GTP Molecules Research Articles

Structural investigation and gene deletion studies of mycobacterial oligoribonuclease reveal modulation of c-di-GMP-mediated phenotypes

Cyclic-di-GMP (c-di-GMP) is a ubiquitous bacterial second messenger required for normal physiology as well as survival under hypoxic and reductive stress conditions of mycobacterial cells. Complete degradation of c-di-GMP is necessary for signal termination and maintaining its homeostasis inside the cells. Homeostasis of c-di-GMP in mycobacteria is brought about by the bifunctional diguanylate cyclase (DGC) that synthesizes c-di-GMP from two molecules of GTP and also catalyses the asymmetric cleavage of c-di-GMP to linear pGpG through its phosphodiesterase activity. However, the mycobacterial enzyme for the last step of degradation from pGpG to GMP has not been characterized thus far. Here, we present the identification of oligoribonuclease (Orn) as the most likely phosphodiesterase to degrade pGpG to GMP through AlphaFold-empowered structural homology that exhibited in vitro phosphodiesterase activity on pGpG substrates. In order to understand the physiological role of Orn in mycobacteria, we created a deletion mutant of orn in M. smegmatis and analysed the phenotypes that are associated with c-di-GMP signaling. We find that orn plays important roles in vivo and is required not only for proper growth of M. smegmatis in normal and stress conditions but also for biofilm formation.

Compartmentalization and regulation of GTP in control of cellular phenotypes.

Genetic or pharmacological inhibition of enzymes involved in GTP biosynthesis has substantial biological effects, underlining the need to better understand the function of GTP levels in regulation of cellular processes and the significance of targeting GTP biosynthesis enzymes for therapeutic intervention. Our current understanding of spatiotemporal regulation of GTP metabolism and its role in physiological and pathological cellular processes is far from complete. Novel methodologies such as genetically encoded sensors of free GTP offered insights into intracellular distribution and function of GTP molecules. In the current Review, we provide analysis of recent discoveries in the field of GTP metabolism and evaluate the key enzymes as molecular targets.

Structure of the MRAS–SHOC2–PP1C phosphatase complex

RAS–MAPK signalling is fundamental for cell proliferation and is altered in most human cancers1–3. However, our mechanistic understanding of how RAS signals through RAF is still incomplete. Although studies revealed snapshots for autoinhibited and active RAF–MEK1–14-3-3 complexes4, the intermediate steps that lead to RAF activation remain unclear. The MRAS–SHOC2–PP1C holophosphatase dephosphorylates RAF at serine 259, resulting in the partial displacement of 14-3-3 and RAF–RAS association3,5,6. MRAS, SHOC2 and PP1C are mutated in rasopathies—developmental syndromes caused by aberrant MAPK pathway activation6–14—and SHOC2 itself has emerged as potential target in receptor tyrosine kinase (RTK)–RAS-driven tumours15–18. Despite its importance, structural understanding of the SHOC2 holophosphatase is lacking. Here we determine, using X-ray crystallography, the structure of the MRAS–SHOC2–PP1C complex. SHOC2 bridges PP1C and MRAS through its concave surface and enables reciprocal interactions between all three subunits. Biophysical characterization indicates a cooperative assembly driven by the MRAS GTP-bound active state, an observation that is extendible to other RAS isoforms. Our findings support the concept of a RAS-driven and multi-molecular model for RAF activation in which individual RAS–GTP molecules recruit RAF–14-3-3 and SHOC2–PP1C to produce downstream pathway activation. Importantly, we find that rasopathy and cancer mutations reside at protein–protein interfaces within the holophosphatase, resulting in enhanced affinities and function. Collectively, our findings shed light on a fundamental mechanism of RAS biology and on mechanisms of clinically observed enhanced RAS–MAPK signalling, therefore providing the structural basis for therapeutic interventions.

Structural and Biochemical Studies of Bacillus subtilis MobB

The biosynthesis of molybdenum cofactor for redox enzymes is carried out by multiple enzymes in bacteria including MobA and MobB. MobA is known to catalyze the attachment of GMP to molybdopterin to form molybdopterin guanine dinucleotide. MobB is a GTP binding protein that enhances the activity of MobA by forming the MobA:MobB complex. However, the mechanism of activity enhancement by MobB is not well understood. The structure of Bacillus subtilis MobB was determined to 2.4 Å resolution and it showed an elongated homodimer with an extended β-sheet. Bound sulfate ions were observed in the Walker A motifs, indicating a possible phosphate-binding site for GTP molecules. The binding assay showed that the affinity between B. subtilis MobA and MobB increased in the presence of GTP, suggesting a possible role of MobB as an enhancer of MobA activity.

Studying GGDEF Domain in the Act: Minimize Conformational Frustration to Prevent Artefacts.

GGDEF-containing proteins respond to different environmental cues to finely modulate cyclic diguanylate (c-di-GMP) levels in time and space, making the allosteric control a distinctive trait of the corresponding proteins. The diguanylate cyclase mechanism is emblematic of this control: two GGDEF domains, each binding one GTP molecule, must dimerize to enter catalysis and yield c-di-GMP. The need for dimerization makes the GGDEF domain an ideal conformational switch in multidomain proteins. A re-evaluation of the kinetic profile of previously characterized GGDEF domains indicated that they are also able to convert GTP to GMP: this unexpected reactivity occurs when conformational issues hamper the cyclase activity. These results create new questions regarding the characterization and engineering of these proteins for in solution or structural studies.

Bacteriophage-mediated interference of the c-di-GMP signalling pathway in Pseudomonas aeruginosa.

C-di-GMP is a key signalling molecule which impacts bacterial motility and biofilm formation and is formed by the condensation of two GTP molecules by a diguanylate cyclase. We here describe the identification and characterization of a family of bacteriophage-encoded peptides that directly impact c-di-GMP signalling in Pseudomonas aeruginosa. These phage proteins target Pseudomonas diguanylate cyclase YfiN by direct protein interaction (termed YIPs, YfiN Interacting Peptides). YIPs induce an increase of c-di-GMP production in the host cell, resulting in a decrease in motility and an increase in biofilm mass in P.aeruginosa. A dynamic analysis of the biofilm morphology indicates a denser biofilm structure after induction of the phage protein. This intracellular signalling interference strategy by a lytic phage constitutes an unexplored phage-based mechanism of metabolic regulation and could potentially serve as inspiration for the development of molecules that interfere with biofilm formation in P.aeruginosa and other pathogens.

N-terminal truncation of VC0395_0300 protein from Vibrio cholerae does not lead to loss of diguanylate cyclase activity

The bacterial secondary messenger bis-(3′,5′)-cyclic-dimeric-guanosine monophosphate (c-di-GMP) has been implicated in the pathogenesis of Vibrio cholerae, due to its significant role in regulating the virulence, biofilm formation and motility of the host organism. The VC0395_0300 protein from V. cholerae, possessing a GGEEF sequence has been established as a diguanylate cyclase (DGC) capable of catalyzing the conversion of two GTP molecules to form cyclic-di-GMP. This in turn, plays a crucial role in allowing the organism to adopt a dual lifestyle, thriving both in human and aquatic systems. The difficulty in procuring sufficient amounts of homogenous soluble protein for structural assessment of the GGDEF domain in VC0395_0300 and the lack of soluble protein yield, prompted the truncation into smaller constructs (Sebox31 and Sebox32) carrying the GGDEF domain. The truncates retained their diguanylate cyclase activity comparable to the wild type, and were able to form biofilms as well. Fluorescence and circular dichroism spectroscopy measurements revealed that the basic structural elements do not show significant changes in the truncated proteins as compared to the full-length. This has also been confirmed using homology modeling and molecular docking of the wild type and truncates. This led us to conclude that the truncated constructs retain their activity in spite of the deletions in the N terminal region. This is supportive of the fact that DGC activity in GGDEF proteins is predominantly dependent on the presence of the conserved GGD(/E)EF domain and its interaction with GTP.

Cell cycle control and environmental response by second messengers in Caulobacter crescentus

BackgroundSecond messengers, c-di-GMP and (p)ppGpp, are vital regulatory molecules in bacteria, influencing cellular processes such as biofilm formation, transcription, virulence, quorum sensing, and proliferation. While c-di-GMP and (p)ppGpp are both synthesized from GTP molecules, they play antagonistic roles in regulating the cell cycle. In C. crescentus, c-di-GMP works as a major regulator of pole morphogenesis and cell development. It inhibits cell motility and promotes S-phase entry by inhibiting the activity of the master regulator, CtrA. Intracellular (p)ppGpp accumulates under starvation, which helps bacteria to survive under stressful conditions through regulating nucleotide levels and halting proliferation. (p)ppGpp responds to nitrogen levels through RelA-SpoT homolog enzymes, detecting glutamine concentration using a nitrogen phosphotransferase system (PTS Ntr). This work relates the guanine nucleotide-based second messenger regulatory network with the bacterial PTS Ntr system and investigates how bacteria respond to nutrient availability.ResultsWe propose a mathematical model for the dynamics of c-di-GMP and (p)ppGpp in C. crescentus and analyze how the guanine nucleotide-based second messenger system responds to certain environmental changes communicated through the PTS Ntr system. Our mathematical model consists of seven ODEs describing the dynamics of nucleotides and PTS Ntr enzymes. Our simulations are consistent with experimental observations and suggest, among other predictions, that SpoT can effectively decrease c-di-GMP levels in response to nitrogen starvation just as well as it increases (p)ppGpp levels. Thus, the activity of SpoT (or its homologues in other bacterial species) can likely influence the cell cycle by influencing both c-di-GMP and (p)ppGpp.ConclusionsIn this work, we integrate current knowledge and experimental observations from the literature to formulate a novel mathematical model. We analyze the model and demonstrate how the PTS Ntr system influences (p)ppGpp, c-di-GMP, GMP and GTP concentrations. While this model does not consider all aspects of PTS Ntr signaling, such as cross-talk with the carbon PTS system, here we present our first effort to develop a model of nutrient signaling in C. crescentus.

Coordinated Activation of ARF1 GTPases by ARF-GEF GNOM Dimers Is Essential for Vesicle Trafficking in Arabidopsis.

Membrane trafficking maintains the organization of the eukaryotic cell and delivers cargo proteins to their subcellular destinations, such as sites of action or degradation. The formation of membrane vesicles requires the activation of the ADP-ribosylation factor ARF GTPase by the SEC7 domain of ARF guanine-nucleotide exchange factors (ARF-GEFs), resulting in the recruitment of coat proteins by GTP-bound ARFs. In vitro exchange assays were done with monomeric proteins, although ARF-GEFs form dimers in vivo. This feature is conserved across eukaryotes, although its biological significance is unknown. Here, we demonstrate the proximity of ARF1•GTPs in vivo by fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy, mediated through coordinated activation by dimers of Arabidopsis (Arabidopsis thaliana) ARF-GEF GNOM, which is involved in polar recycling of the auxin transporter PIN-FORMED1. Mutational disruption of ARF1 spacing interfered with ARF1-dependent trafficking but not with coat protein recruitment. A mutation impairing the interaction of one of the two SEC7 domains of the GNOM ARF-GEF dimer with its ARF1 substrate reduced the efficiency of coordinated ARF1 activation. Our results suggest a model of coordinated activation-dependent membrane insertion of ARF1•GTP molecules required for coated membrane vesicle formation. Considering the evolutionary conservation of ARFs and ARF-GEFs, this initial regulatory step of membrane trafficking might well occur in eukaryotes in general.

Stochastic thermodynamics and modes of operation of a ribosome: A network theoretic perspective.

The ribosome is one of the largest and most complex macromolecular machines in living cells. It polymerizes a protein in a step-by-step manner as directed by the corresponding nucleotide sequence on the template messenger RNA (mRNA) and this process is referred to as "translation" of the genetic message encoded in the sequence of mRNA transcript. In each successful chemomechanical cycle during the (protein) elongation stage, the ribosome elongates the protein by a single subunit, called amino acid, and steps forward on the template mRNA by three nucleotides called a codon. Therefore, a ribosome is also regarded as a molecular motor for which the mRNA serves as the track, its step size is that of a codon and two molecules of GTP and one molecule of ATP hydrolyzed in that cycle serve as its fuel. What adds further complexity is the existence of competing pathways leading to distinct cycles, branched pathways in each cycle, and futile consumption of fuel that leads neither to elongation of the nascent protein nor forward stepping of the ribosome on its track. We investigate a model formulated in terms of the network of discrete chemomechanical states of a ribosome during the elongation stage of translation. The model is analyzed using a combination of stochastic thermodynamic and kinetic analysis based on a graph-theoretic approach. We derive the exact solution of the corresponding master equations. We represent the steady state in terms of the cycles of the underlying network and discuss the energy transduction processes. We identify the various possible modes of operation of a ribosome in terms of its average velocity and mean rate of GTP hydrolysis. We also compute entropy production as functions of the rates of the interstate transitions and the thermodynamic cost for accuracy of the translation process.

Structure of the active GGEEF domain of a diguanylate cyclase from Vibrio cholerae

Cyclic-di-GMP (c-di-GMP) synthesized by diguanylate cyclases has been an important and ubiquitous secondary messenger in almost all bacterial systems. In Vibrio cholerae, c-di-GMP plays an intricate role in the production of the exopolysaccharide matrix, and thereby, in biofilm formation. The formation of the surface biofilm enables the bacteria to survive in aquatic bodies, when not infecting a human host. Diguanylate cyclases are the class of enzymes which synthesize c-di-GMP from two molecules of GTP and are endowed with a GGDEF or, a GGEEF signature domain. The VC0395_0300 protein from V. cholerae, has been established as a diguanylate cyclase with a necessary role in biofilm formation. Here we present the structure of an N-terminally truncated form of VC0395_0300, which retains the active GGEEF domain for diguanylate cyclase activity but lacks 160 residues from the poorly organized N-terminal domain. X-ray diffraction data was collected from a crystal of VC0395_0300(161-321) to a resolution of 1.9 Å. The structure displays remarkable topological similarity with diguanylate cyclases from other bacterial systems, but lacks the binding site for c-di-GMP present in its homologues. Finally, we demonstrate the ability of the truncated diguanylate cyclase VC0395_0300(161-321) to produce c-di-GMP, and its role in biofilm formation for the bacteria.

C-di-GMP Arms an Anti-σ to Control Progression of Multicellular Differentiation in Streptomyces.

SummaryStreptomyces are our primary source of antibiotics, produced concomitantly with the transition from vegetative growth to sporulation in a complex developmental life cycle. We previously showed that the signaling molecule c-di-GMP binds BldD, a master repressor, to control initiation of development. Here we demonstrate that c-di-GMP also intervenes later in development to control differentiation of the reproductive hyphae into spores by arming a novel anti-σ (RsiG) to bind and sequester a sporulation-specific σ factor (σWhiG). We present the structure of the RsiG-(c-di-GMP)2-σWhiG complex, revealing an unusual, partially intercalated c-di-GMP dimer bound at the RsiG-σWhiG interface. RsiG binds c-di-GMP in the absence of σWhiG, employing a novel E(X)3S(X)2R(X)3Q(X)3D motif repeated on each helix of a coiled coil. Further studies demonstrate that c-di-GMP is essential for RsiG to inhibit σWhiG. These findings reveal a newly described control mechanism for σ-anti-σ complex formation and establish c-di-GMP as the central integrator of Streptomyces development.

Navigating oxygen deprivation: liver transcriptomic responses of the red eared slider turtle to environmental anoxia.

The best facultative anaerobes among vertebrates are members of the genera Trachemys (pond slider turtles) and Chrysemys (painted turtles), and are able to survive without oxygen for up to 12 to 18 weeks at ∼3 °C. In this study, we utilized RNAseq to profile the transcriptomic changes that take place in response to 20 hrs of anoxia at 5 °C in the liver of the red eared slide turtle (Trachemys scripta elegans). Sequencing reads were obtained from at least 18,169 different genes and represented a minimum 49x coverage of the C. picta bellii exome. A total of 3,105 genes showed statistically significant changes in gene expression between the two animal groups, of which 971 also exhibited a fold change equal to or greater than 50% of control normoxic values. This study also highlights a number of anoxia-responsive molecular pathways that are may be important to navigating anoxia survival. These pathways were enriched in mRNA found to significantly increase in response to anoxia and included molecular processes such as DNA damage repair and metabolic reprogramming. For example, our results indicate that the anoxic turtle may utilize succinate metabolism to yield a molecule of GTP in addition to the two molecules that results from lactate production, and agrees with other established models of anoxia tolerance. Collectively, our analysis provides a snapshot of the molecular landscape of the anoxic turtle and may provide hints into the how this animal is capable of surviving this extreme environmental stress.

Overexpression of the diguanylate cyclase CdgD blocks developmental transitions and antibiotic biosynthesis in Streptomyces coelicolor.

Cyclic dimeric GMP (c-di-GMP) has emerged as the nucleotide second messenger regulating both development and antibiotic production in high-GC, Gram-positive streptomycetes. Here, a diguanylate cyclase (DGC), CdgD, encoded by SCO5345 from the model strain Streptomyces coelicolor, was functionally identified and characterized to be involved in c-di-GMP synthesis through genetic and biochemical analysis. cdgD overexpression resulted in significantly reduced production of actinorhodin and undecylprodigiosin, as well as completely blocked sporulation or aerial mycelium formation on two different solid media. In the cdgD-overexpression strain, intracellular c-di-GMP levels were 13-27-fold higher than those in the wild-type strain. In vitro enzymatic assay demonstrated that CdgD acts as a DGC, which could efficiently catalyze the synthesis of c-di-GMP from two GTP molecules. Heterologous overproduction of cdgD in two industrial Streptomyces strains could similarly impair developmental transitions as well as antibiotic biosynthesis. Collectively, our results combined with previously reported data clearly demonstrated that c-di-GMP-mediated signalling pathway plays a central and universal role in the life cycle as well as secondary metabolism in streptomycetes.

In silico study of a functional mutation associated with non-small cell lung cancer: G12D mutation of exon 2 in KRAS gene

Biology flourished during the XXth century and was profoundly disrupted during the last decade because of the transition to the post-genomic era, the spread of high-throughput biology, and the advent of a relatively new discipline, namely bioinformatics. This latter, which encompasses the collection, organization and analysis of biological data using the computer tool, has quickly become inseparable from the studies related to the genome understanding. The consequences of the different mutations that may affect our genes are responsible for a change in the protein sequence and are likely to affect, for example, the stability of the protein, its intracellular targeting, its maturation, its assembly in a multimeric structure, the essential sites for its enzymatic activity or for the interaction with ligands. Thus, a number of bioinformatic developments have made it possible to set up in silico prediction tools of the structure of a protein that is aiming at predicting the impact of local mutations on the structure of proteins. Throughout our study, we have been interested in exploring, through in silico bioinformatic study, three analytical, prediction and modeling, software, choosing as exemple the G12D mutation that affects the proto-oncogene KRAS found in numerous algerian patients with bronchopulmonary cancers cells (NSCLC). This study allowed us to integrate these bioinformatic tools into our laboratory of developmental biology and LBDD differentiation at the University of Oran 1 Ahmed Benbella, in Algeria. Thus, we have been able to conclude, even if the found mutation is predicted to be tolerated and has no deleterious effect on the entire Ras protein, that the consequence of this missense mutation depends mainly on the position in the protein and the chemical properties of the amino acid involved in the substitution and which shows a strong affinity with the GTP molecule.

Approved drugs screening against the nsP1 capping enzyme of Venezuelan equine encephalitis virus using an immuno-based assay

Alphaviruses such as the Venezuelan equine encephalitis virus (VEEV) are important human emerging pathogens transmitted by mosquitoes. They possess a unique viral mRNA capping mechanism catalyzed by the viral non-structural protein nsP1, which is essential for virus replication. The alphaviruses capping starts by the methylation of a GTP molecule by the N7-guanine methyltransferase (MTase) activity; nsP1 then forms a covalent link with m7GMP releasing pyrophosphate (GT reaction) and the m7GMP is next transferred onto the 5′-diphosphate end of the viral mRNA to form a cap-0 structure. The cap-0 structure decreases the detection of foreign viral RNAs, prevents RNA degradation by cellular exonucleases, and promotes viral RNA translation into proteins. Additionally, reverse-genetic studies have demonstrated that viruses mutated in nsP1 catalytic residues are both impaired towards replication and attenuated. The nsP1 protein is thus considered an attractive antiviral target for drug discovery. We have previously demonstrated that the guanylylation of VEEV nsP1 can be monitored by Western blot analysis using an antibody recognizing the cap structure. In this study, we developed a high throughput ELISA screening assay to monitor the GT reaction through m7GMP-nsP1 adduct quantitation. This assay was validated using known nsP1 inhibitors before screening 1220 approved compounds. 18 compounds inhibiting the nsP1 guanylylation were identified, and their IC50 determined. Compounds from two series were further characterized and shown to inhibit the nsP1 MTase activity. Conversely, these compounds barely inhibited a cellular MTase demonstrating their specificity towards nsP1. Analogues search and SAR were also initiated to identify the active pharmacophore features. Altogether the results show that this HT enzyme-based assay is a convenient way to select potent and specific hit compounds targeting the viral mRNA capping of Alphaviruses.

Structural characterization of ribT from Bacillus subtilis reveals it as a GCN5-related N-acetyltransferase.

In bacteria, biosynthesis of riboflavin occurs through a series of enzymatic steps starting with one molecule of GTP and two molecules of ribulose-5-phosphate. In Bacillus subtilis (B. subtilis) the genes (ribD/G, ribE, ribA, ribH and ribT) which are involved in riboflavin biosynthesis are organized in an operon referred as rib operon. All the genes of rib operon are characterized functionally except for ribT. The ribT gene with unknown function is found at the distal terminal of rib operon and annotated as a putative N-acetyltransferase. Here, we report the crystal structure of ribT from B. subtilis (bribT) complexed with coenzyme A (CoA) at 2.1 Å resolution determined by single wavelength anomalous dispersion method. Our structural study reveals that bribT is a member of GCN5-related N-acetyltransferase (GNAT) superfamily and contains all the four conserved structural motifs that have been in other members of GNAT superfamily. The members of GNAT family transfers the acetyl group from acetyl coenzyme A (AcCoA) to a variety of substrates. Moreover, the structural analysis reveals that the residues Glu-67 and Ser-107 are suitably positioned to act as a catalytic base and catalytic acid respectively suggesting that the catalysis by bribT may follow a direct transfer mechanism. Surprisingly, the mutation of a non-conserved amino acid residue Cys-112 to alanine or serine affected the binding of AcCoA to bribT, indicating a possible role of Cys-112 in the catalysis.

Explaining the Microtubule Energy Balance: Contributions Due to Dipole Moments, Charges, van der Waals and Solvation Energy.

Microtubules are the main components of mitotic spindles, and are the pillars of the cellular cytoskeleton. They perform most of their cellular functions by virtue of their unique dynamic instability processes which alternate between polymerization and depolymerization phases. This in turn is driven by a precise balance between attraction and repulsion forces between the constituents of microtubules (MTs)—tubulin dimers. Therefore, it is critically important to know what contributions result in a balance of the interaction energy among tubulin dimers that make up microtubules and what interactions may tip this balance toward or away from a stable polymerized state of tubulin. In this paper, we calculate the dipole–dipole interaction energy between tubulin dimers in a microtubule as part of the various contributions to the energy balance. We also compare the remaining contributions to the interaction energies between tubulin dimers and establish a balance between stabilizing and destabilizing components, including the van der Waals, electrostatic, and solvent-accessible surface area energies. The energy balance shows that the GTP-capped tip of the seam at the plus end of microtubules is stabilized only by kcal/mol, which can be completely reversed by the hydrolysis of a single GTP molecule, which releases kcal/mol and destabilizes the seam by an excess of kcal/mol. This triggers the breakdown of microtubules and initiates a disassembly phase which is aptly called a catastrophe.

Crystal Structure and Thermostability Characterization of Enterovirus D68 3Dpol.

Enterovirus D68 (EV-D68) is one of the many nonpolio enteroviruses that cause mild to severe respiratory illness. The nonstructural protein 3Dpol is an RNA-dependent RNA polymerase (RdRP) of EV-D68 which plays a critical role in the replication of the viral genome and represents a promising drug target. Here, we report the first three-dimensional crystal structure of the RdRP from EV-D68 in complex with the substrate GTP to 2.3-Å resolution. The RdRP structure is similar to structures of other viral RdRPs, where the three domains, termed the palm, fingers, and thumb, form a structure resembling a cupped right hand. Particularly, an N-terminal fragment (Gly1 to Phe30) bridges the fingers and the thumb domains, which accounts for the enhanced stability of the full-length enzyme over the truncation mutant, as assessed by our thermal shift assays and the dynamic light scattering studies. Additionally, the GTP molecule bound proximal to the active site interacts with both the palm and fingers domains to stabilize the core structure of 3Dpol Interestingly, using limited proteolysis assays, we found that different nucleoside triphosphates (NTPs) stabilize the polymerase structure by various degrees, with GTP and CTP being the most and least stabilizing nucleosides, respectively. Lastly, we derived a model of the core structure of 3Dpol stabilized by GTP, according to our proteolytic studies. The biochemical and biophysical characterizations conducted in this study help us to understand the stability of EV-D68-3Dpol, which may extend to other RdRPs as well.IMPORTANCE Enterovirus D68 (EV-D68) is an emerging viral pathogen, which caused sporadic infections around the world. In recent years, epidemiology studies have reported an increasing number of patients with respiratory diseases globally due to the EV-D68 infection. Moreover, the infection has been associated with acute flaccid paralysis and cranial nerve dysfunction in children. However, there are no vaccines and antiviral treatments specifically targeting the virus to date. In this study, we solved the crystal structure of the RNA-dependent RNA polymerase of EV-D68 and carried out systematic biophysical and biochemical characterizations on the overall and local structural stability of the wild-type (WT) enzyme and several variants, which yields a clear view on the structure-activity relationship of the EV-D68 RNA polymerase.

Abstract 1246: Development of a novel KRAS-targeting agent: systematic validation using in silico, in solution, cell models, PDX and transgenic mouse models

Abstract Aberrant KRAS signaling plays an important role in the pathogenesis of malignancies including pancreatic (PDAC), colon and lung cancer. Because therapies targeting either downstream or associated factors of KRAS pathway have shown dismal results in clinics, there is a need to identify effective agents targeting active KRAS (GTP-KRAS) protein. In this study, we screened a chemical library for their efficacy to inhibit KRAS activity using in silico methods. Based on their binding energy, we selected a candidate agent (LP1) that exhibited high potential of binding to KRAS protein at a region where KRAS-activator protein “SOS1” binds to, and GDP-GTP exchange takes place. We next investigated if LP1 exhibits the efficacy of KRAS binding in biological solution using recombinant proteins and employing drug development techniques i.e., florescence (FL)-based competition, Isothermal Titration Calorimetry (ITC) and Surface Plasmon Resonance (SPR) assays. The FL-based study show that LP1 inhibits the binding of SOS1 protein and GTP molecules to KRAS protein. The ITC and SPR analysis showed that LP1 binds to the KRAS protein (at 10 μM). We next determined the KRAS-inhibitory potential of LP1 in activated-KRAS cells representing premalignant and malignant models of PDAC, colon and lung cancer. Dissociation study involving incubation of KRAS/SOS1 proteins showed the LP1 inhibits the binding of SOS1 to KRAS. Notably, LP1 therapy significantly decreased the (i) KRAS-GTP protein levels (ii) growth and (iii) proliferation of activated-KRAS single cell and 3D Organoid cultures. We next tested KRAS-inhibitory efficacy of LP1 in PDAC patient-derived (PD) tumor explants and found that LP1 therapy (10 days) significantly decreased growth of, and KRAS-GTP levels in PD explants. Based on the in silico, in solution, cell model and PD-explant data, we determined the efficacy of LP1 as a KRAS inhibitor in in vivo conditions. We first performed the pharmacokinetic study of LP1 in mice and observed that LP1 is physiologically available in the blood (peak of 15-20 μM) after oral and intraperitoneal administrations and detectable up to 24h post-administration. We next tested LP1 therapy in tumor xenograft models and show that LP1 therapy significantly reduces the tumorigenicity of KRAS-activated PDAC and colon cell-derived tumors implanted in athymic mice. We next tested efficacy of LP1 administration on the developmental phases of PDAC disease (from normal to high grade neoplasia/early stage carcinoma) and show that LP1 feeding for six months caused a significant inhibition of PanIN development in KPCG12D transgenic mouse model. Notably the analysis of tumors xenograft models and pancreatic tissues of KPCG12D mice receiving LP1 therapy exhibited reduced KRAS-GTP levels. Taken together, these data show that LP1 is a potent KRAS activity inhibitor and a potential candidate for clinical use against KRAS-driven cancers. Citation Format: Arsheed Ahmad Ganaie, Hifzur R. Siddique, Ishfaq Sheikh, Lei Wang, Aijaz Parray, Jayanth panyam, Peter Villalta, Joshua Liao, Yibin deng, Mohammad saleem. Development of a novel KRAS-targeting agent: systematic validation using in silico, in solution, cell models, PDX and transgenic mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1246. doi:10.1158/1538-7445.AM2017-1246