Scaffold-Hopping Strategy on a Series of Proteasome Inhibitors Led to a Preclinical Candidate for the Treatment of Visceral Leishmaniasis.

There is an urgent need for new treatments for visceral leishmaniasis (VL), a parasitic infection which impacts heavily large areas of East Africa, Asia, and South America. We previously reported on the discovery of GSK3494245/DDD01305143 (1) as a preclinical candidate for VL and, herein, we report on the medicinal chemistry program that led to its identification. A hit from a phenotypic screen was optimized to give a compound with in vivo efficacy, which was hampered by poor solubility and genotoxicity. The work on the original scaffold failed to lead to developable compounds, so an extensive scaffold-hopping exercise involving medicinal chemistry design, in silico profiling, and subsequent synthesis was utilized, leading to the preclinical candidate. The compound was shown to act via proteasome inhibition, and we report on the modeling of different scaffolds into a cryo-EM structure and the impact this has on our understanding of the series' structure-activity relationships.

Role of Oral Antioxidant Supplementation in the Current Management of Diabetic Retinopathy.

Oxidative stress has been postulated as an underlying pathophysiologic mechanism of diabetic retinopathy (DR), the main cause of avoidable blindness in working-aged people. This review addressed the current daily clinical practice of DR and the role of antioxidants in this practice. A systematic review of the studies on antioxidant supplementation in DR patients was presented. Fifteen studies accomplished the inclusion criteria. The analysis of these studies concluded that antioxidant supplementation has a IIB level of recommendation in adult Type 1 and Type 2 diabetes mellitus subjects without retinopathy or mild-to-moderate nonproliferative DR without diabetic macular oedema as a complementary therapy together with standard medical care.

Preclinical candidate for the treatment of visceral leishmaniasis that acts through proteasome inhibition.

Visceral leishmaniasis (VL), caused by the protozoan parasites Leishmania donovani and Leishmania infantum, is one of the major parasitic diseases worldwide. There is an urgent need for new drugs to treat VL, because current therapies are unfit for purpose in a resource-poor setting. Here, we describe the development of a preclinical drug candidate, GSK3494245/DDD01305143/compound 8, with potential to treat this neglected tropical disease. The compound series was discovered by repurposing hits from a screen against the related parasite Trypanosoma cruzi Subsequent optimization of the chemical series resulted in the development of a potent cidal compound with activity against a range of clinically relevant L. donovani and L. infantum isolates. Compound 8 demonstrates promising pharmacokinetic properties and impressive in vivo efficacy in our mouse model of infection comparable with those of the current oral antileishmanial miltefosine. Detailed mode of action studies confirm that this compound acts principally by inhibition of the chymotrypsin-like activity catalyzed by the ß5 subunit of the L. donovani proteasome. High-resolution cryo-EM structures of apo and compound 8-bound Leishmania tarentolae 20S proteasome reveal a previously undiscovered inhibitor site that lies between the ß4 and ß5 proteasome subunits. This induced pocket exploits ß4 residues that are divergent between humans and kinetoplastid parasites and is consistent with all of our experimental and mutagenesis data. As a result of these comprehensive studies and due to a favorable developability and safety profile, compound 8 is being advanced toward human clinical trials.

Metabolic Clustering Analysis as a Strategy for Compound Selection in the Drug Discovery Pipeline for Leishmaniasis.

A lack of viable hits, increasing resistance, and limited knowledge on mode of action is hindering drug discovery for many diseases. To optimize prioritization and accelerate the discovery process, a strategy to cluster compounds based on more than chemical structure is required. We show the power of metabolomics in comparing effects on metabolism of 28 different candidate treatments for Leishmaniasis (25 from the GSK Leishmania box, two analogues of Leishmania box series, and amphotericin B as a gold standard treatment), tested in the axenic amastigote form of Leishmania donovani. Capillary electrophoresis-mass spectrometry was applied to identify the metabolic profile of Leishmania donovani, and principal components analysis was used to cluster compounds on potential mode of action, offering a medium throughput screening approach in drug selection/prioritization. The comprehensive and sensitive nature of the data has also made detailed effects of each compound obtainable, providing a resource to assist in further mechanistic studies and prioritization of these compounds for the development of new antileishmanial drugs.

Unravelling the rate of action of hits in the Leishmania donovani box using standard drugs amphotericin B and miltefosine.

In recent years, the neglected diseases drug discovery community has elected phenotypic screening as the key approach for the identification of novel hit compounds. However, when this approach is applied, important questions related to the mode of action for these compounds remain unanswered. One of such questions is related to the rate of action, a useful piece of information when facing the challenge of prioritising the most promising hit compounds. In the present work, compounds of the "Leishmania donovani box" were evaluated using a rate of action assay adapted from a replicative intracellular high content assay recently developed. The potency of each compound was determined every 24 hours up to 96 hours, and standard drugs amphotericin B and miltefosine were used as references to group these compounds according to their rate of action. Independently of this biological assessment, compounds were also clustered according to their minimal chemical scaffold. Comparison of the results showed a complete correlation between the chemical scaffold and the biological group for the vast majority of compounds, demonstrating how the assay was able to bring information on the rate of action for each chemical series, a property directly linked to the mode of action. Overall, the assay here described permitted us to evaluate the rate of action of the "Leishmania donovani box" using two of the currently available drugs as references and, also, to propose a number of fast-acting chemical scaffolds present in the box as starting points for future drug discovery projects to the wider scientific community. The results here presented validate the use of this assay for the determination of the rate of action early in the discovery process, to assist in the prioritisation of hit compounds.

A Replicative In Vitro Assay for Drug Discovery against Leishmania donovani.

The protozoan parasite Leishmania donovani is the causative agent of visceral leishmaniasis, a disease potentially fatal if not treated. Current available treatments have major limitations, and new and safer drugs are urgently needed. In recent years, advances in high-throughput screening technologies have enabled the screening of millions of compounds to identify new antileishmanial agents. However, most of the compounds identified in vitro did not translate their activities when tested in in vivo models, highlighting the need to develop more predictive in vitro assays. In the present work, we describe the development of a robust replicative, high-content, in vitro intracellular L. donovani assay. Horse serum was included in the assay media to replace standard fetal bovine serum, to completely eliminate the extracellular parasites derived from the infection process. A novel phenotypic in vitro infection model has been developed, complemented with the identification of the proliferation of intracellular amastigotes measured by EdU incorporation. In vitro and in vivo results for miltefosine, amphotericin B, and the selected compound 1 have been included to validate the assay.

Mining the potential of label-free biosensors for seven-transmembrane receptor drug discovery.

Label-free is a broad term used to describe a number of cutting-edge biosensor technologies that have attracted considerable attention in the area of drug discovery for seven-transmembrane G protein-coupled receptors (GPCRs). Label-free biosensors resolve receptor-mediated responses noninvasively in real time and living cells and do so with high textural information and broad signaling-pathway coverage. They should facilitate studies of the receptor's integrated signal transduction biology intractable to classical assays with single pathway focus. Label-free occupies a privilege niche with respect to mechanistic studies in human native cells-healthy or disease-relevant-and the probing of context-dependent pharmacology in relation to whole biological system efficacy. It is expected that implementation of label-free approaches into the drug discovery process will improve clinical predictability of drug candidates at early stages of discovery research by their exquisite capability to sense whole cellular responses akin to tissue bioassays. Here, we present an overview of promises and challenges this rapidly evolving technology offers to drug screening and we also discuss the prospect of advancing drug discovery.

Actualización en el tratamiento de la neuropatía óptica inflamatoria desmielinizante / Updating on the treatment of the demyelinating inflammatory optical neuropathy

Se realizó una revisión bibliográfica con el objetivo de proporcionar una actualización de las drogas que se emplean para retrasar la aparición de esclerosis múltiple en el manejo de la neuropatía óptica inflamatoria desmielinizante. El artículo presenta el origen y la justificación de la terapia esteroidea en este grupo de enfermedad, así como los mecanismos de acción y beneficios de tratamientos más modernos como los inmunomoduladores e inmunosupresores. El trabajo también introduce muchas de las drogas con efectos neuroprotectores que se encuentran en fases experimentales, cuyo uso prevendría la neurodegeneración que se produce a nivel de las células ganglionares retinianas en esta enfermedad neurológica. Las opciones terapéuticas actuales ofrecen variantes de tratamiento adicionales a pacientes con mayores probabilidades de desarrollo de esclerosis múltiple y retrasan la aparición de un segundo brote, así como las secuelas invalidantes que esta suele originar(AU)

A bibliographic review was conducted to provide an updating of drugs used to retard the appearance of multiple sclerosis in the management of the demyelinating inflammatory optical neuropathy. Present paper shows the origin and the justification of the steroid therapy in this disease, as well as the mechanisms of action and benefits of more recent treatments, e.g. the ongoing immunomodulations and immunosuppressive ones and also to introduce many drugs in experimental phase having neuroprotection effects whose use will prevent the neurodegenerative effect produced at level of the retinal ganglion cells in this neurologic disease. The current therapeutical options offer variants of additional treatment to those patients with greater possibilities to development multiple sclerosis and retarding the appearance of a second outbreak, as well as its disabling sequelae(AU)

Actualización en el tratamiento de la neuropatía óptica inflamatoria desmielinizante / Updating on the treatment of the demyelinating inflammatory optical neuropathy

Se realizó una revisión bibliográfica con el objetivo de proporcionar una actualización de las drogas que se emplean para retrasar la aparición de esclerosis múltiple en el manejo de la neuropatía óptica inflamatoria desmielinizante. El artículo presenta el origen y la justificación de la terapia esteroidea en este grupo de enfermedad, así como los mecanismos de acción y beneficios de tratamientos más modernos como los inmunomoduladores e inmunosupresores. El trabajo también introduce muchas de las drogas con efectos neuroprotectores que se encuentran en fases experimentales, cuyo uso prevendría la neurodegeneración que se produce a nivel de las células ganglionares retinianas en esta enfermedad neurológica. Las opciones terapéuticas actuales ofrecen variantes de tratamiento adicionales a pacientes con mayores probabilidades de desarrollo de esclerosis múltiple y retrasan la aparición de un segundo brote, así como las secuelas invalidantes que esta suele originar

A bibliographic review was conducted to provide an updating of drugs used to retard the appearance of multiple sclerosis in the management of the demyelinating inflammatory optical neuropathy. Present paper shows the origin and the justification of the steroid therapy in this disease, as well as the mechanisms of action and benefits of more recent treatments, e.g. the ongoing immunomodulations and immunosuppressive ones and also to introduce many drugs in experimental phase having neuroprotection effects whose use will prevent the neurodegenerative effect produced at level of the retinal ganglion cells in this neurologic disease. The current therapeutical options offer variants of additional treatment to those patients with greater possibilities to development multiple sclerosis and retarding the appearance of a second outbreak, as well as its disabling sequelae

Validation of an optical microplate label-free platform in the screening of chemical libraries for direct binding to a nuclear receptor.

Optical microplate-based biosensors combine the advantages of label-free detection with industry-standard assay laboratory infrastructure and scalability. A plate-based label-free platform allows the same basic platform to be used to quantify molecular interactions of macromolecules and to screen and characterize drug-like small-molecule interactions. The ligand-binding domain of orphan estrogen-related nuclear receptor-γ (ERRγ) is utilized, as a model system of a challenging type of target, to illustrate the rapid development and utility of a range of biochemical assay formats on these biosensors. Formats in which either the domain, or a peptide derived from its cognate corepressor, RIP140, were immobilized were utilized. The direct binding of small drug molecules to the domain was characterized using immobilized domain. Subsequent addition of peptide distinguished whether compounds acted as either antagonists of peptide binding, or as agonists promoting a ternary complex. The format with peptide immobilized gave a more sensitive procedure for establishing the effect of compounds on the domain-peptide interaction. Using a direct-binding format, a diverse chemical library of 1,408 compounds in DMSO was screened for ability to bind to biosensors coated with ERRγ ligand-binding domain. Hits were then characterized using the other biosensor assay formats. The standard requirements for a full primary screening campaign were fulfilled by the acceptable hit-rate, quality-performance parameters, and throughput of the direct-binding assay format. Such a format allows direct screening of targets, such as orphan receptors, without the requirement for prior knowledge of a validated ligand.

Label-free imaging and temporal signature in phenotypic cellular assays: a new approach to high-content screening.

Some drug targets are not amenable to screening because of the lack of a practical or validated biological assay. Likewise, some screening assays may not be predictive of compound activity in a more disease-relevant scenario, or assay development may demand excessive allocation of resources (i.e., time, money or personnel) with limited knowledge of the actual tractability of the target. Label-free methodologies, implemented in microtiter plate format, may help address these issues and complement, simplify, or facilitate assays. Label-free biosensors, based on grating resonance or electrical impedance, are versatile platforms for detecting phenotypic changes in both engineered and native cells. Their non-invasive nature allows for the kinetic monitoring of multiple real-time cellular responses to external stimuli, as well as for the use of successive pharmacological challenges. The temporal signature recorded for a particular stimulus is characteristic of the cell type and the signaling pathway activated upon binding of a ligand to its receptor. Cellular label-free technology is an important technical advance in the study of functional pharmacological selectivity. Described in this overview are some of the hurdles encountered in modern drug discovery and the ways in which label-free technologies can be used to overcome these obstacles.

Statistics and decision making in high-throughput screening.

Screening is about making decisions on the modulating activity of one particular compound on a biological system. When a compound testing experiment is repeated under the same conditions or as close to the same conditions as possible, the observed results are never exactly the same, and there is an apparent random and uncontrolled source of variability in the system under study. Nevertheless, randomness is not haphazard. In this context, we can see statistics as the science of decision making under uncertainty. Thus, the usage of statistical tools in the analysis of screening experiments is the right approach to the interpretation of screening data, with the aim of making them meaningful and converting them into valuable information that supports sound decision making.In the HTS workflow, there are at least three key stages where key decisions have to be made based on experimental data: (1) assay development (i.e. how to assess whether our assay is good enough to be put into screening production for the identification of modulators of the target of interest), (2) HTS campaign process (i.e. monitoring that screening process is performing at the expected quality and assessing possible patterned signs of experimental response that may adversely bias and mislead hit identification) and (3) data analysis of primary HTS data (i.e. flagging which compounds are giving a positive response in the assay, namely hit identification).In this chapter we will focus on how some statistical tools can help to cope with these three aspects. Assessment of assay quality is reviewed in other chapters, so in Section 1 we will briefly make some further considerations. Section 2 will review statistical process control, Section 3 will cover methodologies for detecting and dealing with HTS patterns and Section 4 will describe approaches for statistically guided selection of hits in HTS.

A modular, fully integrated ultra-high-throughput screening system based on confocal fluorescence analysis techniques.

The rapid increase in size of compound libraries, as well as new targets emerging from the Human Genome Project, require progress in ultra-high-throughput screening (uHTS) systems. In a joint effort with scientists and engineers from the biotech and the pharmaceutical industry, a modular, fully integrated system for miniaturized uHTS was developed. The goal was to achieve high data quality in small assay volumes (1-4 microL) combined with reliable and unattended operation. Two new confocal fluorescence readers have been designed. One of the instruments is a 4-channel confocal fluorescence reader, measuring with 4 objectives in parallel. The fluorescence readout is based on single-molecule detection methods, allowing high sensitivity at low tracer concentrations and delivering an information-rich output. The other instrument is a confocal fluorescence imaging reader, where the images are analyzed in terms of generic patterns and quantified in units of intensity per pixel. Both readers are spanning the application range from assays with isolated targets in homogenous solution or membrane vesicle-based assays (4-channel reader) to cell-based assays (imaging reader). Results from a comprehensive test on these assay types demonstrate the high quality and robustness of this screening system.