A medium-firm drug-candidate library of cryptand-like structures on T7 phage: design and selection of a strong binder for Hsp90

2021 ◽  
Vol 19 (1) ◽  
pp. 146-150
Author(s):  
Kazuto Mochizuki ◽  
Lisa Matsukura ◽  
Yuji Ito ◽  
Naoyuki Miyashita ◽  
Masumi Taki
Keyword(s):  
Drug Candidate ◽  
Related Protein ◽  
T7 Phage ◽  
Selection Of

We designed and synthesized a library of cryptand-like structures on the T7 phage; a strong binder for a cancer-related protein was selected from the library.

scholarly journals Regioisomerization of Antimalarial Drug WR99210 Explains the Inactivity of a Commercial Stock

2020 ◽  
Vol 65 (1) ◽  
pp. e01385-20
Author(s):  
T. Parks Remcho ◽  
Sravanthi D. Guggilapu ◽  
Phillip Cruz ◽  
Glenn A. Nardone ◽  
Gavin Heffernan ◽  
...  
Keyword(s):  
Active Site ◽  
Antimalarial Drug ◽  
High Performance ◽  
Drug Candidate ◽  
Resonance Spectroscopy ◽  
Content Type ◽  
Computational Docking ◽  
Layer Chromatography ◽  
Commercial Stock ◽  
Selection Of

ABSTRACTWR99210, a former antimalarial drug candidate now widely used for the selection of Plasmodium transfectants, selectively targets the parasite’s dihydrofolate reductase thymidine synthase bifunctional enzyme (DHFR-TS) but not human DHFR, which is not fused with TS. Accordingly, WR99210 and plasmids expressing the human dhfr gene have become valued tools for the genetic modification of parasites in the laboratory. Concerns over the ineffectiveness of WR99210 from some sources encouraged us to investigate the biological and chemical differences of supplies from two different companies (compounds 1 and 2). Compound 1 proved effective at low nanomolar concentrations against Plasmodium falciparum parasites, whereas compound 2 was ineffective, even at micromolar concentrations. Intact and fragmented mass spectra indicated identical molecular formulae of the unprotonated (free base) structures of compounds 1 and 2; however, the compounds displayed differences by thin-layer chromatography, reverse-phase high-performance liquid chromatography, and UV-visible spectroscopy, indicating important isomeric differences. Structural evaluations by 1H, 13C, and 15N nuclear magnetic resonance spectroscopy confirmed compound 1 as WR99210 and compound 2 as a dihydrotriazine regioisomer. Induced fit computational docking models showed that compound 1 binds tightly and specifically in the P. falciparum DHFR active site, whereas compound 2 fits poorly to the active site in loose and varied orientations. Stocks and concentrates of WR99210 should be monitored for the presence of regioisomer 2, particularly when they are not supplied as the hydrochloride salt or are exposed to basic conditions that may promote rearrangement. Absorption spectroscopy can serve for assays of the unrearranged and rearranged triazines.

scholarly journals In vitro and in vivo efficacy, toxicity, bio-distribution and resistance selection of a novel antibacterial drug candidate

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Jlenia Brunetti ◽  
Chiara Falciani ◽  
Giulia Roscia ◽  
Simona Pollini ◽  
Stefano Bindi ◽  
...  
Keyword(s):  
Drug Candidate ◽  
Antibacterial Drug ◽  
In Vivo Efficacy ◽  
Resistance Selection ◽  
Selection Of

Efficient one-cycle affinity selection of binding proteins or peptides specific for a small-molecule using a T7 phage display pool

2008 ◽  
Vol 16 (22) ◽  
pp. 9837-9846 ◽  
Author(s):  
Yoichi Takakusagi ◽  
Kouji Kuramochi ◽  
Manami Takagi ◽  
Tomoe Kusayanagi ◽  
Daisuke Manita ◽  
...  
Keyword(s):  
Phage Display ◽  
Small Molecule ◽  
Binding Proteins ◽  
Affinity Selection ◽  
T7 Phage Display ◽  
T7 Phage ◽  
Selection Of

scholarly journals Isomerization of antimalarial drug WR99210 explains its inactivity in a commercial stock

2020 ◽  
Author(s):  
T. Parks Remcho ◽  
Sravanthi D. Guggilapu ◽  
Phillip Cruz ◽  
Glenn A. Nardone ◽  
Gavin Heffernan ◽  
...  
Keyword(s):  
Active Site ◽  
Antimalarial Drug ◽  
High Performance ◽  
Mass Spectra ◽  
Drug Candidate ◽  
Resonance Spectroscopy ◽  
Computational Docking ◽  
Layer Chromatography ◽  
Commercial Stock ◽  
Selection Of

ABSTRACTWR99210, a former antimalarial drug candidate now widely used for the selection of Plasmodium transfectants, selectively targets the parasite dihydrofolate reductase thymidine synthase bifunctional enzyme (DHFR-TS) but not human DHFR, which is not fused with TS. Accordingly, WR99210 and plasmids expressing human dhfr have become valued tools for the genetic modification of parasites in the laboratory. Concerns over the ineffectiveness of WR99210 from some sources encouraged us to investigate the biological and chemical differences of supplies from two different companies (compounds 1 and 2). Compound 1 proved effective at low nanomolar concentrations against Plasmodium falciparum parasites, whereas compound 2 was ineffective even at micromolar concentrations. Intact and fragmented mass spectra indicated identical molecular formulae of the unprotonated (free base) structures of 1 and 2; however, the compounds displayed differences by thin layer chromatography, reverse phase high performance liquid chromatography, and ultraviolet-visible spectroscopy, indicating important isomeric differences. Structural evaluations by 1H, 13C, and 15N nuclear magnetic resonance spectroscopy confirmed 1 as WR99210 and 2 as an isomeric dihydrotriazine. Induced fit, computational docking models showed that 1 binds tightly and specifically in the P. falciparum DHFR active site whereas 2 fits poorly to the active site in loose and varied orientations. Stocks and concentrates of WR99210 should be monitored for the presence of isomer 2, particularly when they are not supplied as the hydrochloride salt or are exposed to basic conditions that can promote isomerization. Absorption spectroscopy may serve for assays of the unrearranged and rearranged triazines.

Human Hepatic Cell Cultures: In Vitro and In Vivo Drug Metabolism

2003 ◽  
Vol 31 (3) ◽  
pp. 257-265 ◽  
Author(s):  
María José Gómez-Lechón ◽  
Teresa Donato ◽  
Xavier Ponsoda ◽  
José V. Castell
Keyword(s):  
Drug Metabolism ◽  
Metabolic Profile ◽  
Animal Species ◽  
In Vitro Models ◽  
Human Hepatocytes ◽  
Cellular Systems ◽  
Drug Candidate ◽  
Selection Of

Drug metabolism is the major determinant of drug clearance, and the factor most frequently responsible for inter-individual differences in drug pharmacokinetics. The expression of drug metabolising enzymes shows significant interspecies differences, and variability among human individuals (polymorphic or inducible enzymes) makes the accurate prediction of the metabolism of a new compound in humans difficult. Several key issues need to be addressed at the early stages of drug development to improve drug candidate selection: a) how fast the compound will be metabolised; b) what metabolites will be formed (metabolic profile); c) which enzymes are involved and to what extent; and d) whether drug metabolism will be affected directly (drug-drug interactions) or indirectly (enzyme induction) by the administered compound. Drug metabolism studies are routinely performed in laboratory animals, but they are not sufficiently accurate to predict the metabolic profiles of drugs in humans. Many of these issues can now be addressed by the use of relevant human in vitro models, which speed up the selection of new candidate drugs. Human hepatocytes are the closest in vitro model to the human liver, and they are the only model which can produce a metabolic profile of a drug which is very similar to that found in vivo. However, the use of human hepatocytes is restricted, because limited access to suitable tissue samples prevents their use in high throughput screening systems. The pharmaceutical industry has made great efforts to develop fast and reliable in vitro models to overcome these drawbacks. Comparative studies on liver microsomes and cells from animal species, including humans, are very useful for demonstrating species differences in the metabolic profile of given drug candidates, and are of great value in the judicious and justifiable selection of animal species for later pharmacokinetic and toxicological studies. Cytochrome P450 (CYP)-engineered cells (or microsomes from CYP-engineered cells, for example, Supersomes™) have made the identification of the CYPs involved in the metabolism of a drug candidate more straightforward and much easier. However, the screening of compounds acting as potential CYP inducers can only be conducted in cellular systems fully capable of transcribing and translating CYP genes.

scholarly journals First person – Raja Gopal Reddy Mooli

2020 ◽  
Vol 133 (18) ◽  
pp. jcs253963
Keyword(s):  
Liver Cancer ◽  
Early Career ◽  
First Person ◽  
Postdoctoral Fellow ◽  
Related Protein ◽  
University Of Pittsburgh ◽  
Early Career Researchers ◽  
Selection Of ◽  
Brown Adipogenesis

ABSTRACTFirst Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Raja Gopal Reddy Mooli is first author on ‘An indispensable role for dynamin-related protein 1 in beige and brown adipogenesis’, published in JCS. Raja Gopal Reddy is a postdoctoral fellow in the lab of Sadeesh Ramakrishnan at the Division of Endocrinology and Metabolism, University of Pittsburgh, PA, USA, investigating the role of hypoxic signaling in liver cancer and the crosstalk between liver and beige adipogenesis mechanisms.

scholarly journals Selection of efficient cleavage sites in target RNAs by using a ribozyme expression library.

1995 ◽  
Vol 15 (1) ◽  
pp. 540-551 ◽  
Author(s):  
A Lieber ◽  
M Strauss
Keyword(s):  
Growth Hormone ◽  
Human Growth ◽  
Cleavage Sites ◽  
Expression Library ◽  
Stem Loop ◽  
Random Sequences ◽  
Hormone Synthesis ◽  
Suitable Target ◽  
T7 Phage ◽  
Selection Of

Inactivation of gene expression by antisense mechanisms in general and by ribozymes in particular is a powerful technique for studying the function of a gene product. We have designed a strategy for expression of ribozymes, for selection of accessible cleavage sites in target RNAs, and for isolation of ribozymes from a library of random sequences flanking the unique sequence of a hammerhead. The expression cassette for ribozyme genes is based on adenovirus-associated RNA. Alternatively, we used polymerase III or the T7 phage transcription machinery. The ribozyme sequences are positioned in the center of a stable stem-loop structure, allowing for a correctly folded ribozyme region within the expressed RNA. A library of ribozyme genes with random sequences of 13 nucleotides on both sides of the hammerhead was generated. As an example, ribozymes which are specific for seven sites within the mRNA or nuclear RNA of human growth hormone were selected and identified. Sequencing of ribozyme genes reamplified from the library confirmed not only the predicted cleavage sites but also the presence of different ribozyme variants in the library. In a test of the ribozyme variants for repression of growth hormone synthesis in a cellular assay, the strongest effect (more than 99% inhibition) was found for the variant with the shortest stretch of complementarity (7 and 8 nucleotides on either side) to the target RNA. This basic strategy seems to be applicable to the selection of suitable target sites and to the isolation of corresponding ribozymes for any mRNA of interest.

scholarly journals Selection of a trioxaquine as an antimalarial drug candidate

2008 ◽  
Vol 105 (45) ◽  
pp. 17579-17584 ◽  
Author(s):  
F. Cosledan ◽  
L. Fraisse ◽  
A. Pellet ◽  
F. Guillou ◽  
B. Mordmuller ◽  
...  
Keyword(s):  
Antimalarial Drug ◽  
Drug Candidate ◽  
Selection Of

Cryo electron microscopy structures of Hsp100 proteins: crowbars in or out?This paper is one of a selection of papers published in this special issue entitled 8th International Conference on AAA Proteins and has undergone the Journal's usual peer review process.

2010 ◽  
Vol 88 (1) ◽  
pp. 89-96 ◽  
Author(s):  
Petra Wendler ◽  
Helen R Saibil
Keyword(s):  
Electron Microscopy ◽  
Peer Review ◽  
Atomic Structure ◽  
Review Process ◽  
Peer Review Process ◽  
Experimental Information ◽  
Related Protein ◽  
Special Issue ◽  
Cryo Electron Microscopy ◽  
Selection Of

Independent cryo electron microscopy (cryo-EM) studies of the closely related protein disaggregases ClpB and Hsp104 have resulted in two different models of subunit arrangement in the active hexamer. We compare the EM maps and resulting atomic structure fits, discuss their differences, and relate them to published experimental information in an attempt to discriminate between models. In addition, we present some general assessment criteria for low-resolution cryo-EM maps to offer non-structural biologists tools to evaluate these structures.

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