Structure-based drug design against Trypanosoma brucei methionyl-tRNA synthetase

Infection by the protozoan parasite Trypanosoma brucei causes human African trypanosomiasis, commonly known as sleeping sickness. The disease is fatal without treatment; yet, current therapeutic options for the disease are inadequate due to toxicity, difficulty in administration and emerging resistance. Therefore, methionyl-tRNA synthetase of T. brucei (TbMetRS) is targeted for the development of new antitrypanosomal drugs. We have recently completed a high-throughput screening campaign against TbMetRS using a 364,131 compounds library in The Scripps Research Institute Molecular Screening Center. Here we outline our strategy to integrate the power of crystal structures with high-throughput screening in a drug discovery project. We applied the rapid crystal soaking procedure to obtain structures of TbMetRS in complex with inhibitors reported earlier[1] to approximately 70 high-throughput screening hits. This resulted in more than 20 crystal structures of TbMetRS·hit complexes. These hits cover a large diversity of chemical structures with IC50 values between 200 nM and 10 µM. Based on the solved structures and existing knowledge drawn from other in-house inhibitors, the IC50 value of the most promising hit has been improved. Further development of the compounds into potent TbMetRS inhibitors with desirable pharmacokinetic properties is on-going and will continue to benefit from information derived from crystal structures.

Download Full-text

Identification of Potent Inhibitors of the Trypanosoma brucei Methionyl-tRNA Synthetase via High-Throughput Orthogonal Screening

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057114548832 ◽

2014 ◽

Vol 20 (1) ◽

pp. 122-130 ◽

Cited By ~ 22

Author(s):

Laura Pedró-Rosa ◽

Frederick S. Buckner ◽

Ranae M. Ranade ◽

Christina Eberhart ◽

Franck Madoux ◽

...

Keyword(s):

Growth Inhibition ◽

Trypanosoma Brucei ◽

High Throughput ◽

High Throughput Screening ◽

Adenosine Monophosphate ◽

Trna Synthetase ◽

Cell Viability Assay ◽

Viability Assay ◽

Cytotoxic Compounds ◽

Methionyl Trna Synthetase

Improved therapies for the treatment of Trypanosoma brucei, the etiological agent of the neglected tropical disease human African trypanosomiasis, are urgently needed. We targeted T. brucei methionyl-tRNA synthetase (MetRS), an aminoacyl-tRNA synthase (aaRS), which is considered an important drug target due to its role in protein synthesis, cell survival, and its significant differences in structure from its mammalian ortholog. Previous work using RNA interference of MetRS demonstrated growth inhibition of T. brucei, further validating it as an attractive target. We report the development and implementation of two orthogonal high-throughput screening assays to identify inhibitors of T. brucei MetRS. First, a chemiluminescence assay was implemented in a 1536-well plate format and used to monitor adenosine triphosphate depletion during the aminoacylation reaction. Hit confirmation then used a counterscreen in which adenosine monophosphate production was assessed using fluorescence polarization technology. In addition, a miniaturized cell viability assay was used to triage cytotoxic compounds. Finally, lower throughput assays involving whole parasite growth inhibition of both human and parasite MetRS were used to analyze compound selectivity and efficacy. The outcome of this high-throughput screening campaign has led to the discovery of 19 potent and selective T. brucei MetRS inhibitors.

Download Full-text

Corrigendum

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057116651590 ◽

2016 ◽

Vol 21 (6) ◽

pp. 653-653

Keyword(s):

Trypanosoma Brucei ◽

High Throughput ◽

Trna Synthetase ◽

Methionyl Trna Synthetase

Pedró-Rosa, L.; Buckner, F.; Ranade, R. M.; et al. Identification of Potent Inhibitors of the Trypanosoma brucei Methionyl-tRNA Synthetase via High-Throughput Orthogonal Screening. J Biomol Screen. 2015, 20, 122–130. (Original DOI: 10.1177/1087057114548832 )

Download Full-text

Inhibition Profiling of Urease and Carbonic Anhydrase II by High- Throughput Screening and Molecular Docking Studies of Structurally Diverse Organic Compounds

Letters in Drug Design & Discovery ◽

10.2174/1570180817999201005200505 ◽

2020 ◽

Vol 17 ◽

Author(s):

Majid Ali ◽

Syed Majid Bukhari ◽

Asma Zaidi ◽

Farhan A. Khan ◽

Umer Rashid ◽

...

Keyword(s):

Molecular Docking ◽

Carbonic Anhydrase ◽

Metal Complexes ◽

Organic Compounds ◽

High Throughput ◽

High Throughput Screening ◽

Docking Studies ◽

Carbonic Anhydrase Ii ◽

Molecular Docking Studies ◽

Ic50 Values

Background:: Structurally diverse organic compounds and available drugs were screened against urease and carbonic anhydrase II in a formulation acceptable for high-throughput screening. Objective: The study was conducted to find out potential inhibitors of urease and carbonic anhydrase II. Methods:: Quantification of the possible HITs was carried out by determining their IC50 values. Results and Discussion:: of several screened compounds including derivatives of oxadiazole, coumarins, chromane-2, 4- diones and metal complexes of cysteine-omeprazole showed promising inhibitory activities with IC50 ranging from 47 μM to 412 μM against the urease. The interactions of active compounds with active sites of enzymes were investigated through molecular docking studies which revealed that (R)-1-(4-amino-4-(5-(thiophen-2-yl)-1,3,4-oxadiazol-2-yl) butyl) guanidine possessing IC50 of 47 μM, interacts with one of the nickel metal atom of urease besides further interactions as predictable hydrogen bonds with KCX490, Asp633, His492, His407 and His409 along with Ala440 and 636. Bi-ligand metal complexes of 4-aminoantipyrine based Schiff bases showed activation of urease with AC50 ranging from 68 μM to 112 μM. Almost 21 compounds with varying functional groups including pyrimidines, oxadiazoles, imidazoles, hydrazides and tin based compounds were active carbonic anhydrase II inhibitors presenting 98 μM to 390 μM IC50 values. Several N-substituted sulfonamide derivatives were inactive against carbonic anhydrase II. Conclusion:: Among all the screened compounds, highly active inhibitor of carbonic anhydrase II was (4-(3- hydroxyphenyl)-6-phenyl-2-thioxo-1,2,3,4-tetrahydropyrimidin-5-yl)phenyl) methanone with IC50 of 98.0 μM. This particular compound showed metallic interaction with Zn ion of carbonic anhydrase II through hydroxyl group of phenyl ring.

Download Full-text

An HTRF based high-throughput screening for discovering chemical compounds that inhibit the interaction between Trypanosoma brucei Pex5p and Pex14p

Biochemistry and Biophysics Reports ◽

10.1016/j.bbrep.2016.05.004 ◽

2016 ◽

Vol 6 ◽

pp. 260-265 ◽

Cited By ~ 1

Author(s):

Yuichi Watanabe ◽

Kosuke Kawaguchi ◽

Syuken Saito ◽

Takayoshi Okabe ◽

Kiyoaki Yonesu ◽

...

Keyword(s):

Trypanosoma Brucei ◽

High Throughput ◽

High Throughput Screening ◽

Chemical Compounds

Download Full-text

Integration of an In Situ MALDI-Based High-Throughput Screening Process: A Case Study with Receptor Tyrosine Kinase c-MET

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/2472555217727701 ◽

2017 ◽

Vol 22 (10) ◽

pp. 1203-1210 ◽

Cited By ~ 5

Author(s):

Katrin Beeman ◽

Jens Baumgärtner ◽

Manuel Laubenheimer ◽

Karlheinz Hergesell ◽

Martin Hoffmann ◽

...

Keyword(s):

Drug Discovery ◽

High Throughput ◽

High Throughput Screening ◽

Maldi Ms ◽

Kinase Assay ◽

Label Free ◽

Screening Process ◽

Label Free Detection ◽

Ic50 Values

Mass spectrometry (MS) is known for its label-free detection of substrates and products from a variety of enzyme reactions. Recent hardware improvements have increased interest in the use of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS for high-throughput drug discovery. Despite interest in this technology, several challenges remain and must be overcome before MALDI-MS can be integrated as an automated “in-line reader” for high-throughput drug discovery. Two such hurdles include in situ sample processing and deposition, as well as integration of MALDI-MS for enzymatic screening assays that usually contain high levels of MS-incompatible components. Here we adapt our c-MET kinase assay to optimize for MALDI-MS compatibility and test its feasibility for compound screening. The pros and cons of the Echo (Labcyte) as a transfer system for in situ MALDI-MS sample preparation are discussed. We demonstrate that this method generates robust data in a 1536-grid format. We use the MALDI-MS to directly measure the ratio of c-MET substrate and phosphorylated product to acquire IC50 curves and demonstrate that the pharmacology is unaffected. The resulting IC50 values correlate well between the common label-based capillary electrophoresis and the label-free MALDI-MS detection method. We predict that label-free MALDI-MS-based high-throughput screening will become increasingly important and more widely used for drug discovery.

Download Full-text

Uncertainty-Informed Deep Transfer Learning of PFAS Toxicity

10.26434/chemrxiv.14397140 ◽

2021 ◽

Author(s):

Jeremy Feinstein ◽

ganesh sivaraman ◽

Kurt Picel ◽

Brian Peters ◽

Alvaro Vazquez-Mayagoitia ◽

...

Keyword(s):

Machine Learning ◽

Transfer Learning ◽

High Throughput ◽

High Throughput Screening ◽

Organic Chemical ◽

Learning Method ◽

Toxicity Data ◽

Chemical Structures ◽

Machine Learning Methods ◽

Computational Methodology

In this article, we present our recent study on computational methodology for predicting the toxicity of PFAS known as “forever chemicals” based on chemical structures through evaluation of multiple machine learning methods. To address the scarcity of PFAS toxicity data, a deep “transfer learning” method has been investigated by leveraging toxicity information over the entire organic chemical domain and an uncertainty-informed workflow by incorporating SelectiveNet architecture, which can support future guidance of high throughput screening with knowledge of chemical structures, has been developed.

Download Full-text

Adapting Structure-Based Drug Design in the Paradigm of Combinatorial Chemistry and High-Throughput Screening

ACS Symposium Series - Rational Drug Design ◽

10.1021/bk-1999-0719.ch015 ◽

1999 ◽

pp. 226-238 ◽

Cited By ~ 4

Author(s):

Arup K. Ghose ◽

Veharkad N. Viswanadhan ◽

John J. Wendoloski

Keyword(s):

Drug Design ◽

High Throughput ◽

Combinatorial Chemistry ◽

High Throughput Screening ◽

Structure Based Drug Design

Download Full-text

Crystal and molecular structures of boldenone and four boldenone steroid esters

Zeitschrift für Kristallographie - Crystalline Materials ◽

10.1515/zkri-2019-0030 ◽

2019 ◽

Vol 234 (10) ◽

pp. 671-683 ◽

Cited By ~ 5

Author(s):

Alexandru Turza ◽

Maria O. Miclăuș ◽

Aurel Pop ◽

Gheorghe Borodi

Keyword(s):

Crystal Structures ◽

High Throughput ◽

High Throughput Screening ◽

Molecular Structures ◽

Anabolic Androgenic Steroid ◽

Crystal And Molecular Structures ◽

Hirshfeld Surfaces ◽

Intermolecular Contacts ◽

Lattice Energies ◽

Steroid Skeleton

Abstract Androsta-1,4-dien-17β-ol-3-one, also known as boldenone, is an anabolic-androgenic steroid derived from testosterone. The crystal structures of boldenone base, boldenone acetate, boldenone propionate, boldenone cypionate and a boldenone acetate polymorph obtained by high throughput screening were investigated. Hirshfeld surfaces and fingerprint plots breakdown revealed that the molecular packing in the crystals are driven by dominant H⋯H intermolecular contacts, followed by O⋯H/H⋯O contacts and to a lesser degree C⋯H/H⋯C contacts. The steroid skeleton rings, for all the reported compounds, adopt the following conformation: planar in A, chair in B and C, whereas C(13) envelope conformations are found for the five-membered D rings. The total lattice energies were calculated as a sum of four terms (Coulombic, polarization, dispersion, repulsion).

Download Full-text

Staphylococcus aureus DNA ligase: characterization of its kinetics of catalysis and development of a high-throughput screening compatible chemiluminescent hybridization protection assay

Biochemical Journal ◽

10.1042/bj20040054 ◽

2004 ◽

Vol 383 (3) ◽

pp. 551-559 ◽

Cited By ~ 7

Author(s):

Sheraz GUL ◽

Richard BROWN ◽

Earl MAY ◽

Marie MAZZULLA ◽

Martin G. SMYTH ◽

...

Keyword(s):

Staphylococcus Aureus ◽

High Throughput ◽

High Throughput Screening ◽

Dna Ligase ◽

Protection Assay ◽

Dna Ligases ◽

Enzyme Substrate ◽

Ic50 Values ◽

Acridinium Ester

DNA ligases are key enzymes involved in the repair and replication of DNA. Prokaryotic DNA ligases uniquely use NAD+ as the adenylate donor during catalysis, whereas eukaryotic enzymes use ATP. This difference in substrate specificity makes the bacterial enzymes potential targets for therapeutic intervention. We have developed a homogeneous chemiluminescence-based hybridization protection assay for Staphylococcus aureus DNA ligase that uses novel acridinium ester technology and demonstrate that it is an alternative to the commonly used radiometric assays for ligases. The assay has been used to determine a number of kinetic constants for S. aureus DNA ligase catalysis. These included the Km values for NAD+ (2.75±0.1 μM) and the acridinium-ester-labelled DNA substrate (2.5±0.2 nM). A study of the pH-dependencies of kcat, Km and kcat/Km has revealed values of kinetically influential ionizations within the enzyme–substrate complexes (kcat) and free enzyme (kcat/Km). In each case, the curves were shown to be composed of one kinetically influential ionization, for kcat, pKa=6.6±0.1 and kcat/Km, pKa=7.1±0.1. Inhibition characteristics of the enzyme against two Escherichia coli DNA ligase inhibitors have also been determined with IC50 values for these being 3.30±0.86 μM for doxorubicin and 1.40±0.07 μM for chloroquine diphosphate. The assay has also been successfully miniaturized to a sufficiently low volume to allow it to be utilized in a high-throughput screen (384-well format; 20 μl reaction volume), enabling the assay to be used in screening campaigns against libraries of compounds to discover leads for further drug development.

Download Full-text