Discovery of Novel Trypanosoma brucei Phosphodiesterase B1 Inhibitors by Virtual Screening against the Unliganded TbrPDEB1 Crystal Structure

Trypanosoma brucei cyclic nucleotide phosphodiesterase B1 (TbrPDEB1) and TbrPDEB2 have recently been validated as new therapeutic targets for human African Trypanosomiasis by both genetic and pharmacological means. In this study we report the crystal structure of the catalytic domain of the unliganded TbrPDEB1 and its use for the in silico screening for new TbrPDEB1 inhibitors with novel scaffolds. The TbrPDEB1 crystal structure shows the characteristic folds of human PDE enzymes, but also contains the parasite-specific P-pocket found in the structures of Leishmania major PDEB1 and Trypanosoma cruzi PDEC. The unliganded TbrPDEB1 X-ray structure was subjected to a structure-based in silico screening approach that combines molecular docking simulations with a protein-ligand interaction fingerprint (IFP) scoring method. This approach identified, six novel TbrPDEB1 inhibitors with IC50 values of 10–80 μM, which may be further optimized as potential selective TbrPDEB inhibitors

Phenyldihydropyrazolones as Novel Lead Compounds Against Trypanosoma cruzi

As over 6 million people are infected with Chagas disease and only limited therapeutic options are available, there is an urgent need for novel drugs. The involvement of cyclic nucleotide phosphodiesterases (PDE) in the lifecycle and biological fitness of a number of protozoan parasites has been described and several of these enzymes are thought to be viable drug targets. Within this context, a PDE-focused library was screened for its ability to affect the viability of Trypanosoma cruzi parasites. 5-(3-(Benzyloxy)-4-methoxyphenyl)-2-isopropyl-4,4-dimethyl-2,4-dihydro-3H-pyrazol-3-one (4), previously reported as a human PDE4 inhibitor, was identified as a hit. Upon optimization on three positions of the phenylpyrazolone scaffold, 2-isopropyl-5-(4-methoxy-3-(pyridin-3-yl)phenyl)-4,4-dimethyl-2,4-dihydro-3H-pyrazol-3-one (34) proved to be the most active compound against intracellular forms of T. cruzi (pIC50 = 6.4) with a 100-fold selectivity with respect to toxicity toward human MRC-5 cells. Evaluation on different life stages and clinically relevant T. cruzi strains revealed that the phenylpyrazolones are not active against the bloodstream form of the Y strain but show submicromolar activity against the intracellular form of the Y- and Tulahuen strains as well as against the nitro-drug-resistant Colombiana strain. In vitro screening of phenylpyrazolones against TcrPDEB1, TcrPDEC, and TcrCYP51 showed that there was a poor correlation between enzyme inhibition and the observed phenotypic effect. Among the most potent compounds, both TcrCYP51 and non-TcrCYP51 inhibitors are identified, which were both equally able to inhibit T. cruzi in vitro

Targeting a Subpocket in Trypanosoma brucei Phosphodiesterase B1 (TbrPDEB1) Enables the Structure-Based Discovery of Selective Inhibitors with Trypanocidal Activity

Several trypanosomatid cyclic nucleotide phosphodiesterases (PDEs) possess a unique, parasite-specific cavity near the ligand-binding region that is referred to as the P-pocket. One of these enzymes, Trypanosoma brucei PDE B1 (TbrPDEB1), is considered a drug target for the treatment of African sleeping sickness. Here, we elucidate the molecular determinants of inhibitor binding and reveal that the P-pocket is amenable to directed design. By iterative cycles of design, synthesis, and pharmacological evaluation and by elucidating the structures of inhibitor-bound TbrPDEB1, hPDE4B, and hPDE4D complexes, we have developed 4a,5,8,8a-tetrahydrophthalazinones as the first selective TbrPDEB1 inhibitor series. Two of these, 8 (NPD-008) and 9 (NPD-039), were potent (Ki = 100 nM) TbrPDEB1 inhibitors with antitrypanosomal effects (IC50 = 5.5 and 6.7 ?M, respectively). Treatment of parasites with 8 caused an increase in intracellular cyclic adenosine monophosphate (cAMP) levels and severe disruption of T. brucei cellular organization, chemically validating trypanosomal PDEs as therapeutic targets in trypanosomiasis

On the Versatility of Microwave-Assisted Chemistry : Exemplified by Applications in Medicinal Chemistry, Heterocyclic Chemistry and Biochemistry

Today, the demand for speed in drug discovery is constantly increasing, particularly in the iterative processes of hit validation and expansion and lead optimization. Irradiation with microwaves (MWs) has been applied in the area of organic synthesis to accelerate chemical reactions and to facilitate the generation of new chemical entities since 1986. In the work presented in this thesis, the use of MW-mediated heating has been expanded to address three fields of drug discovery, namely hit expansion, chemical library generation and genomics. In the first project, potential inhibitors of malaria aspartic proteases were designed and synthesized, partly by MW-assisted organic chemistry, and evaluated with regard to their inhibitory efficacy on five malaria aspartic proteases and their selectivity over two human aspartic proteases. The synthetic work included the development of fast and convenient methods of MW-assisted formation of thiazolidines and epoxy esters. Some of the resulting structures proved to be efficacious inhibitors of the aspartic protease that degrades haemoglobin in all four malaria parasites infecting man. No inhibitor affected the human aspartic proteases. Expedient, two-step, single-operation synthetic routes to heterocycles of medicinal interest were developed in the second and third projects. In the former, the use of a versatile synthon, Ph3PCCO, provided α,β-unsaturated lactones, lactams and amides within 5–10 minutes. In the latter project, saturated lactams were formed from amines and lactones in 35 minutes, in the absence of strong additives. These two MW-mediated protocols allowed the reduction of the reaction time from several hours or days to minutes. In the fourth project, a fully automated MW-assisted protocol for the important enzyme-catalysed polymerase chain reaction (PCR) was established. In addition, the PCR reaction could be performed in unusually large volumes, 2.5 mL and 15 mL, with yields corresponding to those from conventional PCR. Good amplification rates suggested that the thermophilic enzyme, Taq polymerase, was not affected by the MW radiation

Simple colorimetric trypanothione reductase-based assay for high-throughput screening of drugs against Leishmania intracellular amastigotes

Critical to the search for new anti-leishmanial drugs is the availability of high-throughput screening (HTS) methods to test chemical compounds against the relevant stage for pathogenesis, the intracellular amastigotes. Recent progress in automated microscopy and genetic recombination has produced powerful tools for drug discovery. Nevertheless, a simple and efficient test for measuring drug activity against Leishmania clinical isolates is lacking. Here we describe a quantitative colorimetric assay in which the activity of a Leishmania native enzyme is used to assess parasite viability. Enzymatic reduction of disulfide trypanothione, monitored by a microtiter plate reader, was used to quantify the growth of Leishmania parasites. An excellent correlation was found between the optical density at 412 nm and the number of parasites inoculated. Pharmacological validation of the assay was performed against the conventional alamarBlue method for promastigotes and standard microscopy for intracellular amastigotes. The activity of a selected-compound panel, including several anti-leishmanial reference drugs, demonstrated high consistency between the newly developed assay and the reference method and corroborated previously published data. Quality assessment with standard measures confirmed the robustness and reproducibility of the assay, which performed in compliance with HTS requirements. This simple and rapid assay provides a reliable, accurate method for screening anti-leishmanial agents, with high throughput. The basic equipment and manipulation required to perform the assay make it easy to implement, simplifying the method for scoring inhibitor assay

The development of an artificial intelligence-based digital pathology for neglected tropical diseases : a platform specific analysis of the World Health Organization diagnostic target product profile for soil-transmitted helminthiasis

The World Health Organization (WHO) recently published target product profiles (TPPs) for neglected tropical diseases (NTDs) to inform and accelerate the development of diagnostics tools necessary to achieve targets in the decade ahead. These TPPs describe the minimal and ideal requirements for various diagnostic needs related to NTD specific use-cases. An early step towards the manufacture and implementation of new diagnostics is to critically review the TPPs and translate these into an initial design and ultimately into user requirement specifications (URS). Artificial intelligence-based digital pathology (AI-DP) may overcome critical shortcomings of current standards for most NTDs reliant on microscopy, such as poor reproducibility and error-prone manual read-out. Furthermore, a digitalised workflow can create opportunities to reduce operational costs via increased throughput and automated data capture, analysis, and reporting. Despite these promising benefits, a critical review of the NTD TPPs with consideration to an AI-DP diagnostic solution is lacking. We present a systematic analysis of one of the WHO TPPs with the aim to inform the development of a URS for an AI-DP solution for NTDs. As a case study we focused on monitoring and evaluation (M&E) of programs designed to control soil-transmitted helminths (STHs). To this end, we start by outlining a brief overview of diagnostic needs for STHs, after which we systematically analyse the recently published WHO TPPs, highlighting the technical considerations for an AI-DP diagnostic solution to meet the minimal requirements for this TPP. Finally, we further reflect on the feasibility of an AI-DP informing STH programs towards the WHO 2030 targets in due time

Fragment-based screening in tandem with phenotypic screening provides novel antiparasitic hits

Methods to discover biologically active small molecules include target-based and phenotypic screening approaches. One of the main difficulties in drug discovery is elucidating and exploiting the relationship between drug activity at the protein target and disease modification, a phenotypic endpoint. Fragment-based drug discovery is a target-based approach that typically involves the screening of a relatively small number of fragment-like (molecular weight <300) molecules that efficiently cover chemical space. Here, we report a fragment screening on TbrPDEB1, an essential cyclic nucleotide phosphodiesterase (PDE) from Trypanosoma brucei, and human PDE4D, an off-target, in a workflow in which fragment hits and a series of close analogs are subsequently screened for antiparasitic activity in a phenotypic panel. The phenotypic panel contained T. brucei, Trypanosoma cruzi, Leishmania infantum, and Plasmodium falciparum, the causative agents of human African trypanosomiasis (sleeping sickness), Chagas disease, leishmaniasis, and malaria, respectively, as well as MRC-5 human lung cells. This hybrid screening workflow has resulted in the discovery of various benzhydryl ethers with antiprotozoal activity and low toxicity, representing interesting starting points for further antiparasitic optimization

Fragment-Based Screening in Tandem with Phenotypic Screening Provides Novel Antiparasitic Hits

Methods to discover biologically active small molecules include target-based and phenotypic screening approaches. One of the main difficulties in drug discovery is elucidating and exploiting the relationship between drug activity at the protein target and disease modification, a phenotypic endpoint. Fragment-based drug discovery is a target-based approach that typically involves the screening of a relatively small number of fragment-like (molecular weight <300) molecules that efficiently cover chemical space. Here, we report a fragment screening on TbrPDEB1, an essential cyclic nucleotide phosphodiesterase (PDE) from Trypanosoma brucei, and human PDE4D, an off-target, in a workflow in which fragment hits and a series of close analogs are subsequently screened for antiparasitic activity in a phenotypic panel. The phenotypic panel contained T. brucei, Trypanosoma cruzi, Leishmania infantum, and Plasmodium falciparum, the causative agents of human African trypanosomiasis (sleeping sickness), Chagas disease, leishmaniasis, and malaria, respectively, as well as MRC-5 human lung cells. This hybrid screening workflow has resulted in the discovery of various benzhydryl ethers with antiprotozoal activity and low toxicity, representing interesting starting points for further antiparasitic optimization

Discovery of Novel <i>Trypanosoma brucei</i> Phosphodiesterase B1 Inhibitors by Virtual Screening against the Unliganded TbrPDEB1 Crystal Structure

<i>Trypanosoma brucei</i> cyclic nucleotide phosphodiesterase B1 (TbrPDEB1) and TbrPDEB2 have recently been validated as new therapeutic targets for human African trypanosomiasis by both genetic and pharmacological means. In this study we report the crystal structure of the catalytic domain of the unliganded TbrPDEB1 and its use for the in silico screening for new TbrPDEB1 inhibitors with novel scaffolds. The TbrPDEB1 crystal structure shows the characteristic folds of human PDE enzymes but also contains the parasite-specific P-pocket found in the structures of <i>Leishmania major</i> PDEB1 and <i>Trypanosoma cruzi</i> PDEC. The unliganded TbrPDEB1 X-ray structure was subjected to a structure-based in silico screening approach that combines molecular docking simulations with a protein–ligand interaction fingerprint (IFP) scoring method. This approach identified six novel TbrPDEB1 inhibitors with IC₅₀ values of 10–80 μM, which may be further optimized as potential selective TbrPDEB inhibitors