Ligand binding studies, preliminary structure–activity relationship and detailed mechanistic characterization of 1-phenyl-6,6-dimethyl-1,3,5-triazine-2,4-diamine derivatives as inhibitors of Escherichia coli dihydrofolate reductase

Srinivasan, Bharath; Tonddast-Navaei, Sam; Skolnick, Jeffrey

doi:10.1016/j.ejmech.2015.08.021

Cited by 23 publications

(37 citation statements)

References 59 publications

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“…As above, the LB plots showed parallel lines further indicative of uncompetitive inhibition (Fig 3 D) and an ordered binding event whereby NADPH binding facilitates inhibitor binding. Further, this data is in agreement with reported mechanisms of DHFR inhibitors by folate-analogs 16, 23 . It has been demonstrated that the binding of and inhibition by folate-analogs is conditional to NADPH-binding 16, 23 .…”

Section: Resultssupporting

confidence: 92%

“…However, as has been demonstrated in previous studies 16, 23 and inspection of curves at low inhibitor concentrations in Fig 3 A and Fig 3 C shows, no substrate-inhibition is seen in the absence of the inhibitor. This led us to hypothesize that the observed behavior might be indicative of a unique two-site binding inhibition mechanism displayed by ononetin.…”

Section: Resultssupporting

confidence: 87%

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Rational Design of Novel Allosteric Dihydrofolate Reductase Inhibitors Showing Antibacterial Effects on Drug-Resistant Escherichia coli Escape Variants

et al. 2017

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In drug-discovery, systematic variations of substituents on a common scaffold and bioisosteric replacements are often used to generate diversity and obtain molecules with better biological effects. However, this could saturate the small-molecule diversity pool resulting in drug-resistance. On the other hand, conventional drug-discovery relies on targeting known pockets on protein surfaces leading to drug resistance by mutations of critical pocket residues. Here, we present a two-pronged strategy of designing novel drugs that target unique pockets on a protein’s surface to overcome the above problems. Dihydrofolate reductase, DHFR, is a critical enzyme involved in thymidine and purine nucleotide biosynthesis. Several classes of compounds that are structural-analogues of the substrate dihydrofolate have been explored for their anti-folate activity. Here, we describe 10 novel small-molecule inhibitors of Escherichia coli DHFR, EcDHFR, belonging to the stilbenoid, deoxybenzoin and chalcone family of compounds discovered by a combination of pocket-based virtual ligand screening and systematic scaffold-hopping. These inhibitors show a unique uncompetitive or noncompetitive inhibition mechanism, distinct from those reported for all known inhibitors of DHFR, indicative of binding to a unique pocket distinct from either substrate or cofactor-binding pockets. Furthermore, we demonstrate that rescue mutants of EcDHFR, with reduced affinity to all known classes of DHFR inhibitors, are inhibited at the same concentration as the wild-type. These compounds also exhibit antibacterial activity against E. coli harboring the drug-resistant variant of DHFR. This discovery is the first report on a novel class of inhibitors targeting a unique pocket on EcDHFR.

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Section: Resultssupporting

confidence: 92%

Section: Resultssupporting

confidence: 87%

Rational Design of Novel Allosteric Dihydrofolate Reductase Inhibitors Showing Antibacterial Effects on Drug-Resistant Escherichia coli Escape Variants

et al. 2017

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“…This is to ensure their utility for high-throughput experimental differential scanning fluorimetry [17, 31, 33]. Fig 4A–B shows the thermal figure, the stability curves for the wild-type FLNGLY1 and the R402XFLNGLY1.…”

Section: Resultsmentioning

confidence: 99%

“…Experimental validation of the VLS hits was carried out using a high-throughput thermal shift assay methodology following established guidelines [17, 31–33]. Briefly, protein samples were dispensed into 96-well PCR plates with sypro-orange, SO, as the extrinsic fluorophore dye and thermal melting was carried out by heating the samples from 25° C–74° C using a 1° C/min heating ramp in a RealPlex quantitative PCR instrument (Eppendorf, NY, USA).…”

Section: Methodsmentioning

confidence: 99%

Novel small molecule binders of human N-glycanase 1, a key player in the endoplasmic reticulum associated degradation pathway

Srinivasan

Zhou

Mitra

et al. 2016

Bioorganic & Medicinal Chemistry

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Peptide:N-glycanase (NGLY1) is an enzyme responsible for cleaving oligosaccharide moieties from misfolded glycoproteins to enable their proper degradation. Deletion and truncation mutations in this gene are responsible for an inherited disorder of the endoplasmic reticulum-associated degradation pathway. However, the literature is unclear whether the disorder is a result of mutations leading to loss-of-function, loss of substrate specificity, loss of protein stability or a combination of these factors. In this communication, without burdening ourselves with the mechanistic underpinning of disease causation because of mutations on the NGLY1 protein, we demonstrate the successful application of virtual ligand screening (VLS) combined with experimental high-throughput validation to the discovery of novel small-molecules that show binding to the transglutaminase domain of NGLY1. Attempts at recombinant expression and purification of six different constructs led to successful expression of five, with three constructs purified to homogeneity. Most mutant variants failed to purify possibly because of misfolding and the resultant exposure of surface hydrophobicity that led to protein aggregation. For the purified constructs, our threading/structure-based VLS algorithm, FINDSITEcomb, was employed to predict ligands that may bind to the protein. Then, the predictions were assessed by high-throughput differential scanning fluorimetry. This led to the identification of nine different ligands that bind to the protein of interest and provide clues to the nature of pharmacophore that facilitates binding. This is the first study that has identified novel ligands that bind to the NGLY1 protein as a possible starting point in the discovery of ligands with potential therapeutic applications in the treatment of the disorder caused by NGLY1 mutants.

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“…A single data point was acquired for each degree increment. The reaction was carried out in a volume of 20 μl, with 100 mM HEPES pH 7.3, 150 mM NaCl and 5 μM of the respective protein 8, 11, 26, 27 . The experiments were performed with biological replicates and the mean value was considered for further analysis.…”

mentioning

confidence: 99%

Pocket detection and interaction-weighted ligand-similarity search yields novel high-affinity binders for Myocilin-OLF, a protein implicated in glaucoma

Srinivasan

Tonddast-Navaei

Skolnick

2017

Bioorganic & Medicinal Chemistry Letters

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Traditional structure and ligand based virtual screening approaches rely on the availability of structural and ligand binding information. To overcome this limitation, hybrid approaches were developed that relied on extraction of ligand binding information from proteins sharing similar folds and hence, evolutionarily relationship. However, they cannot target a chosen pocket in a protein. To address this, a pocket centric virtual ligand screening approach is required. Here, we employ a new, iterative implementation of a pocket and ligand-similarity based approach to virtual ligand screening to predict small molecule binders for the olfactomedin domain of human myocilin implicated in glaucoma. Small-molecule binders of the protein might prevent the aggregation of the protein, commonly seen during glaucoma. First round experimental assessment of the predictions using differential scanning fluorimetry with myoc-OLF yielded 7 hits with a success rate of 12.7%; the best hit had an apparent dissociation constant of 99 nM. By matching to the key functional groups of the best ligand that were likely involved in binding, the affinity of the best hit was improved by almost 10,000 fold from the high nanomolar to the low picomolar range. Thus, this study provides preliminary validation of the methodology on a medically important glaucoma associated protein.

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Ligand binding studies, preliminary structure–activity relationship and detailed mechanistic characterization of 1-phenyl-6,6-dimethyl-1,3,5-triazine-2,4-diamine derivatives as inhibitors of Escherichia coli dihydrofolate reductase

Cited by 23 publications

References 59 publications

Rational Design of Novel Allosteric Dihydrofolate Reductase Inhibitors Showing Antibacterial Effects on Drug-Resistant Escherichia coli Escape Variants

Rational Design of Novel Allosteric Dihydrofolate Reductase Inhibitors Showing Antibacterial Effects on Drug-Resistant Escherichia coli Escape Variants

Novel small molecule binders of human N-glycanase 1, a key player in the endoplasmic reticulum associated degradation pathway

Pocket detection and interaction-weighted ligand-similarity search yields novel high-affinity binders for Myocilin-OLF, a protein implicated in glaucoma

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