Characterization of the NiRAN domain from RNA-dependent RNA polymerase provides insights into a potential therapeutic target against SARS-CoV-2

AbstractApart from the canonical fingers, palm and thumb domains, the RNA dependent RNA polymerases (RdRp) from the viral order Nidovirales possess two additional domains. Of these, the function of the Nidovirus RdRp associated nucleotidyl transferase domain (NiRAN) remains unanswered. The elucidation of the 3D structure of RdRp from the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), provided the first ever insights into the domain organisation and possible functional characteristics of the NiRAN domain. Using in silico tools, we predict that the NiRAN domain assumes a kinase or phosphotransferase like fold and binds nucleoside triphosphates at its proposed active site. Additionally, using molecular docking we have predicted the binding of three widely used kinase inhibitors and five well characterized anti-microbial compounds at the NiRAN domain active site along with their drug-likeliness as well as DFT properties. For the first time ever, using basic biochemical tools, this study shows the presence of a kinase like activity exhibited by the SARS-CoV-2 RdRp. Interestingly, the proposed kinase inhibitors and a few of the predicted nucleotidyl transferase inhibitors significantly inhibited the aforementioned enzymatic activity. In line with the current global COVID-19 pandemic urgency and the emergence of newer strains with significantly higher infectivity, this study provides a new anti-SARS-CoV-2 drug target and potential lead compounds for drug repurposing against SARS-CoV-2.

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Characterization of the NiRAN domain from RNA-dependent RNA polymerase provides insights into a potential therapeutic target against SARS-CoV-2

PLoS Computational Biology ◽

10.1371/journal.pcbi.1009384 ◽

2021 ◽

Vol 17 (9) ◽

pp. e1009384

Author(s):

Abhisek Dwivedy ◽

Richard Mariadasse ◽

Mohammed Ahmad ◽

Sayan Chakraborty ◽

Deepsikha Kar ◽

...

Keyword(s):

Active Site ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

3D Structure ◽

Drug Repurposing ◽

Significant Inhibition ◽

Lead Compounds ◽

Nucleotidyl Transferase ◽

Domain Organisation ◽

Infected Cells

Apart from the canonical fingers, palm and thumb domains, the RNA dependent RNA polymerases (RdRp) from the viral order Nidovirales possess two additional domains. Of these, the function of the Nidovirus RdRp associated nucleotidyl transferase domain (NiRAN) remains unanswered. The elucidation of the 3D structure of RdRp from the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), provided the first ever insights into the domain organisation and possible functional characteristics of the NiRAN domain. Using in silico tools, we predict that the NiRAN domain assumes a kinase or phosphotransferase like fold and binds nucleoside triphosphates at its proposed active site. Additionally, using molecular docking we have predicted the binding of three widely used kinase inhibitors and five well characterized anti-microbial compounds at the NiRAN domain active site along with their drug-likeliness. For the first time ever, using basic biochemical tools, this study shows the presence of a kinase like activity exhibited by the SARS-CoV-2 RdRp. Interestingly, a well-known kinase inhibitor- Sorafenib showed a significant inhibition and dampened viral load in SARS-CoV-2 infected cells. In line with the current global COVID-19 pandemic urgency and the emergence of newer strains with significantly higher infectivity, this study provides a new anti-SARS-CoV-2 drug target and potential lead compounds for drug repurposing against SARS-CoV-2.

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In silico characterization of the NiRAN domain of RNA-dependent RNA polymerase provides insights into a potential therapeutic target against SARS-CoV2

10.31219/osf.io/wd6zu ◽

2020 ◽

Cited By ~ 1

Author(s):

Abhisek Dwivedy ◽

Richard Mariadasse ◽

Mohammed Ahmed ◽

Deepsikha Kar ◽

Jeyaraman Jeyakanthan ◽

...

Keyword(s):

Active Site ◽

In Silico ◽

3D Structure ◽

Drug Repurposing ◽

The Novel ◽

Lead Compounds ◽

Nucleotidyl Transferase ◽

Domain Organisation ◽

Novel Coronavirus ◽

In Silico Characterization

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Identification and Characterization of a Rhodopsin Kinase Gene in the Suckers of Octopus vulgaris: Looking around Using Arms?

Biology ◽

10.3390/biology10090936 ◽

2021 ◽

Vol 10 (9) ◽

pp. 936

Author(s):

Al-Sayed Al-Soudy ◽

Valeria Maselli ◽

Stefania Galdiero ◽

Michael J. Kuba ◽

Gianluca Polese ◽

...

Keyword(s):

3D Structure ◽

Complete Characterization ◽

Octopus Vulgaris ◽

Kinase Gene ◽

Light Sensing ◽

Rhodopsin Kinase ◽

First Time ◽

Identification And Characterization

In their foraging behavior octopuses rely on arm search movements outside the visual field of the eyes. In these movements the environment is explored primarily by the suckers that line the entire length of the octopus arm. In this study, for the first time, we report the complete characterization of a light-sensing molecule, Ov-GRK1, in the suckers, skin and retina of Octopus vulgaris. We sequenced the O. vulgaris GRK1 gene, defining a phylogenetic tree and performing a 3D structure model prediction. Furthermore, we found differences in relative mRNA expression in different sucker types at several arm levels, and localized it through in situ hybridization. Our findings suggest that the suckers in octopus arms are much more multimodal than was previously shown, adding the potential for light sensing to the already known mechanical and chemical sensing abilities.

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Molecular Identification and Characterization of an Essential Pyruvate Transporter from Trypanosoma brucei

Journal of Biological Chemistry ◽

10.1074/jbc.m113.473157 ◽

2013 ◽

Vol 288 (20) ◽

pp. 14428-14437 ◽

Cited By ~ 14

Author(s):

Marco A. Sanchez

Keyword(s):

Trypanosoma Brucei ◽

Drug Target ◽

Life Cycle Stage ◽

Bloodstream Form ◽

Potential Drug Target ◽

High Affinity ◽

Physiological Relevance ◽

First Time ◽

Identification And Characterization

Pyruvate export is an essential physiological process for the bloodstream form of Trypanosoma brucei as the parasite would otherwise accumulate this end product of glucose metabolism to toxic levels. In the studies reported here, genetic complementation in Saccharomyces cerevisiae has been employed to identify a gene (TbPT0) that encodes this vital pyruvate transporter from T. brucei. Expression of TbPT0 in S. cerevisiae reveals that TbPT0 is a high affinity pyruvate transporter. TbPT0 belongs to a clustered multigene family consisting of five members, whose expression is up-regulated in the bloodstream form. Interestingly, TbPT family permeases are related to polytopic proteins from plants but not to characterized monocarboxylate transporters from mammals. Remarkably, inhibition of the TbPT gene family expression in bloodstream parasites by RNAi is lethal, confirming the physiological relevance of these transporters. The discovery of TbPT0 reveals for the first time the identity of the essential pyruvate transporter and provides a potential drug target against the mammalian life cycle stage of T. brucei.

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Characterization of non-active site TrkA selective kinase inhibitors and implications on obtaining kinase selectivity

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s0108767317098221 ◽

2017 ◽

Vol 73 (a1) ◽

pp. a179-a179

Author(s):

Hua-Poo Su ◽

Keith Rickert ◽

Christine Burlein ◽

Kartik Narayan ◽

Marina Bukhtiyarova ◽

...

Keyword(s):

Active Site ◽

Kinase Inhibitors

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A cautionary tale of structure-guided inhibitor development against an essential enzyme in the aspartate-biosynthetic pathway

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004714023979 ◽

2014 ◽

Vol 70 (12) ◽

pp. 3244-3252 ◽

Cited By ~ 6

Author(s):

Alexander G. Pavlovsky ◽

Bharani Thangavelu ◽

Pravin Bhansali ◽

Ronald E. Viola

Keyword(s):

Active Site ◽

Enzyme Inhibitor ◽

Bacterial Survival ◽

Cautionary Tale ◽

Lead Compounds ◽

Semialdehyde Dehydrogenase ◽

Fragment Library ◽

Aspartate Pathway ◽

Essential Enzyme

The aspartate pathway is essential for the production of the amino acids required for protein synthesis and of the metabolites needed in bacterial development. This pathway also leads to the production of several classes of quorum-sensing molecules that can trigger virulence in certain microorganisms. The second enzyme in this pathway, aspartate β-semialdehyde dehydrogenase (ASADH), is absolutely required for bacterial survival and has been targeted for the design of selective inhibitors. Fragment-library screening has identified a new set of inhibitors that, while they do not resemble the substrates for this reaction, have been shown to bind at the active site of ASADH. Structure-guided development of these lead compounds has produced moderate inhibitors of the target enzyme, with some selectivity observed between the Gram-negative and Gram-positive orthologs of ASADH. However, many of these inhibitor analogs and derivatives have not yet achieved the expected enhanced affinity. Structural characterization of these enzyme–inhibitor complexes has provided detailed explanations for the barriers that interfere with optimal binding. Despite binding in the same active-site region, significant changes are observed in the orientation of these bound inhibitors that are caused by relatively modest structural alterations. Taken together, these studies present a cautionary tale for issues that can arise in the systematic approach to the modification of lead compounds that are being used to develop potent inhibitors.

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Glucose-6-phosphate dehydrogenase–6-phosphogluconolactonase: a unique bifunctional enzyme from Plasmodium falciparum

Biochemical Journal ◽

10.1042/bj20110170 ◽

2011 ◽

Vol 436 (3) ◽

pp. 641-650 ◽

Cited By ~ 32

Author(s):

Esther Jortzik ◽

Boniface M. Mailu ◽

Janina Preuss ◽

Marina Fischer ◽

Lars Bode ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Pentose Phosphate Pathway ◽

Drug Target ◽

Pentose Phosphate ◽

Bifunctional Enzyme ◽

Functional Differences ◽

Glucose 6 Phosphate Dehydrogenase ◽

First Time ◽

Human Rbcs

The survival of malaria parasites in human RBCs (red blood cells) depends on the pentose phosphate pathway, both in Plasmodium falciparum and its human host. G6PD (glucose-6-phosphate dehydrogenase) deficiency, the most common human enzyme deficiency, leads to a lack of NADPH in erythrocytes, and protects from malaria. In P. falciparum, G6PD is combined with the second enzyme of the pentose phosphate pathway to create a unique bifunctional enzyme named GluPho (glucose-6-phosphate dehydrogenase–6-phosphogluconolactonase). In the present paper, we report for the first time the cloning, heterologous overexpression, purification and kinetic characterization of both enzymatic activities of full-length PfGluPho (P. falciparum GluPho), and demonstrate striking structural and functional differences with the human enzymes. Detailed kinetic analyses indicate that PfGluPho functions on the basis of a rapid equilibrium random Bi Bi mechanism, where the binding of the second substrate depends on the first substrate. We furthermore show that PfGluPho is inhibited by S-glutathionylation. The availability of recombinant PfGluPho and the major differences to hG6PD (human G6PD) facilitate studies on PfGluPho as an excellent drug target candidate in the search for new antimalarial drugs.

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SYNTHESIS AND CHARACTERIZATION OF A HIGHLY CRYSTALLINE NOVEL MESOPOROUS C- AND N-CODOPED TiO2 NANOPHOTOCATALYST

NANO ◽

10.1142/s1793292008001210 ◽

2008 ◽

Vol 03 (05) ◽

pp. 367-372 ◽

Cited By ~ 5

Author(s):

SRINIVASAN ANANDAN ◽

YASURO IKUMA ◽

KATSUYOSHI KAKINUMA ◽

KOICHI NIWA

Keyword(s):

High Surface ◽

3D Structure ◽

High Surface Area ◽

Titanium Tetraisopropoxide ◽

Tem Analysis ◽

Light Region ◽

C And N ◽

First Time ◽

Uniform Pore Size

Here we report, for the first time, the synthesis of highly crystalline novel mesoporous carbon/nitrogen ( C / N )-codoped TiO 2 (MCNT) using ethylene diamine and carbon tetrachloride as the source for nitrogen and carbon, KIT-6 as the template, and titanium tetraisopropoxide as the TiO 2 source. The obtained material has been unambiguously characterized by various sophisticated techniques. XRD and TEM analysis revealed that the MCNT material possesses highly crystalline 3D structure with a continuous network of mesoporous channels as well as structure corresponding to TiO 2. UV-DRS analysis indicated that light absorption shifted to lower energy and stronger absorption in the visible light region. XPS revealed that C and N were doped effectively and C or N dopants might be present in the chemical environment of Ti – O – N or Ti – O – C . It has been found that the material is highly crystalline and possesses high surface area, pore volume and uniform pore size distribution. Owing to its textural characteristics, it could be useful for various applications, such as photocatalysis, fuel cells, bulky biomolecule adsorption and nanotechnology.

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Snapshots during the catalytic cycle of a histidine acid phytase reveal an induced-fit structural mechanism

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.015925 ◽

2020 ◽

Vol 295 (51) ◽

pp. 17724-17737

Author(s):

Isabella M. Acquistapace ◽

Monika A. Zi¸etek ◽

Arthur W. H. Li ◽

Melissa Salmon ◽

Imke Kühn ◽

...

Keyword(s):

Active Site ◽

Bifidobacterium Longum ◽

Catalytic Cycle ◽

Inositol Polyphosphate ◽

Positional Specificity ◽

Structural Mechanism ◽

Mechanism Of Hydrolysis ◽

Histidine Phosphatases ◽

First Time

Highly engineered phytases, which sequentially hydrolyze the hexakisphosphate ester of inositol known as phytic acid, are routinely added to the feeds of monogastric animals to improve phosphate bioavailability. New phytases are sought as starting points to further optimize the rate and extent of dephosphorylation of phytate in the animal digestive tract. Multiple inositol polyphosphate phosphatases (MINPPs) are clade 2 histidine phosphatases (HP2P) able to carry out the stepwise hydrolysis of phytate. MINPPs are not restricted by a strong positional specificity making them attractive targets for development as feed enzymes. Here, we describe the characterization of a MINPP from the Gram-positive bacterium Bifidobacterium longum (BlMINPP). BlMINPP has a typical HP2P-fold but, unusually, possesses a large α-domain polypeptide insertion relative to other MINPPs. This insertion, termed the U-loop, spans the active site and contributes to substrate specificity pockets underpopulated in other HP2Ps. Mutagenesis of U-loop residues reveals its contribution to enzyme kinetics and thermostability. Moreover, four crystal structures of the protein along the catalytic cycle capture, for the first time in an HP2P, a large ligand-driven α-domain motion essential to allow substrate access to the active site. This motion recruits residues both downstream of a molecular hinge and on the U-loop to participate in specificity subsites, and mutagenesis identified a mobile lysine residue as a key determinant of positional specificity of the enzyme. Taken together, these data provide important new insights to the factors determining stability, substrate recognition, and the structural mechanism of hydrolysis in this industrially important group of enzymes.

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The crystal structure of human Aldehyde Oxidase in native and inhibited forms

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314081728 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C1828-C1828

Author(s):

Catarina Coelho ◽

Tobias Hartmann ◽

Alessandro Foti ◽

Teresa Santos-Silva ◽

Silke Leimkühler ◽

...

Keyword(s):

Active Site ◽

3D Structure ◽

Aldehyde Oxidase ◽

Drug Metabolizing Enzyme ◽

Resolution Analysis ◽

Metabolizing Enzyme ◽

Novel Drug ◽

First Time ◽

Important Drug

Aldehyde oxidases (AOX; E.C. 1.2.3.1) are molybdo-flavoenzymes with broad substrate specificity, oxidizing aldehydes and N-heterocycles. AOX belongs to the xanthine oxidase (XO) family of Mo-containing enzymes. The true physiological function of AOX is still unknown, although it is recognized to play a role in the metabolism of compounds with medicinal and toxicological relevance [1]. AOX importance has increased in recent years since it is substituting Cyt-P450 as the central drug-metabolizing system in humans. We have solved the 3D structure of mouse AOX3 to 2.9 Å resolution [2] that was the first structure of an aldehyde oxidase, providing important evidences on substrate and inhibitor specificities between AOX and XO. The complement of AOX proteins in mammals varies from one in humans (hAOX1) to four in rodents (mAOX1, mAOX3, mAOX4 and mAOX3L1) as a result of evolutionary genetic events. Due to this unusual complement of AOX genes in different animal species, conclusions regarding protein metabolism in humans cannot be taken exclusively from the mouse model. Using the human aldehyde oxidase (hAOX1) purified after heterologous expression in Escherichia coli we were able to crystallize it and solve its 3D structure to 2.7 Å resolution (submitted). In addition to the native protein we also solved the structure of an inhibited form of the enzyme to 2.6Å resolution. Analysis of the protein active site and comparison with the structure of the mouse isoform (mAOX3) allowed us to identity, for the first time, the unique features that characterize hAOX1 as an important drug-metabolizing enzyme. In spite of the similarities of both enzymes, they show marked and relevant differences at the Mo active site, substrate tunnel as well as at the FAD site. The ensemble of these structures provides important insights into the role of aldehyde oxidases, contributing to elucidate the clinical metabolism implications of hAOX1 in humans which has particular relevance for novel drug design studies.

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