounds. One compound (35 (UAMC-03011)) with potent anti-trypanosomal activity and no cytotoxicity was selected for further study because of its good microsomal stability and high selectivity for Trypanosoma brucei over a panel including Trypanosoma cruzi, L.eishmania infantum, and Plasmodium falciparum. In vivo pharmacokinetic parameters were determined and the compound was studied in an acute in vivo mouse disease model. One of the important learnings of this study was that the rate of trypanocidal activity is an important parameter during the lead optimization process.

Parkinson’s disease (PD), an age-related neurodegenerative disorder that results from a progressive loss of dopaminergic neurons has an enormous economical and human cost. Unfortunately, only symptomatic treatment such as dopamine replacement therapy is available. Therefore, drugs with new mechanisms of action able to protect against neuronal cell death are an urgent need. We here report the in vivo efficacy on dopaminergic neuronal protection in a PD mouse model and the lack of toxicity in zebrafish and Ames test of benzothiazole-based casein kinase-1δ (CK-1δ) nanomolar inhibitors. On the basis of these results, we propose protein kinase CK-1δ inhibitors as the possible disease-modifying drugs for PD, benzothiazole 4 being a promising drug candidate for further development as a new therapy of this neurodegenerative disease., This work was supported by grants from MINECO (SAF2012-37979-C03-01 to A.M. and SAF2014-52940-R to A.P.-C.) and from MICINN (grant SAF2010-16365 to A.P.-C.). I.G.S. and D.I.P. acknowledge a pre- and postdoctoral fellowship from MICINN (FPI program) and CSIC (JAE program), respectively. CIBERNED is funded by the Instituto de Salud Carlos III. J.A.M.-G. is a fellow from CIBERNED.

Background Chagas’ disease, caused by the protozoan parasite Trypanosoma cruzi, needs urgent alternative therapeutic options as the treatments currently available display severe limitations, mainly related to efficacy and toxicity. Objectives As phosphodiesterases (PDEs) have been claimed as novel targets against T. cruzi, our aim was to evaluate the biological aspects of 12 new phthalazinone PDE inhibitors against different T. cruzi strains and parasite forms relevant for human infection. Methods In vitro trypanocidal activity of the inhibitors was assessed alone and in combination with benznidazole. Their effects on parasite ultrastructural and cAMP levels were determined. PDE mRNA levels from the different T. cruzi forms were measured by quantitative reverse transcription PCR. Results Five TcrPDEs were found to be expressed in all parasite stages. Four compounds displayed strong effects against intracellular amastigotes. Against bloodstream trypomastigotes (BTs), three were at least as potent as benznidazole. In vitro combination therapy with one of the most active inhibitors on both parasite forms (NPD-040) plus benznidazole demonstrated a quite synergistic profile (xΣ FICI = 0.58) against intracellular amastigotes but no interaction (xΣ FICI = 1.27) when BTs were assayed. BTs treated with NPD-040 presented disrupted Golgi apparatus, a swollen flagellar pocket and signs of autophagy. cAMP measurements of untreated parasites showed that amastigotes have higher ability to efflux this second messenger than BTs. NPD-001 and NPD-040 increase the intracellular cAMP content in both BTs and amastigotes, which is also released into the extracellular milieu. Conclusions The findings demonstrate the potential of PDE inhibitors as anti-T. cruzi drug candidates.

The caseinolytic protease proteolytic subunit (ClpP) is a serine protease playing an important role in proteostasis of eukaryotic organelles and prokaryotic cells. Alteration of ClpP function has been proved to affect the virulence and infectivity of a number of pathogens. Increased bacterial resistance to antibiotics has become a global problem and new classes of antibiotics with novel mechanisms of action are needed. In this regard, ClpP has emerged as an attractive and potentially viable option to tackle pathogen fitness without suffering cross-resistance to established antibiotic classes and, when not an essential target, without causing an evolutionary selection pressure. This opens a greater window of opportunity for the host immune system to clear the infection by itself or by co-administration with commonly prescribed antibiotics. A comprehensive overview of the function, regulation and structure of ClpP across the different organisms is given. Discussion about mechanism of action of this protease in bacterial pathogenesis and human diseases are outlined, focusing on the compounds developed in order to target the activation or inhibition of ClpP.

Human African trypanosomiasis (HAT), also known as sleeping sickness is a parasitic disease transmitted by the bite of the 'Glossina' insect, commonly known as the tsetse fly. This disease affects mostly poor populations living in remote rural areas of Africa. Untreated, it is usually fatal. Currently, safe and effective treatments against this disease are lacking. Phenotypic screening of triazine non-nucleoside HIV-1 reverse transcriptase inhibitors (monomers) resulted in potent and selective antitrypanosomal compounds. This serendipitous discovery and the presence of dimers in many compounds active against these neglected tropical diseases prompted us to investigate antitrypanosomal activity of triazine dimers. Optimization of the triazine dimers resulted in 3,3'-(((ethane-1,2-diylbis(azanediyl))bis(4-(mesityloxy)-1,3,5-triazine-6,2-diyl))bis(azanediyl))dibenzonitrile (compound 38), a compound with very potent in vitro and moderate in vivo antitrypanosomal activity.

46 p.-7 fig.-1 tab., Leucine-rich repeat kinase 2 (LRRK2) is one of the most pursued targets for Parkinson’s disease (PD) therapy. Moreover, it has recently described its role in regulating Wnt signaling and thus, it may be involved in adult neurogenesis. This new hypothesis could give rise to double disease-modifying agents firstly by the benefits of inhibiting LRRK2 and secondly by promoting adult neurogenesis. Herein we report, the design, synthesis, biological evaluation, SAR and potential binding mode of indolinelike LRRK2 inhibitors and their preliminary neurogenic effect in neural precursor cells isolated from adult mice ventricular-subventricular zone. These results open new therapeutic horizons for the use of LRRK2 inhibitors as neuroregenerative agents.Moreover, the indolinone derivatives here prepared, inhibitors of the kinase activity of LRRK2, may be considered as pharmacological probes to study the potential neuroregeneration of the damaged brain., Financial support from MINECO (grant no. SAF2012-37979-C03-01 and RTC-2015-3439-1 to A. M. and BFU2014-57494-R to A. V. M), MECD (FPU13-003262 to J. Z.D.).

[Background] Mutations in the progranulin gene (GRN) are the most common cause of frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP). TDP-43 pathology is characterized by the hyperphosphorylation of the protein at Serine 409/410 residues. Casein kinase-1δ (CK-1δ) was reported to phosphorylate TDP-43 directly. Previous works from our laboratory described the presence of CDK6/pRb-dependent cell cycle alterations, and cytosolic accumulation of TDP-43 protein in lymphoblast from FTLD-TDP patients carriers of a loss-of function mutation in GRN gene (c.709-1G > A). In this work, we have investigated the effects of two brain penetrant CK-1δ inhibitors (IGS-2.7 and IGS-3.27) designed and synthetized in our laboratory on cell proliferation, TDP-43 phosphorylation and subcellular localization, as well as their effects on the known nuclear TDP-43 function repressing the expression of CDK6., [Results] We report here that both CK-1δ inhibitors (IGS-2.7 and IGS-3.27) normalized the proliferative activity of PGRN-deficient lymphoblasts by preventing the phosphorylation of TDP-43 fragments, its nucleo-cytosol translocation and the overactivation of the CDK6/pRb cascade. Moreover, ours results show neuroprotective effects of CK-1δ inhibitors in a neuronal cell model of induced TDP-43 phosphorylation., [Conclusions] Our results suggest that modulating CK-1δ activity could be considered a novel therapeutic approach for the treatment of FTLD-TDP and other TDP-43 proteinopathies., This work has been supported by grants from The Spanish Ministry of Economy and Competitiveness (projects SAF2012-37979-C03-01 to Ana Martinez and SAF2011-28603 to Angeles Martín-Requero). Moreover, Dr. Angeles Martín-Requero was supported by Ramón Areces foundation and Dr. López de Munain received research support from Ilundain Foundation.

The use of FeCl2 as the catalyst yields mixtures of E/Z-olefins except in the case of substrates (VI), where only the (Z) isomers are formed.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease where motor neurons in cortex, brain stem, and spinal cord die progressively, resulting in muscle wasting, paralysis, and death. Currently, effective therapies for ALS are lacking; however, identification of pathological TAR DNA-binding protein 43 (TDP-43) as the hallmark lesion in sporadic ALS suggests new therapeutic targets for pharmacological intervention. Pathological TDP-43 phosphorylation appears to drive the onset and progression of ALS and may result from upregulation of the protein kinase CK-1 in affected neurons, resulting in postranslational TDP-43 modification. Consequently, brain penetrant specific CK-1 inhibitors may provide a new therapeutic strategy for treating ALS and other TDP-43 proteinopathies. Using a chemical genetic approach, we report the discovery and further optimization of a number of potent CK-1δ inhibitors. Moreover, these small heterocyclic molecules are able to prevent TDP-43 phosphorylation in cell cultures, to increase Drosophila lifespan by reduction of TDP-43 neurotoxicity, and are predicted to cross the blood-brain barrier. Thus, N-(benzothiazolyl)-2-phenyl-acetamides are valuable drug candidates for further studies and may be a new therapeutic approach for ALS and others pathologies in which TDP-43 is involved. © 2014 American Chemical Society.

It is a common practice in drug discovery organizations to screen new chemical entities in order to predict future drug-drug interactions. For this purpose, there are two main assay strategies, one based on recombinant cytochrome P450 (rCYP) enzymes and fluorescent detection, and other on human liver microsomes (HLM) and liquid chromatography coupled to mass spectrometry. Many authors have reported a poor correlation between both technologies, giving rise to concerns about the usefulness of fluorometric methods for predicting drug-drug interactions. In this study, we investigated the role that compound aqueous kinetic solubility may play in this lack of correlation. We found that drug discovery compounds with unacceptable kinetic solubility, measured by a turbidimetric solutibility assay, tended to yield higher IC50 values in in vitro models based on human liver microsomes, whereas compounds with kinetic solubility values higher than 50 μM showed very similar IC50 values in both in vitro models. Our results show that the turbidimetric solubility assay is a useful tool to identify those discovery compounds that may require further investigation in order to avoid overlooking future drug-drug interactions