In silico & In vitro study to estimate Plasma Protein Binding of anti-parasitic compounds for Sleeping sickness (Human African trypanosomiasis)

Human African trypanosomiasis (HAT), also known as sleeping sickness, is a disease caused by a group of parasites called Trypanosoma brucei (Tb). The two main types causing HAT are T. brucei gambiense and T. brucei rhodesiense. T. brucei gambiense is the most common form of HAT, accounting for ninety seven percent of all reported cases of sleeping sickness. According to WHO, HAT is endemic in 36 sub-Saharan African countries. The disease can lead to death during the second stage if left untreated. Several drugs have been developed for the first stage such as pentamidine and suramin, and for the second stage such as melarsoprol, nifurtimox-eflornithine combination therapy (NECT). In 2019, fexinidazole was introduced as an oral treatment for the first stage and non-severe second stage of HAT. Several antiparasitic compounds prepared by our collaborator’s research group at the University of Graz, Austria showed varying levels of activity against Tb in vitro, whereas the compounds had only a moderate in vivo effect if at all. The suggested reason for the poor in vivo activities is that the compounds may bind tightly to plasma proteins, or they are metabolized before reaching the target sites for therapeutic effect. The prediction of plasma protein binding is of paramount importance in the pharmacokinetics characterization of drugs, as it causes significant changes in volume of distribution, clearance and drug half-life. Human serum albumin (HSA), an abundant plasma protein, can bind a remarkable variety of drugs impacting their delivery and efficacy and ultimately altering the drug’s pharmacokinetic and pharmacodynamic properties. In this current investigation, the overall aim was to investigate whether a strong HSA binding could be a probable reason for the poor in vivo activity of the provided antiparasitic compounds. The interaction of the antiparasitic compounds with HSA was studied computationally by docking them in the HSA drug binding site I and II. The compounds with the highest docking score were additionally studied using molecular dynamics simulations to evaluate the stability of the binding interactions. Moreover, the HSA binding affinity of the compounds was estimated by calculating the binding free energies using the MM-GBSA approach. In addition, experimental HSA binding studies using Microscale thermophoresis (MST) were conducted for some of the compounds. The results of the in silico studies suggest that majority of the investigated compounds may bind to HSA with varying affinity whereas a few of them did not show favorable binding interactions with HSA. Further, none of the compounds studied in vitro by MST showed HSA binding. In sum, plasma protein binding may be the reason for the in vivo inactivity for some of the investigated antiparasitic compounds

Computational Prediction and Experimental Validation of ADMET Properties for Potential Therapeutics

The drug development process in the United States is an expensive and lengthy process, usually taking a decade or more to gain approval for a drug candidate. The majority of proposed, early stage therapeutics fail, even though the typical process narrows from hundreds or thousands of small molecules down to one late stage candidate. One reason for failure is due to the drugs poor or unexpected absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties. Researchers attempt to predict ADMET properties as a way to help prioritize compounds for lead development to minimize expense and time. It was the overall goal of this project to further the prediction of two ADMET properties (absorption and distribution) through the development and application of quantitative structure-activity (QSAR) relationship computational models predicting human intestinal absorption (HIA), Caco-2 permeability (in vivo & in vitro measurements of absorption), and protein binding (measurement of distribution). These combined models would then be paired with additional experimental methods to help prioritize compounds for future ligand discovery efforts in our lab group and for our collaborators. Five computational QSAR models for each of these three properties were created using different molecular descriptor types and solvation models in an effort to examine which approach resulted in optimal performance. The model development process and validation stages of these QSAR models is outlined herein, along with analysis and discussion of commonly mispredicted compounds. Performance was similar across all models (independent of the molecular descriptor used and the solvation models applied. Future efforts at model development will depend on the size of the dataset to be analyzed. If the dataset is small, the i3D-Born solvation models will be used because these models better represent physiological conditions and performed slightly better than the other models. However, if the dataset is large, the 2D descriptor models will be used as these models do not require that a time and resource-intensive conformational search be performed and because it performed nearly as well as the i3D-Born solvation models. There were no common structural features consistently found associated with mispredicted structures. As such we are unable, at this time to pinpoint classes of compounds to avoid in future effortsThe experimental methods outlined in this work focused on developing methods to determine protein binding, specifically determining a fast, inexpensive workflow to classify the difference between high and low protein binding small molecules. Two techniques were used to determine protein binding of small molecules to bovine serum albumin (BSA): fluorescence polarization (FP) competition, and Nano Differential Scanning Fluorimetry (NanoDSF). FP assays quantifies the change in polarization of a target fluorophore between its protein bound and free states, an equilibrium that can be impacted by the presence of small molecule competitors. This method can be performed in a quantitative manner, but it also requires more time and more expensive and specialized instrumentation. In contrast, NanoDSF determines the melting temperature of BSA in the presence (higher) or in the absence (lower) small molecules by determining the intrinsic fluorescence of tryptophan and tyrosine residues while applying a temperature gradient. This method is qualitative, at least in our approach, but is very fast and requires much less expensive instrumentation. In our hands both techniques were successful in distinguishing differences between small molecules exhibiting low and high BSA binding. In summary, this project was successful in that we 1) developed computational tools capable of correctly predicting ADMET properties including HIA, Caco-2 permeability, and protein binding and 2) developed experimental workflows to quantitatively and qualitatively separate small molecules into low and high affinity BSA binders. With these in silico models and in vitro methods established, future research in our group and with our collaborators can make use of these tools to help prioritize compounds in ligand/ inhibitor discovery efforts

Steroid-sensitive nephrotic syndrome candidate gene CLVS1 regulates podocyte oxidative stress and endocytosis

We performed next-generation sequencing in patients with familial steroid-sensitive nephrotic syndrome (SSNS) and identified a homozygous segregating variant (p.H310Y) in the gene encoding clavesin-1 (CLVS1) in a consanguineous family with 3 affected individuals. Knockdown of the clavesin gene in zebrafish (clvs2) produced edema phenotypes due to disruption of podocyte structure and loss of glomerular filtration barrier integrity that could be rescued by WT CLVS1 but not the p.H310Y variant. Analysis of cultured human podocytes with CRISPR/Cas9-mediated CLVS1 knockout or homozygous H310Y knockin revealed deficits in clathrin-mediated endocytosis and increased susceptibility to apoptosis that could be rescued with corticosteroid treatment, mimicking the steroid responsiveness observed in patients with SSNS. The p.H310Y variant also disrupted binding of clavesin-1 to α-tocopherol transfer protein, resulting in increased reactive oxygen species (ROS) accumulation in CLVS1-deficient podocytes. Treatment of CLVS1-knockout or homozygous H310Y-knockin podocytes with pharmacological ROS inhibitors restored viability to control levels. Taken together, these data identify CLVS1 as a candidate gene for SSNS, provide insight into therapeutic effects of corticosteroids on podocyte cellular dynamics, and add to the growing evidence of the importance of endocytosis and oxidative stress regulation to podocyte function

Relativistic DFT calculation and their effect on the accuracy of results

This study explores the significance of density functional theory (DFT) calculations with relativistic effects for two ethylenediaminetetraacetate (edta) type complexes: trans(O5)-[M(eddadp)]- (M = Rh3+, Co3+). Relativistic effects affect the electronic structure of a molecule and, thus, its chemical and spectroscopic properties. With the use of scalar relativistic corrections (SR-ZORA), as implemented in the ADF package, with the B3LYP functional, the TZP basis set and the COSMO solvation model, structural analyses show improved predictions for the geometries of both complexes. In the case of the Rh3+ complex, the differences in metal-ligand bond lengths with and without the relativistic effects were small. In the case of the Co3+ complex, the changes in metal-ligand bond lengths due to the relativistic effects were slightly more pronounced. Compared to experimental values, excitation energies are better when including relativistic corrections, especially for the Rh3+ complex. These results indicate the importance of relativistic DFT calculations for heavy element compounds

Targeting tumors using peptides

To penetrate solid tumors, low molecular weight (Mw < 10 KDa) compounds have an edge over antibodies: their higher penetration because of their small size. Because of the dense stroma and high interstitial fluid pressure of solid tumors, the penetration of higher Mw compounds is unfavored and being small thus becomes an advantage. This review covers a wide range of peptidic ligands—linear, cyclic, macrocyclic and cyclotidic peptides—to target tumors: We describe the main tools to identify peptides experimentally, such as phage display, and the possible chemical modifications to enhance the properties of the identified peptides. We also review in silico identification of peptides and the most salient non-peptidic ligands in clinical stages. We later focus the attention on the current validated ligands available to target different tumor compartments: blood vessels, extracelullar matrix, and tumor associated macrophages. The clinical advances and failures of these ligands and their therapeutic conjugates will be discussed. We aim to present the reader with the state-of-the-art in targeting tumors, by using low Mw molecules, and the tools to identify new ligands.Fil: Scodeller, Pablo David. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. University of Tartu; EstoniaFil: Asciutto, Eliana Karina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de San Martín. Escuela de Ciencia y Tecnología; Argentin

Detailed investigations of proximal tubular function in Imerslund-Grasbeck syndrome

BACKGROUND: Imerslund-Gräsbeck Syndrome (IGS) is a rare genetic disorder characterised by juvenile megaloblastic anaemia. IGS is caused by mutations in either of the genes encoding the intestinal intrinsic factor-vitamin B(12) receptor complex, cubam. The cubam receptor proteins cubilin and amnionless are both expressed in the small intestine as well as the proximal tubules of the kidney and exhibit an interdependent relationship for post-translational processing and trafficking. In the proximal tubules cubilin is involved in the reabsorption of several filtered plasma proteins including vitamin carriers and lipoproteins. Consistent with this, low-molecular-weight proteinuria has been observed in most patients with IGS. The aim of this study was to characterise novel disease-causing mutations and correlate novel and previously reported mutations with the presence of low-molecular-weight proteinuria. METHODS: Genetic screening was performed by direct sequencing of the CUBN and AMN genes and novel identified mutations were characterised by in silico and/or in vitro investigations. Urinary protein excretion was analysed by immunoblotting and high-resolution gel electrophoresis of collected urines from patients and healthy controls to determine renal phenotype. RESULTS: Genetic characterisation of nine IGS patients identified two novel AMN frameshift mutations alongside a frequently reported AMN splice site mutation and two CUBN missense mutations; one novel and one previously reported in Finnish patients. The novel AMN mutations were predicted to result in functionally null AMN alleles with no cell-surface expression of cubilin. Also, the novel CUBN missense mutation was predicted to affect structural integrity of the IF-B(12) binding site of cubilin and hereby most likely cubilin cell-surface expression. Analysis of urinary protein excretion in the patients and 20 healthy controls revealed increased urinary excretion of cubilin ligands including apolipoprotein A-I, transferrin, vitamin D-binding protein, and albumin. This was, however, only observed in patients where plasma membrane expression of cubilin was predicted to be perturbed. CONCLUSIONS: In the present study, mutational characterisation of nine IGS patients coupled with analyses of urinary protein excretion provide additional evidence for a correlation between mutation type and presence of the characteristic low-molecular-weight proteinuria

Magnetic nanoparticles as a versatile solid-support for fusion protein purification and antimicrobial assays

Magnetic nano-and microparticles are unique platforms for the development of bioseparation and antimicrobial devices. This work explored the application of magnetic particles for the purification of fusion proteins through the use of magnetic adsorbents coupled to novel affinity ligands towards peptidic and proteic tags. Furthermore, and in view of the novelty of these ligands, molecular modeling and simulation techniques were employed to explain the key structuralfeatures involved inthe binding of two affinity pairs: GFP/LA-A4C7 and RK-GFP/LR-A7C1.The results showed that the interaction between GFP and LA-A4C7 is mainly hydrophobicwhile the interaction between RK-GFP and LR-A7C1 is mostly driven byhydrogen bonds. Moreover, the same modeling techniques have been used to idealize a theoretical second generation library with view of maximizing the estimated free energy of binding and the correspondent affinity constant. When immobilizing the biomimetic ligands LA-A4C7 and LR-A7C1 onto magnetic nanoparticles, it was possibleto bind the protein of interest and recover pure elution fractions. The best elution condition for GFP elution was 0.1mM glycine-NaOH pH9 50% (v/v) ethylene glycoland the best elution condition for RK-GFP elution was PBS pH 7.4, 500mM arginine, which are in accordance with the theoretical results described previously. Final binding constants for the studied systems (Ka=0.83×105M-1and Qmax=4mg/g for GFP/LA-A4C7, Ka=3.21×105M-1and Qmax=2mg/g for RK-GFP/LR-A7C1) show promising results for an affinity-based protein purification system.Magnetic particleswere also functionalized with (RW)3, an peptidewith antimicrobial properties, by different routes. We were able to develop a novel antimicrobial nanodevice based on the EDC-coupling of (RW)3that has shown antimicrobial activity against Escherichiacoliand Bacillussubtilis

Protein-Ligand Interactions: Target Identification and Drug Discovery

Bioactive compounds and drugs are designed and screened on the basis of specific molecular targets as well as via the identification of active ingredients from traditional medicine or by serendipitous discovery. The development of novel therapeutic strategies not only requires a deep knowledge of the molecular processes and the cellular pathways involved in each pathological condition and disease, but also the specific protein targets and the effects of drug binding on protein conformation and activity. Understanding of how drugs can modify and modulate specific cellular pathways and functions will be helpful during the process of drug development and clinical trials