Genotoxicity test battery - An assessment of its utility in early drug development.

Formal requirements for genotoxicity testing of drug candidates to support clinical entry have been in place since the issue of initial regulatory guidance over 25 years ago and subsequent update a decade ago. An evaluation of such testing, supporting first clinical entry of 108 small molecule drug candidates over the last decade, showed that the most common approach (75 % of tested compounds) was for a Good Laboratory Practice test battery in the form of 2 in vitro (a bacterial reverse mutation and a mammalian cell) assays and one in vivo assay. The majority of other tested compounds involved in vitro testing only in bacterial reverse mutation and mammalian cell assays. Testing using a bacterial reverse mutation assay and an in vivo assessment of genotoxicity with 2 different tissues was limited to 2 occasions. For in vitro mammalian cell testing, the chromosome aberration test was most commonly used (70 % occasions), followed by a micronucleus test (16 % occasions) or a mouse lymphoma assay (14 % occasions). For in vivo evaluation, the most common test was a rodent bone marrow micronucleus test (87 % occasions). A positive in vitro mammalian cell assay result was seen on 13 % occasions but was not confirmed with further in vivo testing and the drug candidates were taken into the clinic. In conclusion, the present evaluation showed that the current test battery paradigm for genotoxicity testing has an integral part in supporting clinical entry to confirm candidate drugs taken into the clinic are unlikely to have genotoxic activity.

Recent advancements in anti-leishmanial research: Synthetic strategies and structural activity relationships.

Leishmaniasis is a parasitic neglected tropical disease caused by various species of Leishmania parasite. Despite tremendous advancements in the therapeutic sector and drug development strategies, still the existing anti-leishmanial agents are associated with some clinical issues like drug resistance, toxicity and selectivity. Therefore, several research groups are continuously working towards the development of new therapeutic candidates to overcome these issues. Many potential heterocyclic moieties have been explored for this purpose including triazoles, chalcones, chromone, thiazoles, thiosemicarbazones, indole, quinolines, etc. It is evident from the literature that the majority of anti-leishmanial agents act by interacting with key regulators including PTR-I, DHFR, LdMetAP1, MAPK, 14 α-demethylase and pteridine reductase-I, etc. Also, these tend to induce the production of ROS which causes damage to parasites. In the present compilation, authors have summarized various significant synthetic procedures for anti-leishmanial agents reported in recent years. A brief description of the pharmacological potentials of synthesized compounds along with important aspects related to structural activity relationship has been provided. Important docking outcomes highlighting the possible mode of interaction for the reported compounds have also been included. This review would be helpful to the scientific community to design newer strategies and also to develop novel therapeutic candidates against leishmaniasis.

Identification of new lead molecules against anticancer drug target TFIIH subunit P8 using biophysical and molecular docking studies.

The identification of molecules, which could modulate protein-protein interactions (PPIs), is of primary interest to medicinal chemists. Using biophysical methods during the current study, we have screened 76 compounds (grouped into 16 mixtures) against the p8 subunit of the general transcription factor (TFIIH), which has recently been validated as an anti-cancer drug target. 10% of the tested compounds showed interactions with p8 protein in STD-NMR experiments. These results were further validated by molecular docking studies where interactions between compounds and important amino acid residues were identified, including Lys20 in the hydrophobic core of p8, and Asp42 and 43 in the ß3 strand. Moreover, these compounds were able to destabilize the p8 protein by negatively shifting the Tm (≥2 °C) in thermal shift assay. Thus, this study has identified 8 compounds which are likely negative modulators of p8 protein stability, and could be further considered as potential anticancer agents.

Modulatory and Toxicological Perspectives on the Effects of the Small Molecule Kinetin.

Plant hormones are small regulatory molecules that exert pharmacological actions in mammalian cells such as anti-oxidative and pro-metabolic effects. Kinetin belongs to the group of plant hormones cytokinin and has been associated with modulatory functions in mammalian cells. The mammalian adenosine receptor (A2a-R) is known to modulate multiple physiological responses in animal cells. Here, we describe that kinetin binds to the adenosine receptor (A2a-R) through the Asn253 residue in an adenosine dependent manner. To harness the beneficial effects of kinetin for future human use, we assess its acute toxicity by analyzing different biochemical and histological markers in rats. Kinetin at a dose below 1 mg/kg had no adverse effects on the serum level of glucose or on the activity of serum alanine transaminase (ALT) or aspartate aminotransferase (AST) enzymes in the kinetin treated rats. Whereas, creatinine levels increased after a kinetin treatment at a dose of 0.5 mg/kg. Furthermore, 5 mg/kg treated kinetin rats showed normal renal corpuscles, but a mild degeneration was observed in the renal glomeruli and renal tubules, as well as few degenerated hepatocytes were also observed in the liver. Kinetin doses below 5 mg/kg did not show any localized toxicity in the liver and kidney tissues. In addition to unraveling the binding interaction between kinetin and A2a-R, our findings suggest safe dose limits for the future use of kinetin as a therapeutic and modulatory agent against various pathophysiological conditions.

Discovery and Biological Evaluation of a Novel Highly Potent Selective Butyrylcholinsterase Inhibitor.

To discover novel BChE inhibitors, a hierarchical virtual screening protocol followed by biochemical evaluation was applied. The most potent compound 8012-9656 (eqBChE IC50 = 0.18 ± 0.03 µM, hBChE IC50 = 0.32 ± 0.07 µM) was purchased and synthesized. It inhibited BChE in a noncompetitive manner and could occupy the binding pocket forming diverse interactions with the target. 8012-9656 was proven to be safe in vivo and in vitro and showed comparable performance in ameliorating the scopolamine-induced cognition impairment to tacrine. Additionally, treatment with 8012-9656 could almost entirely recover the Aß1-42 (icv)-impaired cognitive function to the normal level and showed better behavioral performance than donepezil. The evaluation of the Aß1-42 total amount confirmed its anti-amyloidogenic profile. Moreover, 8012-9656 possessed blood-brain barrier (BBB) penetrating ability, a long T1/2, and low intrinsic clearance. Hence, the novel potential BChE inhibitor 8012-9656 can be considered as a promising lead compound for further investigation of anti-AD agents.

Identification of Phosphate-Containing Compounds as New Inhibitors of 14-3-3/c-Abl Protein-Protein Interaction.

The 14-3-3/c-Abl protein-protein interaction (PPI) is related to carcinogenesis and in particular to pathogenesis of chronic myeloid leukemia (CML). Previous studies have demonstrated that molecules able to disrupt this interaction improve the nuclear translocation of c-Abl, inducing apoptosis in leukemia cells. Through an X-ray crystallography screening program, we have identified two phosphate-containing compounds, inosine monophosphate (IMP) and pyridoxal phosphate (PLP), as binders of human 14-3-3σ, by targeting the protein amphipathic groove. Interestingly, they also act as weak inhibitors of the 14-3-3/c-Abl PPI, demonstrated by NMR, SPR, and FP data. A 37-compound library of PLP and IMP analogues was investigated using a FP assay, leading to the identification of three further molecules acting as weak inhibitors of the 14-3-3/c-Abl complex formation. The antiproliferative activity of IMP, PLP, and the three derivatives was tested against K-562 cells, showing that the parent compounds had the most pronounced effect on tumor cells. PLP and IMP were also effective in promoting the c-Abl nuclear translocation in c-Abl overexpressing cells. Further, these compounds demonstrated low cytotoxicity on human Hs27 fibroblasts. In conclusion, our data suggest that 14-3-3σ targeting compounds represent promising hits for further development of drugs against c-Abl-dependent cancers.

LMP2 Inhibitors as a Potential Treatment for Alzheimer's Disease.

The immunoproteasome (iP), an inducible proteasome variant harboring three immunosubunits, low molecular mass polypeptide-2 (LMP2), multicatalytic endopeptidase complex subunit-1, and low molecular mass polypeptide-7 (LMP7), is involved in multiple facets of inflammatory responses. We recently reported that YU102, a dual inhibitor of the iP subunit LMP2 and the constitutive proteasome catalytic subunit ß1, ameliorates cognitive impairments in mouse models of Alzheimer's disease (AD) independently of amyloid deposits. To investigate whether inhibition of LMP2 is sufficient to improve the cognitive functions of AD mice, here we prepared 37 YU102 analogues and identified a potent LMP2 inhibitor DB-310 (28) (IC50: 80.6 nM) with improved selectivity and permeability in cells overexpressing ABCB1 transporters. We show that DB-310 induces suppression of IL-1α production in microglia cells and improves cognitive functions in the Tg2576 transgenic mouse model of AD. This study supports that inhibition of LMP2 is a promising therapeutic strategy for treatment of AD.

Discovery of Novel Latency-Associated Nuclear Antigen Inhibitors as Antiviral Agents Against Kaposi's Sarcoma-Associated Herpesvirus.

With the aim to develop novel antiviral agents against Kaposi's Sarcoma Herpesvirus (KSHV), we are targeting the latency-associated nuclear antigen (LANA). This protein plays an important role in viral genome maintenance during latent infection. LANA has the ability to tether the viral genome to the host nucleosomes and, thus, ensures latent persistence of the viral genome in the host cells. By inhibition of the LANA-DNA interaction, we seek to eliminate or reduce the load of the viral DNA in the host. To achieve this goal, we screened our in-house library using a dedicated fluorescence polarization (FP)-based competition assay, which allows for the quantification of LANA-DNA-interaction inhibition by small organic molecules. We successfully identified three different compound classes capable of disrupting this protein-nucleic acid interaction. We characterized these compounds by IC50 dose-response evaluation and confirmed the compound-LANA interaction using surface plasmon resonance (SPR) spectroscopy. Furthermore, two of the three hit scaffolds showed only marginal cytotoxicity in two human cell lines. Finally, we conducted STD-NMR competition experiments with our new hit compounds and a previously described fragment-sized inhibitor. Based on these results, future compound linking approaches could serve as a promising strategy for further optimization studies in order to generate highly potent KSHV inhibitors.

Elucidating the aryl hydrocarbon receptor antagonism from a chemical-structural perspective.

The aryl hydrocarbon receptor (AhR) plays an important role in several biological processes such as reproduction, immunity and homoeostasis. However, little is known on the chemical-structural and physicochemical features that influence the activity of AhR antagonistic modulators. In the present report, in vitro AhR antagonistic activity evaluations, based on a chemical-activated luciferase gene expression (AhR-CALUX) bioassay, and an extensive literature review were performed with the aim of constructing a structurally diverse database of contaminants and potentially toxic chemicals. Subsequently, QSAR models based on Linear Discriminant Analysis and Logistic Regression, as well as two toxicophoric hypotheses were proposed to model the AhR antagonistic activity of the built dataset. The QSAR models were rigorously validated yielding satisfactory performance for all classification parameters. Likewise, the toxicophoric hypotheses were validated using a diverse set of 350 decoys, demonstrating adequate robustness and predictive power. Chemical interpretations of both the QSAR and toxicophoric models suggested that hydrophobic constraints, the presence of aromatic rings and electron-acceptor moieties are critical for the AhR antagonism. Therefore, it is hoped that the deductions obtained in the present study will contribute to elucidate further on the structural and physicochemical factors influencing the AhR antagonistic activity of chemical compounds.

Small Molecule Antipsychotic Aripiprazole Potentiates Ozone-Induced Inflammation in Airway Epithelium.

Inhaled ground level ozone (O3) has well described adverse health effects, which may be augmented in susceptible populations. While conditions, such as pre-existing respiratory disease, have been identified as factors enhancing susceptibility to O3-induced health effects, the potential for chemical interactions in the lung to sensitize populations to pollutant-induced responses has not yet been studied. In the airways, inhaled O3 reacts with lipids, such as cholesterol, to generate reactive and electrophilic oxysterol species, capable of causing cellular dysfunction and inflammation. The enzyme regulating the final step of cholesterol biosynthesis, 7-dehydrocholesterol reductase (DHCR7), converts 7-dehydrocholesterol (7-DHC) to cholesterol. Inhibition of DHCR7 increases the levels of 7-DHC, which is much more susceptible to oxidation than cholesterol. Chemical analysis established the capacity for a variety of small molecule antipsychotic drugs, like Aripiprazole (APZ), to inhibit DHCR7 and elevate circulating 7-DHC. Our results show that APZ and the known DHCR7 inhibitor, AY9944, increase 7-DHC levels in airway epithelial cells and potentiate O3-induced IL-6 and IL-8 expression and cytokine release. Targeted immune-related gene array analysis demonstrates that APZ significantly modified O3-induced expression of 16 genes, causing dysregulation in expression of genes associated with leukocyte recruitment and inflammatory response. Additionally, we find that APZ increases O3-induced IL-6 and IL-8 expression in human nasal epithelial cells from male but not female donors. Overall, the evidence we provide describes a novel molecular mechanism by which chemicals, such as APZ, that perturb cholesterol biosynthesis affect O3-induced biological responses.

Benzoic acid-derived nitrones: A new class of potential acetylcholinesterase inhibitors and neuroprotective agents.

The discovery of new chemical entities endowed with potent and selective acetylcholinesterase (AChE) and/or butyrylcholinesterase (BChE) inhibitory activity is still a relevant subject for Alzheimer's disease therapy. Therefore, a small library of benzoic based amide nitrones (compounds 24 to 42) was synthesized and screened toward cholinesterase enzymes. SAR studies showed that the tert-butyl moiety is the most favourable nitrone pattern. In general, tert-butyl derivatives effectively inhibited AChE, being compound 33 the most potent (IC50 = 8.3 ±â€¯0.3 µM; Ki 5.2 µM). The data pointed to a non-competitive inhibition mechanism of action, which was also observed for the standard donepezil. None of compounds showed BChE inhibitory activity. Molecular modelling studies provided insights into the enzyme-inhibitor interactions and rationalised the experimental data, confirming that the binding mode of nitrones 33 and 38 towards AChE has the most favourable binding free energy. The tert-butylnitrones 33 and 38 were not cytotoxic on different cell lines (SH-SY5Y and HepG2). Moreover, compound 33 was able to prevent t-BHP-induced oxidative stress in SH-SY5Y differentiated cells. Due to its AChE selectivity and promising cytoprotective properties, as well as its appropriate drug-like profile pointing toward blood-brain barrier permeability, compound 33 is proposed as a valid lead for a further optimization step.

In-silico design and synthesis of N9-substituted ß-Carbolines as PLK-1 inhibitors and their in-vitro/in-vivo tumor suppressing evaluation.

A new series of ß-Carboline/Schiff bases was designed, synthesized, characterised and biologically evaluated as inhibitors of PLK-1. The synthesized compounds exhibited strong to moderate cytotoxic activities against NCI-60 panel cell assay. Compound SB-2 was the most potent, particularly against colon with GI50 of 3-45 µM on NCI-60 panel cell lines. SB-2 selectively inhibited PLK-1 at 15 µM on KinomeScan screening. It also showed a dose-dependent cell cycle arrest at S/G2 phase on HCT-116 and induced apoptosis by the activation of procaspase-3 and cleaved PARP. Further, the antitumor studies on DLA and EAC model revealed that SB-2, at 100 mg/kg/bd.wt significantly increased their average lifespan. Further, a decrease in the body weight of the tumor-bearing mice was also observed when compared to the tumor controlled mice. SB-2 thus shows good potential as antitumor agent.

Identification of Thienopyrimidine Scaffold as an Inhibitor of the ABC Transport Protein ABCC1 (MRP1) and Related Transporters Using a Combined Virtual Screening Approach.

A virtual screening protocol with combination of similarity search and pharmacophore modeling was applied to virtually screen a large compound library to gain new scaffolds regarding ABCC1 inhibition. Biological investigation of promising candidates revealed four compounds as ABCC1 inhibitors, three of them with scaffolds not associated with ABCC1 inhibition until now. The best hit molecule-a thienopyrimidine-was a moderately potent, competitive inhibitor of the ABCC1-mediated transport of calcein AM which also sensitized ABCC1-overexpressing cells toward daunorubicin. Further evaluation showed that it was a moderately potent, competitive inhibitor of the ABCB1-mediated transport of calcein AM, and noncompetitive inhibitor of the ABCG2-mediated pheophorbide A transport. In addition, the thienopyrimidine could also sensitize ABCB1- as well as ABCG2-overexpressing cells toward daunorubicin and SN-38, respectively, in concentration ranges that qualified it as one of the ten best triple ABCC1/ABCB1/ABCG2 inhibitors in the literature. Besides, three more new multitarget inhibitors were identified by this virtual screening approach.

Identification of Modulators That Activate the Constitutive Androstane Receptor From the Tox21 10K Compound Library.

The constitutive androstane receptor (CAR; NR1I3) is a nuclear receptor involved in all phases of drug metabolism and disposition. However, recently it's been implicated in energy metabolism, tumor progression, and cancer therapy as well. It is, therefore, important to identify compounds that induce human CAR (hCAR) activation to predict drug-drug interactions and potential therapeutic usage. In this study, we screen the Tox21 10,000 compound collection to characterize hCAR activators. A potential novel structural cluster of compounds was identified, which included nitazoxanide and tenonitrozole, whereas known structural clusters, such as flavones and prazoles, were also detected. Four compounds, neticonazole, diphenamid, phenothrin, and rimcazole, have been identified as novel hCAR activators, one of which, rimcazole, shows potential selectivity toward hCAR over its sister receptor, the pregnane X receptor (PXR). All 4 compounds translocated hCAR from the cytoplasm into the nucleus demonstrating the first step to CAR activation. Profiling these compounds as hCAR activators would enable an estimation of drug-drug interactions, as well as identify prospective therapeutically beneficial drugs.

Screening the ToxCast Phase 1, Phase 2, and e1k Chemical Libraries for Inhibitors of Iodothyronine Deiodinases.

Deiodinase enzymes play an essential role in converting thyroid hormones between active and inactive forms by deiodinating the pro-hormone thyroxine (T4) to the active hormone triiodothyronine (T3) and modifying T4 and T3 to inactive forms. Chemical inhibition of deiodinase activity has been identified as an important endpoint to include in screening chemicals for thyroid hormone disruption. To address the lack of data regarding chemicals that inhibit the deiodinase enzymes, we developed robust in vitro assays that utilized human deiodinase types 1, 2, and 3 and screened over 1800 unique chemicals from the U.S. EPA's ToxCast phase 1_v2, phase 2, and e1k libraries. Initial testing at a single concentration identified 411 putative deiodinase inhibitors that produced inhibition of 20% or greater in at least 1 of the 3 deiodinase assays, including chemicals that have not previously been shown to inhibit deiodinases. Of these, 228 chemicals produced enzyme inhibition of 50% or greater; these chemicals were further tested in concentration-response to determine relative potency. Comparisons across these deiodinase assays identified 81 chemicals that produced selective inhibition, with 50% inhibition or greater of only 1 of the deiodinases. This set of 3 deiodinase inhibition assays provides a significant contribution toward expanding the limited number of in vitro assays used to identify chemicals with the potential to interfere with thyroid hormone homeostasis. In addition, these results set the groundwork for development and evaluation of structure-activity relationships for deiodinase inhibition, and inform targeted selection of chemicals for further testing to identify adverse outcomes of deiodinase inhibition.

Screening Method for Identifying Toxicants Capable of Inducing Astrocyte Senescence.

Cellular senescence is a tumor-suppressive mechanism which leads to near irreversible proliferative arrest. However, senescent cells can cause tissue dysfunction, in large part because they express a senescence-associated secretory phenotype (SASP) involving secretion of, amongst other factors, proinflammatory cytokines known to compromise neuronal health. Therefore, established neurotoxicants may cause neurotoxicity in vivo, in part by triggering mitotic cells in the brain to undergo senescence and adopt an inflammatory SASP which in turn could cause deleterious effects to surrounding neurons. To begin to address this hypothesis, we examined whether we could screen known neurotoxicants for their ability to cause astrocytes (a mitotic cell type especially important for maintaining neuronal health) to undergo senescence. For this purpose, we utilized inducible pluripotent stem cell-derived human astrocytes and screened an 80 compound neurotoxicant library provided by the Biomolecular Screening Branch of the NIEHS National Toxicology Program. Here we present a screening method based on induction of the senescent marker, senescent-associated beta-galactosidase (SA-ß-gal). We describe in detail an automated method for the unbiased quantitation of percentage of SA-ß-gal + astrocytes. Although our results suggest that conducting an SA-ß-gal senescence screen using human inducible pluripotent stem cell-derived astrocytes may be feasible, they also highlight challenges that likely preclude its adaptation to high-throughput. We also explore the possibility of using primary mouse astrocytes for this purpose and explain why this platform is problematic and very unlikely to yield meaningful results, even in small screens with compound replicates.

Chemoproteomics-Enabled Covalent Ligand Screening Reveals ALDH3A1 as a Lung Cancer Therapy Target.

Chemical genetics is a powerful approach for identifying therapeutically active small molecules, but identifying the mechanisms of action underlying hit compounds remains challenging. Chemoproteomic platforms have arisen to tackle this challenge and enable rapid mechanistic deconvolution of small-molecule screening hits. Here, we have screened a cysteine-reactive covalent ligand library to identify hit compounds that impair cell survival and proliferation in nonsmall cell lung carcinoma cells, but not in primary human bronchial epithelial cells. Through this screen, we identified a covalent ligand hit, DKM 3-42, which impaired both in situ and in vivo lung cancer pathogenicity. We used activity-based protein profiling to discover that the primary target of DKM 3-42 was the catalytic cysteine in aldehyde dehydrogenase 3A1 (ALDH3A1). We performed further chemoproteomics-enabled covalent ligand screening directly against ALDH3A1, and identified a more potent and selective lead covalent ligand, EN40, which inhibits ALDH3A1 activity and impairs lung cancer pathogenicity. We show here that ALDH3A1 represents a potentially novel therapeutic target for lung cancers that express ALDH3A1 and put forth two selective ALDH3A1 inhibitors. Overall, we show the utility of combining chemical genetics screening of covalent ligand libraries with chemoproteomic approaches to rapidly identify anticancer leads and targets.

The current limits in virtual screening and property prediction.

Beyond finding inhibitors that show high binding affinity to the respective target, there is the challenge of optimizing their properties with respect to metabolic and toxicological issues, as well as further off-target effects. To reduce the experimental effort of synthesizing and testing actual substances in corresponding assays, virtual screening has become an indispensable toolbox in preclinical development. The scope of application covers the prediction of molecular properties including solubility, metabolic liability and binding to antitargets, such as the hERG channel. Furthermore, prediction of binding sites and drugable targets are emerging aspects of virtual screening. Issues involved with the currently applied computational models including machine learning algorithms are outlined, such as limitations to the accuracy of prediction and overfitting.

Cationic and Neutral N-Heterocyclic Carbene Gold(I) Complexes: Cytotoxicity, NCI-60 Screening, Cellular Uptake, Inhibition of Mammalian Thioredoxin Reductase, and Reactive Oxygen Species Formation.

A structurally diverse library of 14 gold(I) cationic bis(NHC) and neutral mono(NHC) complexes (NHC: N-heterocyclic carbene) was synthesized and characterized in this work. Four of them were new cationic gold(I) complexes containing functionalized NHCs, and their X-ray crystal structures are presented herein. All of the complexes were investigated for their anticancer activities in four cancer cell lines, including a cisplatin-resistant variant, and a noncancerous cell line. Seven of the cationic gold(I) complexes were found to display high and specific cytotoxic activities toward cancer cells. Two of them were even able to overcome cisplatin resistance. Two highly potent cationic complexes (11 and 15) were also submitted to the NCI-60 cancer panel for further cytotoxicity evaluation. Complex 15 showed a surprisingly high potency toward leukemia among the nine examined cancer subtypes, particularly toward the CCRF-CEM leukemia cell line with a concentration for 50 % inhibition of growth down to 79.4 nm. In addition, cationic complex 13, which demonstrated a remarkable cytotoxicity against hepatocellular carcinoma, was selected to obtain insight into the mechanistic aspects in HepG2 cells. Cellular uptake measurements were indicative of good bioavailability. By various biochemical assays, this complex was found to effectively inhibit thioredoxin reductase and its cytotoxicity toward HepG2 cells was found to be reactive oxygen species dependent.

Development of a Focused Library of Triazole-Linked Privileged-Structure-Based Conjugates Leading to the Discovery of Novel Phenotypic Hits against Protozoan Parasitic Infections.

Protozoan infections caused by Plasmodium, Leishmania, and Trypanosoma spp. contribute significantly to the burden of infectious diseases worldwide, causing severe morbidity and mortality. The inadequacy of available treatments calls for cost- and time-effective drug discovery endeavors. To this end, we envisaged the triazole linkage of privileged structures as an effective drug design strategy to generate a focused library of high-quality compounds. The versatility of this approach was combined with the feasibility of a phenotypic assay, integrated with early ADME-tox profiling. Thus, an 18-membered library was efficiently assembled via Huisgen cycloaddition of phenothiazine, biphenyl, and phenylpiperazine scaffolds. The resulting 18 compounds were then tested against seven parasite strains, and counter-screened for selectivity against two mammalian cell lines. In parallel, hERG and cytochrome P450 (CYP) inhibition, and mitochondrial toxicity were assessed. Remarkably, 10-((1-(3-([1,1'-biphenyl]-3-yloxy)propyl)-1H-1,2,3-triazol-5-yl)methyl)-10H-phenothiazine (7) and 10-(3-(1-(3-([1,1'-biphenyl]-3-yloxy)propyl)-1H-1,2,3-triazol-4-yl)propyl)-10H-phenothiazine (12) showed respective IC50 values of 1.8 and 1.9 µg mL-1 against T. cruzi, together with optimal selectivity. In particular, compound 7 showed a promising ADME-tox profile. Thus, hit 7 might be progressed as an antichagasic lead.