Alkynamide phthalazinones as a new class of TbrPDEB1 inhibitors (Part 2)

Inhibitors against Trypanosoma brucei phosphodiesterase B1 (TbrPDEB1) and B2 (TbrPDEB2) have gained interest as new treatments for human African trypanosomiasis. The recently reported alkynamide tetrahydrophthalazinones, which show submicromolar activities against TbrPDEB1 and anti-T. brucei activity, have been used as starting point for the discovery of new TbrPDEB1 inhibitors. Structure-based design indicated that the alkynamide-nitrogen atom can be readily decorated, leading to the discovery of 37, a potent TbrPDEB1 inhibitor with submicromolar activities against T. brucei parasites. Furthermore, 37 is more potent against TbrPDEB1 than hPDE4 and shows no cytotoxicity on human MRC-5 cells. The crystal structures of the catalytic domain of TbrPDEB1 co-crystalized with several different alkynamides show a bidentate interaction with key-residue Gln874, but no interaction with the parasite-specific P-pocket, despite being (uniquely) a more potent inhibitor for the parasite PDE. Incubation of blood stream form trypanosomes by 37 increases intracellular cAMP levels and results in the distortion of the cell cycle and cell death, validating phosphodiesterase inhibition as mode of action.     

Alkynamide phthalazinones as a new class of TbrPDEB1 inhibitors

Several 3′,5′-cyclic nucleotide phosphodiesterases (PDEs) have been validated as good drug targets for a large variety of diseases. Trypanosoma brucei PDEB1 (TbrPDEB1) has been designated as a promising drug target for the treatment of human African trypanosomiasis. Recently, the first class of selective nanomolar TbrPDEB1 inhibitors was obtained by targeting the parasite specific P-pocket. However, these biphenyl-substituted tetrahydrophthalazinone-based inhibitors did not show potent cellular activity against Trypanosoma brucei (T. brucei) parasites, leaving room for further optimization. Herein, we report the discovery of a new class of potent TbrPDEB1 inhibitors that display improved activities against T. brucei parasites. Exploring different linkers between the reported tetrahydrophthalazinone core scaffold and the amide tail group resulted in the discovery of alkynamide phthalazinones as new TbrPDEB1 inhibitors, which exhibit submicromolar activities versus T. brucei parasites and no cytotoxicity to human MRC-5 cells. Elucidation of the crystal structure of alkynamide 8b (NPD-048) bound to the catalytic domain of TbrPDEB1 shows a bidentate interaction with the key-residue Gln874 and good directionality towards the P-pocket. Incubation of trypanosomes with alkynamide 8b results in an increase of intracellular cAMP, validating a PDE-mediated effect in vitro and providing a new interesting compound series for further studies towards selective TbrPDEB1 inhibitors with potent phenotypic activity.     

Gene Conversion Transfers the GAF-A Domain of Phosphodiesterase TbrPDEB1 to One Allele of TbrPDEB2 of Trypanosoma brucei

Background

Chromosome 9 of Trypanosoma brucei contains two closely spaced, very similar open reading frames for cyclic nucleotide specific phosphodiesterases TbrPDEB1 and TbrPDEB2. They are separated by 2379 bp, and both code for phosphodiesterases with two GAF domains in their N-terminal moieties and a catalytic domain at the C-terminus.

Methods and Findings

The current study reveals that in the Lister427 strain of T. brucei, these two genes have undergone gene conversion, replacing the coding region for the GAF-A domain of TbrPDEB2 by the corresponding region of the upstream gene TbrPDEB1. As a consequence, these strains express two slightly different versions of TbrPDEB2. TbrPDEB2a represents the wild-type phosphodiesterase, while TbrPDEB2b represents the product of the converted gene. Earlier work on the subcellular localization of TbrPDEB1 and TbrPDEB2 had demonstrated that TbrPDEB1 is predominantly located in the flagellum, whereas TbrPDEB2 partially locates to the flagellum but largely remains in the cell body. The current findings raised the question of whether this dual localization of TbrPDEB2 may reflect the two alleles. To resolve this, TbrPDEB2 of strain STIB247 that is homozygous for TbrPDEB2a was tagged in situ, and its intracellular localization was analyzed.

Conclusions

The results obtained were very similar to those found earlier with Lister427, indicating that the dual localization of TbrPDEB2 reflects its true function and is not simply due to the presence of the two different alleles. Notably, the gene conversion event is unique for the Lister427 strain and all its derivatives. Based on this finding, a convenient PCR test has been developed that allows the stringent discrimination between Lister-derived strains that are common in many laboratories and other isolates. The technique is likely very useful to resolve questions about potential mix-ups of precious field isolates with the ubiquitous Lister strain.     

Synthesis and assessment of catechol diether compounds as inhibitors of trypanosomal phosphodiesterase B1 (TbrPDEB1)

Human African trypanosomiasis (HAT) is a parasitic neglected tropical disease that affects 10,000 patients each year. Current treatments are sub-optimal, and the disease is fatal if not treated. Herein, we report our continuing efforts to repurpose the human phosphodiesterase 4 (hPDE4) inhibitor piclamilast to target trypanosomal phosphodiesterase TbrPDEB1. We prepared a range of substituted heterocyclic replacements for the 4-amino-3,5-dichloro-pyridine headgroup of piclamilast, and found that these compounds exhibited weak inhibitory activity of TbrPDEB1.     

Synthesis and evaluation of analogs of the phenylpyridazinone NPD-001 as potent trypanosomal TbrPDEB1 phosphodiesterase inhibitors and in vitro trypanocidals

Trypanosomal phosphodiesterases B1 and B2 (TbrPDEB1 and TbrPDEB2) play an important role in the life cycle of Trypanosoma brucei, the causative parasite of human African trypanosomiasis (HAT), also known as African sleeping sickness. Knock down of both enzymes leads to cell cycle arrest and is lethal to the parasite. Recently, we reported the phenylpyridazinone, NPD-001, with low nanomolar IC₅₀ values on both TbrPDEB1 (IC₅₀: 4 nM) and TbrPDEB2 (IC₅₀: 3 nM) (J. Infect. Dis. 2012, 206, 229). In this study, we now report on the first structure activity relationships of a series of phenylpyridazinone analogs as TbrPDEB1 inhibitors. A selection of compounds was also shown to be anti-parasitic. Importantly, a good correlation between TbrPDEB1 IC₅₀ and EC₅₀ against the whole parasite was observed. Preliminary analysis of the SAR of selected compounds on TbrPDEB1 and human PDEs shows large differences which shows the potential for obtaining parasite selective PDE inhibitors. The results of these studies support the pharmacological validation of the Trypanosome PDEB family as novel therapeutic approach for HAT and provide as well valuable information for the design of potent TbrPDEB1 inhibitors that could be used for the treatment of this disease.     

N-Benzyloxycarbonyl-S-(2,4-dinitrophenyl)glutathione dibutyl diester is inhibitory to melarsoprol resistant cell lines overexpressing the T. bruceiMRPA transporter

A series of glutathione derivatives 1–4, modified at the N,S and/or COOH sites, with in vitro antitrypanosomal activity were tested against bloodstream form Trypanosoma brucei 247 wild type and a T. b. brucei 247 strain over-expressing the multiple drug resistance protein (MRPA) by 50–100x to assess the susceptibility of these compounds to resistance by the TbMRP protein. Of the compounds tested, only compound 1 inhibited both bloodstream form T. brucei and T. bruceiMRPA, with a resistance factor of 1.4, indicating it to be an inhibitor of this protein and proteins acting in synergy with the transporter, whilst 2 & 3 and its derivatives showed reduced inhibitory activity against T. bruceiMRPA, indicating them to be substrates and susceptible to resistance.     

Optimization of 2-(1H-imidazo-2-yl)piperazines series of Trypanosoma brucei growth inhibitors as potential treatment for the second stage of HAT

A previous publication from our laboratory reported the identification of a new class of 2-(1H-imidazo-2-yl)piperazines as potent T. brucei growth inhibitors as potential treatment for Human African Trypanosomiasis (HAT). This work describes the structure–activity relationship (SAR) around the hit compound 1, which led to the identification of the optimized compound 18, a single digit nanomolar inhibitor (EC₅₀ 7 nM), not cytotoxic and with optimal in vivo profile that made it a suitable candidate for efficacy studies in a mouse model mimicking the second stage of disease.     

Pyrazolopyridines as potent PDE4B inhibitors: 5-Heterocycle SAR

Following the discovery of 4-(substituted amino)-1-alkyl-pyrazolo[3,4-b]pyridine-5-carboxamides as potent and selective phosphodiesterase 4B inhibitors, [Hamblin, J. N.; Angell, T.; Ballentine, S., et al. Bioorg. Med. Chem. Lett. 2008, 18, 4237] the SAR of the 5-position was investigated further. A range of substituted heterocycles showed good potencies against PDE4. Optimisation using X-ray crystallography and computational modelling led to the discovery of 16, with sub-nM inhibition of LPS-induced TNF-α production from isolated human peripheral blood mononuclear cells.     

Compounds containing 2-substituted imidazole ring for treatment against human African trypanosomiasis

A series of compounds containing 2-substituted imidazoles has been synthesized from imidazole and tested for its biological activity against human African trypanosomiasis (HAT). The 2-substituted 5-nitroimidazoles such as fexinidazole (7a) and 1-[4-(1-methyl-5-nitro-1H-imidazol-2-ylmethoxy)-pyridin-2-yl-piperazine (9e) exhibited potent activity against T. brucei in vitro with low cytotoxicity and good solubility. The presence of the NO₂ group at the 5-position of the imidazole ring in 2-substituted imidazoles is the crucial factor to inhibit T. brucei.     

Design and synthesis of pyrazolo[3,4-d]pyrimidinone derivatives: Discovery of selective phosphodiesterase-5 inhibitors

A novel series of 1,6-disubstituted pyrazolo[3,4-d]pyrimidin-7-one derivatives, 2a-h, 4a-d, 5 and 6, were successfully synthesized, which showed promising, and potent inhibition of phosphodiesterase 5 (PDE5). The inhibitory activities of 5, 4b, 2a, 2d, 2f, 4d and 4a against PDE5 were similar to that of sildenafil (100%). These compounds exhibited potent relaxant effects on isolated rat cavernosum tissue with pEC₅₀ values ranging from 8.31 to 5.16 µM. Pyrazolo[3,4-d]pyrimidin-7-one scaffolds have been rationally designed via consecutive molecular modelling studies prior to their synthesis and biological evaluation. In addition, the results of the pharmacophore-based virtual screening revealed that 1v0p_PVB might have promising activity as a PDE-5 inhibitor.     

Tetrahydroquinoline and tetrahydroisoquinoline derivatives as potential selective PDE4B inhibitors

Tetrahydroquinoline and tetrahydroisoquinoline derivatives containing 2-phenyl-5-furan moiety were designed and synthesized as phosphodiesterase type 4 (PDE4) inhibitors. The bioassay results showed that title compounds showed good inhibitory activity against PDE4B and blockade of LPS (lipopolysaccharide) induced TNF-α release, which also exhibited considerable in vivo activity in animal models of asthma/COPD (chronic obstructive pulmonary disease) and sepsis induced by LPS. The bioactivity of compounds containing tetrahydroquinoline (series 4) was higher than that of tetrahydroisoquinoline derivatives (series 3). Compound 4?m with 4-methoxybenzene moiety exhibited the best potential selective activity against PDE4B. The primary structure–activity relationship study and docking results showed that the tetrahydroquinoline moiety of compound 4?m played a key role to form hydrogen bonds and π-π stacking interaction with PDE4B protein while the rest part of the molecule extended into the catalytic domain to block the access of cAMP and formed the foundation for inhibition of PDE4B. Based on LPS induced sepsis model for the measurement of TNF-α inhibition in Swiss Albino mice and neutrophilia inhibition for asthma and COPD in Sprague Dawley rats with the potential molecules, compound 4?m would be great promise as a hit inhibitor in the future study.     

Synthesis and evaluation of human phosphodiesterases (PDE) 5 inhibitor analogs as trypanosomal PDE inhibitors. Part 2. Tadalafil analogs

In this Letter we describe our ongoing target repurposing efforts focused on discovery of inhibitors of the essential trypanosomal phosphodiesterase TbrPDEB1. This enzyme has been implicated in virulence of Trypanosoma brucei, the causative agent of human African trypanosomiasis (HAT). We outline the synthesis and biological evaluation of analogs of tadalafil, a human PDE5 inhibitor currently utilized for treatment of erectile dysfunction, and report that these analogs are weak inhibitors of TbrPDEB1.     

Discovery of 2-(1H-imidazo-2-yl)piperazines as a new class of potent and non-cytotoxic inhibitors of Trypanosoma brucei growth in vitro

The identification of a new series of growth inhibitors of Trypanosoma brucei rhodesiense, causative agent of Human African Trypanosomiasis (HAT), is described. A selection of compounds from our in-house compound collection was screened in vitro against the parasite leading to the identification of compounds with nanomolar inhibition of T. brucei growth. Preliminary SAR on the hit compound led to the identification of compound 34 that shows low nanomolar parasite growth inhibition (T. brucei EC₅₀ 5?nM), is not cytotoxic (HeLa CC₅₀?>?25,000?nM) and is selective over other parasites, such as Trypanosoma cruzi and Plasmodium falciparum (T. cruzi EC₅₀ 8120?nM, P. falciparum EC₅₀ 3624?nM).     

Novel 1,2-dihydroquinazolin-2-ones: Design,synthesis, and biological evaluation against Trypanosoma brucei

In 2014, a published report of the high-throughput screen of >42,000 kinase inhibitors from GlaxoSmithKline against T. brucei identified 797 potent and selective hits. From this rich data set, we selected NEU-0001101 (1) for hit-to-lead optimization. Through our preliminary compound synthesis and SAR studies, we have confirmed the previously reported activity of 1 in a T. brucei cell proliferation assay and have identified alternative groups to replace the pyridyl ring in 1. Pyrazole 24 achieves improvements in both potency and lipophilicity relative to 1, while also showing good in vitro metabolic stability. The SAR developed on 24 provides new directions for further optimization of this novel scaffold for anti-trypanosomal drug discovery.     

Calmodulin inhibitors from the fungus Emericella sp.

Two new xanthones identified as 15-chlorotajixanthone hydrate (1) and 14-methoxytajixanthone (2) were isolated from an Emericella sp. strain 25379 along with shamixanthone (3) and tajixanthone hydrate (4). The stereostructures of 1 and 2 were elucidated by spectroscopic and molecular modeling methods. The absolute configuration at the stereogenic centers of 1 was established according to CD measurements. In the case of 2, however, the absolute configuration at C-20 and C-25 was designated as S and R, respectively, by Mosher ester methodology. Thereafter, the configuration at C-14 and C-15 of 2 was established as S and S, respectively by comparing the optical rotation and ¹H–¹H coupling constant experimental values with those obtained through molecular modeling calculations at DFT B3LYP/DGDZVP level of theory for diasteroisomers 2a–2d. The activation of the calmodulin-sensitive cAMP phosphodiesterase (PDE1) was inhibited in the presence of 1–4 in a concentration-dependent manner. The effect of compounds 2 (IC₅₀ = 5.54 μM) and 4 (IC₅₀ = 5.62 μM) was comparable with that of chlorpromazine (CPZ; IC₅₀ = 7.26 μM), a well known CaM inhibitor used as a positive control. The inhibition mechanism of both compounds was competitive with respect to CaM according to a kinetic study. A docking analysis with 2 and 4 using the AutoDock 4.0 program revealed that they interacted with CaM in the same pocket as trifluoropiperazine (TFP).     

Discovery of halo-nitrobenzamides with potential application against human African trypanosomiasis

A series of halo-nitrobenzamide were synthesized and evaluated for their ability to block proliferation of Trypanosoma brucei brucei. A number of these compounds had significant activity against the parasite, particularly 2-chloro-N-(4-chlorophenyl)-5-nitrobenzamide 17 which exhibited low micromolar inhibitory potency against T. brucei and selectivity towards both malaria and mammalian cells.     

Synthesis and SAR of alkanediamide-linked bisbenzamidines with anti-trypanosomal and anti-pneumocystis activity

A series of alkanediamide-linked bisbenzamidines was synthesized and tested in vitro against a drug-sensitive strain of Trypanosoma brucei brucei, a drug-resistant strain of Trypanosoma brucei rhodesiense and Pneumocystis carinii. Bisbenzamidines linked with longer alkanediamide chains were potent inhibitors of both strains of T. brucei. However, bisbenzamidines linked with shorter alkanediamide chains were the most potent compounds against P. carinii. N,N′-Bis[4-(aminoiminomethyl)phenyl] hexanediamide, 4 displayed potent inhibition (IC₅₀ = 2–3 nM) against T. brucei and P. carinii, and was non-cytotoxic in the A549 human lung carcinoma cell line. The inhibitory bioactivity was significantly reduced when the amidine groups in 4 were moved from the para to the meta positions or replaced with amides.     

N-Methylanilide and N-methylbenzamide derivatives as phosphodiesterase 10A (PDE10A) inhibitors

PDE10A is a recently identified phosphodiesterase with a quite remarkable localization since the protein is abundant only in brain tissue. Based on this unique localization, research has focused extensively on using PDE10A modulators as a novel therapeutic approach for dysfunction in the basal ganglia circuit including Parkinson’s disease, Huntington’s disease, schizophrenia, addiction and obsessive compulsive disorder. Medicinal chemistry efforts identified the N-methyl-N-[4-(quinolin-2-ylmethoxy)-phenyl]-isonicotinamide (8) as a nanomolar PDE10A inhibitor. A subsequent Lead-optimization program identified analogous N-methylanilides and their corresponding N-methylbenzamides (29) as potent PDE10A inhibitors, concurrently some interesting and unexpected binding modes were identified.     

Evaluation of benzoic acid derivatives as sirtuin inhibitors

Employing a genetically modified yeast strain as a screening tool, 4-dimethylaminobenzoic acid (5) was isolated from the marine sediment-derived Streptomyces sp. CP27-53 as a weak yeast sirtuin (Sir2p) inhibitor. Using this compound as a scaffold, a series of disubstituted benzene derivatives were evaluated to elucidate the structure activity relationships for Sir2p inhibition. The results suggested that 4-alkyl or 4-alkylaminobenzoic acid is the key structure motif for Sir2p inhibitory activity. The most potent Sir2p inhibitor, 4-tert-butylbenzoic acid (20), among the tested compounds in this study turned out to be a weak but selective SIRT1 inhibitor. The calculated binding free energies between the selected compounds and the catalytic domain of SIRT1 were well correlated to their measured SIRT1 inhibitory activities.     

An alternative assay to discover potential calmodulin inhibitors using a human fluorophore-labeled CaM protein

This article describes the development of a new fluorescent-engineered human calmodulin, hCaM M124C-mBBr, useful in the identification of potential calmodulin (CaM) inhibitors. An hCaM mutant containing a unique cysteine residue at position 124 on the protein was expressed, purified, and chemically modified with the fluorophore monobromobimane (mBBr). The fluorophore-labeled protein exhibited stability and functionality to the activation of calmodulin-sensitive cAMP phosphodiesterase (PDE1) similar to wild-type hCaM. The hCaM M124C-mBBr is highly sensitive to detecting inhibitor interaction given that it showed a quantum efficiency of 0.494, approximately 20 times more than the value for wild-type hCaM, and a large spectral change (∼80% quenching) when the protein is in the presence of saturating inhibitor concentrations. Two natural products previously shown to act as CaM inhibitors, malbrancheamide (1) and tajixanthone hydrate (2), and the well-known CaM inhibitor chlorpromazine (CPZ) were found to quench the hCaM M124C-mBBr fluorescence, and the IC₅₀ values were comparable to those obtained for the wild-type protein. These results support the use of hCaM M124C-mBBr as a fluorescence biosensor and a powerful analytical tool in the high-throughput screening demanded by the pharmaceutical and biotechnology industries.