scholarly journals Structural Proteomics-Driven Targeted Design of Favipiravir-Binding Site in The RdRp of SARS-CoV-2 Unravels Susceptible Hotspots and Resistance Mutations

2021 ◽  
Author(s):  
Aditya K. Padhi ◽  
Jagneshwar Dandapat ◽  
Vladimir N. Uversky ◽  
Timir Tripathi
Keyword(s):  
Binding Site ◽  
Protein Design ◽  
Antigenic Drift ◽  
Viral Rna ◽  
Structural Proteomics ◽  
Binding Affinities ◽  
Sequencing Data ◽  
Resistance Mutations ◽  
Structural Modifications ◽  
High Propensity

Favipiravir is a broad-spectrum inhibitor of viral RNA-dependent RNA polymerase (RdRp) currently being used to manage COVID-19 in several countries. By acting as a substrate for RdRp, favipiravir gets incorporated into the nascent viral RNA and prevents strand extension. A high mutation rate of SARS-CoV-2 RdRp may facilitate antigenic drift as an answer to the host immune response, thereby generating resistance of virus to favipiravir. Therefore, it is extremely crucial to predict potential mutational sites in the RdRp and the emergence of structural modifications contributing to drug resistance. Here, we used high-throughput interface-based protein design to generate >100,000 designs and identify mutation hotspot residues in the favipiravir-binding site of RdRp. Several mutants had lower binding affinities to favipiravir, out of which hotspot residues with a high propensity to undergo positive selection were identified. The results showed that the designs retained an average of 97 to 98% sequence identity, suggesting that SARS-CoV-2 can develop favipiravir resistance with just a few mutations. Notably, we observed that out of 134 mutations predicted designs, 63 specific mutations were already present in the CoV-GLUE database, thus attaining ~47% correlation match with the clinical sequencing data. The findings improve our understanding of the potential signatures of adaptation in SARS-CoV-2 against favipiravir and management of COVID-19. Furthermore, they can help develop exhaustive strategies for robust antiviral design and discovery.

scholarly journals Mutation in Abl kinase with altered drug-binding kinetics indicates a novel mechanism of imatinib resistance

2021 ◽  
Vol 118 (46) ◽  
pp. e2111451118
Author(s):  
Agatha Lyczek ◽  
Benedict-Tilman Berger ◽  
Aziz M. Rangwala ◽  
YiTing Paung ◽  
Jessica Tom ◽  
...  
Keyword(s):  
Binding Site ◽  
Kinase Domain ◽  
Protein Kinase Inhibitors ◽  
Drug Binding ◽  
The Body ◽  
Binding Kinetics ◽  
Wild Type ◽  
Resistance Mutations ◽  
Imatinib Resistance ◽  
Abl Kinase

Protein kinase inhibitors are potent anticancer therapeutics. For example, the Bcr-Abl kinase inhibitor imatinib decreases mortality for chronic myeloid leukemia by 80%, but 22 to 41% of patients acquire resistance to imatinib. About 70% of relapsed patients harbor mutations in the Bcr-Abl kinase domain, where more than a hundred different mutations have been identified. Some mutations are located near the imatinib-binding site and cause resistance through altered interactions with the drug. However, many resistance mutations are located far from the drug-binding site, and it remains unclear how these mutations confer resistance. Additionally, earlier studies on small sets of patient-derived imatinib resistance mutations indicated that some of these mutant proteins were in fact sensitive to imatinib in cellular and biochemical studies. Here, we surveyed the resistance of 94 patient-derived Abl kinase domain mutations annotated as disease relevant or resistance causing using an engagement assay in live cells. We found that only two-thirds of mutations weaken imatinib affinity by more than twofold compared to Abl wild type. Surprisingly, one-third of mutations in the Abl kinase domain still remain sensitive to imatinib and bind with similar or higher affinity than wild type. Intriguingly, we identified three clinical Abl mutations that bind imatinib with wild type–like affinity but dissociate from imatinib considerably faster. Given the relevance of residence time for drug efficacy, mutations that alter binding kinetics could cause resistance in the nonequilibrium environment of the body where drug export and clearance play critical roles.

scholarly journals Plasmodium falciparum DHODH inhibitor complexes reveal flexible binding site

2014 ◽  
Vol 70 (a1) ◽  
pp. C1793-C1793
Author(s):  
Paul Rowland ◽  
Onkar SINGH ◽  
Leila Ross ◽  
Francisco Gamo ◽  
Maria Lafuente-Monasterio ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Binding Site ◽  
Crystal Structures ◽  
Point Mutations ◽  
Crystal Form ◽  
Drug Binding ◽  
Binding Modes ◽  
Resistance Mutations ◽  
Drug Binding Site

Malaria is a preventable and treatable disease, yet annually there are still hundreds of thousands of malaria-related deaths. The disease is caused by infection with mosquito-borne Plasmodium parasites. With hundreds of millions of cases each year there is a very high potential for drug resistance and this has compromised many existing therapies. One target under investigation is the enzyme dihydroorotate dehydrogenase (DHODH) which catalyses the rate-limiting step of pyrimidine biosynthesis and is an essential enzyme in the malaria parasite. There are currently several Plasmodium-selective DHODH inhibitors under development. To investigate the potential for drug resistance against DHODH inhibitors in vitro resistance selections were carried out using known inhibitors from different structural classes [1]. These studies identified point mutations in the drug binding site which lead to reduced sensitivity to the inhibitors, and in some cases increased sensitivity to a different inhibitor, suggesting a novel combination therapy approach to combat resistance. To help understand the significance of the inhibitor binding site mutations we determined the crystal structures of P. falciparum DHODH in complex with the inhibitors Genz-669178, IDI-6253 and IDI-6273. Co-crystallisation experiments led to a new crystal form in each case. Here we describe the crystal structures, the binding modes of the inhibitors and the great flexibility of the binding site, which is able to adjust to accommodate different inhibitor series. The structural role of the resistance mutations is also discussed.

scholarly journals Biological effects of simple changes in functionality on rhodium metalloinsertors

2013 ◽  
Vol 371 (1995) ◽  
pp. 20120117 ◽  
Author(s):  
Alyson G. Weidmann ◽  
Alexis C. Komor ◽  
Jacqueline K. Barton
Keyword(s):  
Cellular Proliferation ◽  
Biological Effects ◽  
High Specificity ◽  
Selective Inhibition ◽  
Binding Affinities ◽  
Structural Modifications ◽  
Dna Mismatch ◽  
Dna Mismatches ◽  
Sensitive Structure ◽  
Function Relationship

DNA mismatch repair (MMR) is crucial to ensuring the fidelity of the genome. The inability to correct single base mismatches leads to elevated mutation rates and carcinogenesis. Using metalloinsertors–bulky metal complexes that bind with high specificity to mismatched sites in the DNA duplex–our laboratory has adopted a new chemotherapeutic strategy through the selective targeting of MMR-deficient cells, that is, those that have a propensity for cancerous transformation. Rhodium metalloinsertors display inhibitory effects selectively in cells that are deficient in the MMR machinery, consistent with this strategy. However, a highly sensitive structure–function relationship is emerging with the development of new complexes that highlights the importance of subcellular localization. We have found that small structural modifications, for example a hydroxyl versus a methyl functional group, can yield profound differences in biological function. Despite similar binding affinities and selectivities for DNA mismatches, only one metalloinsertor shows selective inhibition of cellular proliferation in MMR-deficient versus -proficient cells. Studies of whole-cell, nuclear and mitochondrial uptake reveal that this selectivity depends upon targeting DNA mismatches in the cell nucleus.

scholarly journals Characterization of the High-Affinity Drug Ligand Binding Site of Mouse Recombinant TSPO

2019 ◽  
Vol 20 (6) ◽  
pp. 1444 ◽  
Author(s):  
Soria Iatmanen-Harbi ◽  
lucile Senicourt ◽  
Vassilios Papadopoulos ◽  
Olivier Lequin ◽  
Jean-Jacques Lacapere
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Conformational Changes ◽  
Protoporphyrin Ix ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Translocator Protein ◽  
Binding Affinities ◽  
Ligand Selectivity ◽  
High Affinity

The optimization of translocator protein (TSPO) ligands for Positron Emission Tomography as well as for the modulation of neurosteroids is a critical necessity for the development of TSPO-based diagnostics and therapeutics of neuropsychiatrics and neurodegenerative disorders. Structural hints on the interaction site and ligand binding mechanism are essential for the development of efficient TSPO ligands. Recently published atomic structures of recombinant mammalian and bacterial TSPO1, bound with either the high-affinity drug ligand PK 11195 or protoporphyrin IX, have revealed the membrane protein topology and the ligand binding pocket. The ligand is surrounded by amino acids from the five transmembrane helices as well as the cytosolic loops. However, the precise mechanism of ligand binding remains unknown. Previous biochemical studies had suggested that ligand selectivity and binding was governed by these loops. We performed site-directed mutagenesis to further test this hypothesis and measured the binding affinities. We show that aromatic residues (Y34 and F100) from the cytosolic loops contribute to PK 11195 access to its binding site. Limited proteolytic digestion, circular dichroism and solution two-dimensional (2-D) NMR using selective amino acid labelling provide information on the intramolecular flexibility and conformational changes in the TSPO structure upon PK 11195 binding. We also discuss the differences in the PK 11195 binding affinities and the primary structure between TSPO (TSPO1) and its paralogous gene product TSPO2.

scholarly journals Viral Kinetics and Resistance Development in Children Treated with Neuraminidase Inhibitors: The Influenza Resistance Information Study (IRIS)

2019 ◽  
Vol 71 (5) ◽  
pp. 1186-1194 ◽  
Author(s):  
Rueshandra Roosenhoff ◽  
Vaughan Reed ◽  
Andy Kenwright ◽  
Martin Schutten ◽  
Charles A Boucher ◽  
...  
Keyword(s):  
Viral Load ◽  
Influenza A ◽  
Antiviral Treatment ◽  
Vaccination Status ◽  
Viral Clearance ◽  
Viral Rna ◽  
Baseline Viral Load ◽  
Influenza B ◽  
Resistance Mutations ◽  
Viral Burden

Abstract Background We studied the effect of age, baseline viral load, vaccination status, antiviral therapy, and emergence of drug resistance on viral shedding in children infected with influenza A or B virus. Methods Samples from children (aged ≤13 years) enrolled during the 7 years of the prospective Influenza Resistance Information Study were analyzed using polymerase chain reaction to determine the influenza virus (sub-)type, viral load, and resistance mutations. Disease severity was assessed; clinical symptoms were recorded. The association of age with viral load and viral clearance was examined by determining the area under the curve for viral RNA shedding using logistic regression and Kaplan-Meier analyses. Results A total of 2131 children infected with influenza (683, A/H1N1pdm09; 825, A/H3N2; 623, influenza B) were investigated. Age did not affect the mean baseline viral load. Children aged 1−5 years had prolonged viral RNA shedding (±1–2 days) compared with older children and up to 1.2-fold higher total viral burden. Besides, in older age (odds ratio [OR], 1.08; confidence interval [CI], 1.05–1.12), prior vaccination status (OR, 1.72; CI, 1.22–2.43) and antiviral treatment (OR, 1.74; CI, 1.43–2.12) increased the rate of viral clearance. Resistance mutations were detected in 49 children infected with influenza A virus (34, A/H1N1pdm09; 15, A/H3N2) treated with oseltamivir, most of whom were aged <5 years (n = 39). Conclusions Children aged 1−5 years had a higher total viral burden with prolonged virus shedding and had an increased risk of acquiring resistance mutations following antiviral treatment. Clinical Trials Registration NCT00884117.

scholarly journals The predicted 3D structure of the human D2 dopamine receptor and the binding site and binding affinities for agonists and antagonists

2004 ◽  
Vol 101 (11) ◽  
pp. 3815-3820 ◽  
Author(s):  
M. Y. S. Kalani ◽  
N. Vaidehi ◽  
S. E. Hall ◽  
R. J. Trabanino ◽  
P. L. Freddolino ◽  
...  
Keyword(s):  
Binding Site ◽  
Dopamine Receptor ◽  
3D Structure ◽  
Binding Affinities ◽  
D2 Dopamine Receptor

scholarly journals MiniCD4 protein resistance mutations affect binding to the HIV-1 gp120 CD4 binding site and decrease entry efficiency

Retrovirology ◽  
2012 ◽  
Vol 9 (1) ◽  
pp. 36 ◽  
Author(s):  
Katrijn Grupping ◽  
Philippe Selhorst ◽  
Johan Michiels ◽  
Katleen Vereecken ◽  
Leo Heyndrickx ◽  
...  
Keyword(s):  
Binding Site ◽  
Resistance Mutations ◽  
Protein Resistance ◽  
Cd4 Binding Site ◽  
Hiv 1 ◽  
Entry Efficiency

scholarly journals Determination of the range in binding-site densities of rat skin heparin chains with high binding affinities for antithrombin

1988 ◽  
Vol 251 (1) ◽  
pp. 141-145 ◽  
Author(s):  
A A Horner ◽  
M Kusche ◽  
U Lindahl ◽  
C B Peterson
Keyword(s):  
Chemical Analysis ◽  
Binding Site ◽  
Binding Affinity ◽  
Intrinsic Fluorescence ◽  
Binding Affinities ◽  
Rat Skin ◽  
High Binding ◽  
Site Density ◽  
High Binding Affinity

Rat skin heparin proteoglycans vary markedly in the proportions of their constituent polysaccharide chains that have high binding affinity for antithrombin. As the proportion of such chains in a proteoglycan rises, their degree of affinity for antithrombin also increases [Horner (1987) Biochem. J. 244, 693-698]. The antithrombin-binding-site densities of such chains have now been determined, by measuring heparin-induced enhancement of the intrinsic fluorescence of antithrombin and by chemical analysis for the disaccharide sequence glucuronosyl-N-sulphoglucosaminyl (3,6-di-O-sulphate), which is unique to this site in heparin [Lindahl, Bäckström, Thunberg & Leder (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 6551-6555]. Antithrombin-binding-site density ranged from one to five sites per chain.

scholarly journals Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

2014 ◽  
Vol 70 (7) ◽  
pp. 1801-1811 ◽  
Author(s):  
Thanuja Gangi Setty ◽  
Christine Cho ◽  
Sowmya Govindappa ◽  
Michael A. Apicella ◽  
S. Ramaswamy
Keyword(s):  
Sialic Acid ◽  
Binding Site ◽  
Pathogenic Bacteria ◽  
Pasteurella Multocida ◽  
Binding Proteins ◽  
Molecular Mimicry ◽  
Binding Pocket ◽  
Sialic Acids ◽  
Host Immune System ◽  
Binding Affinities

Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteriaFusobacterium nucleatum,Pasteurella multocidaandVibrio choleraeand their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of theHaemophilus influenzaeprotein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states.

scholarly journals Resistance mutations generate divergent antibiotic susceptibility profiles against translation inhibitors

2016 ◽  
Vol 113 (29) ◽  
pp. 8188-8193 ◽  
Author(s):  
Alexis I. Cocozaki ◽  
Roger B. Altman ◽  
Jian Huang ◽  
Ed T. Buurman ◽  
Steven L. Kazmirski ◽  
...  
Keyword(s):  
Binding Site ◽  
Single Molecule ◽  
Antibiotic Susceptibility ◽  
Ribosomal Subunit ◽  
Structural Defects ◽  
Resistance Mutations ◽  
Antibiotic Resistant ◽  
E Coli ◽  
X Ray Crystallography ◽  
Susceptibility Profiles

Mutations conferring resistance to translation inhibitors often alter the structure of rRNA. Reduced susceptibility to distinct structural antibiotic classes may, therefore, emerge when a common ribosomal binding site is perturbed, which significantly reduces the clinical utility of these agents. The translation inhibitors negamycin and tetracycline interfere with tRNA binding to the aminoacyl-tRNA site on the small 30S ribosomal subunit. However, two negamycin resistance mutations display unexpected differential antibiotic susceptibility profiles. Mutant U1060A in 16S Escherichia coli rRNA is resistant to both antibiotics, whereas mutant U1052G is simultaneously resistant to negamycin and hypersusceptible to tetracycline. Using a combination of microbiological, biochemical, single-molecule fluorescence transfer experiments, and X-ray crystallography, we define the specific structural defects in the U1052G mutant 70S E. coli ribosome that explain its divergent negamycin and tetracycline susceptibility profiles. Unexpectedly, the U1052G mutant ribosome possesses a second tetracycline binding site that correlates with its hypersusceptibility. The creation of a previously unidentified antibiotic binding site raises the prospect of identifying similar phenomena in antibiotic-resistant pathogens in the future.

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