Structural Studies of Inhibitors with Clinically Relevant Influenza Endonuclease Variants

Vital to the treatment of influenza is the use of antivirals such as Oseltamivir (Tamiflu) and Zanamivir (Relenza); however, antiviral resistance is becoming an increasing problem for these therapeutics. The RNA-dependent RNA polymerase acidic N-terminal (PAN) endonuclease, a critical component of influenza viral replication machinery, is an antiviral target that was recently validated with the approval of Baloxavir Marboxil (BXM). Despite its clinical success, BXM has demonstrated susceptibility to resistance mutations, specifically the I38T, E23K, and A36 V mutants of PAN. To better understand the effects of these mutations on BXM resistance and improve the design of more robust therapeutics, this study examines key differences in protein–inhibitor interactions with two inhibitors and the I38T, E23K, and A36 V mutants. Differences in inhibitor binding were evaluated by measuring changes in binding to PAN using two biophysical methods. The binding mode of two distinct inhibitors was determined crystallographically with both wild-type and mutant forms of PAN. Collectively, these studies give some insight into the mechanism of antiviral resistance of these mutants

Reorientation of the Methyl Group in MAs(III) is the Rate-Limiting Step in the ArsM As(III) <i>S</i>‑Adenosylmethionine Methyltransferase Reaction

The most common biotransformation of trivalent inorganic arsenic (As­(III)) is methylation to mono-, di-, and trimethylated species. Methylation is catalyzed by As­(III) <i>S</i>-adenosylmethionine (SAM) methyltransferase (termed ArsM in microbes and AS3MT in animals). Methylarsenite (MAs­(III)) is both the product of the first methylation step and the substrate of the second methylation step. When the rate of the overall methylation reaction was determined with As­(III) as the substrate, the first methylation step was rapid, whereas the second methylation step was slow. In contrast, when MAs­(III) was used as the substrate, the rate of methylation was as fast as the first methylation step when As­(III) was used as the substrate. These results indicate that there is a slow conformational change between the first and second methylation steps. The structure of CmArsM from the thermophilic alga Cyanidioschyzon merolae sp. 5508 was determined with bound MAs­(III) at 2.27 Å resolution. The methyl group is facing the solvent, as would be expected when MAs­(III) is bound as the substrate rather than facing the SAM-binding site, as would be expected for MAs­(III) as a product. We propose that the rate-limiting step in arsenic methylation is slow reorientation of the methyl group from the SAM-binding site to the solvent, which is linked to the conformation of the side chain of a conserved residue Tyr70

Structure of an As(III) <i>S</i>-Adenosylmethionine Methyltransferase: Insights into the Mechanism of Arsenic Biotransformation

Enzymatic methylation of arsenic is a detoxification process in microorganisms but in humans may activate the metalloid to more carcinogenic species. We describe the first structure of an As­(III) <i>S</i>-adenosylmethionine methyltransferase by X-ray crystallography that reveals a novel As­(III) binding domain. The structure of the methyltransferase from the thermophilic eukaryotic alga <i>Cyanidioschyzon merolae</i> reveals the relationship between the arsenic and <i>S</i>-adenosylmethionine binding sites to a final resolution of ∼1.6 Å. As­(III) binding causes little change in conformation, but binding of SAM reorients helix α4 and a loop (residues 49–80) toward the As­(III) binding domain, positioning the methyl group for transfer to the metalloid. There is no evidence of a reductase domain. These results are consistent with previous suggestions that arsenic remains trivalent during the catalytic cycle. A homology model of human As­(III) <i>S</i>-adenosylmethionine methyltransferase with the location of known polymorphisms was constructed. The structure provides insights into the mechanism of substrate binding and catalysis

Structure of an As(III) <i>S</i>-Adenosylmethionine Methyltransferase: Insights into the Mechanism of Arsenic Biotransformation

Enzymatic methylation of arsenic is a detoxification process in microorganisms but in humans may activate the metalloid to more carcinogenic species. We describe the first structure of an As­(III) <i>S</i>-adenosylmethionine methyltransferase by X-ray crystallography that reveals a novel As­(III) binding domain. The structure of the methyltransferase from the thermophilic eukaryotic alga <i>Cyanidioschyzon merolae</i> reveals the relationship between the arsenic and <i>S</i>-adenosylmethionine binding sites to a final resolution of ∼1.6 Å. As­(III) binding causes little change in conformation, but binding of SAM reorients helix α4 and a loop (residues 49–80) toward the As­(III) binding domain, positioning the methyl group for transfer to the metalloid. There is no evidence of a reductase domain. These results are consistent with previous suggestions that arsenic remains trivalent during the catalytic cycle. A homology model of human As­(III) <i>S</i>-adenosylmethionine methyltransferase with the location of known polymorphisms was constructed. The structure provides insights into the mechanism of substrate binding and catalysis

Structural Basis of Analog Specificity in PKG I and II

Cyclic GMP analogs, 8-Br, 8-pCPT, and PET-cGMP, have been widely used for characterizing cellular functions of cGMP-dependent protein kinase (PKG) I and II isotypes. However, interpreting results obtained using these analogs has been difficult due to their low isotype specificity. Additionally, each isotype has two binding sites with different cGMP affinities and analog selectivities, making understanding the molecular basis for isotype specificity of these compounds even more challenging. To determine isotype specificity of cGMP analogs and their structural basis, we generated the full-length regulatory domains of PKG I and II isotypes with each binding site disabled, determined their affinities for these analogs, and obtained cocrystal structures of both isotypes bound with cGMP analogs. Our affinity and activation measurements show that PET-cGMP is most selective for PKG I, whereas 8-pCPT-cGMP is most selective for PKG II. Our structures of cyclic nucleotide binding (CNB) domains reveal that the B site of PKG I is more open and forms a unique π/π interaction through Arg285 at β4 with the PET moiety, whereas the A site of PKG II has a larger β5/β6 pocket that can better accommodate the bulky 8-pCPT moiety. Our structural and functional results explain the selectivity of these analogs for each PKG isotype and provide a starting point for the rational design of isotype selective activators

Additional file 1: Figure S1. of Structure of the catalytic domain of the colistin resistance enzyme MCR-1

Amino acid sequence alignment of phosphoethanolamine transferases MCR-1, EptC (C. jejuni), and LptA (N. meningitidis). (TIF 10272 kb

Carboxylic Acid Isostere Derivatives of Hydroxypyridinones as Core Scaffolds for Influenza Endonuclease Inhibitors

Among the most important influenza virus targets is the RNA-dependent RNA polymerase acidic N-terminal (PAN) endonuclease, which is a critical component of the viral replication machinery. To inhibit the activity of this metalloenzyme, small-molecule inhibitors employ metal-binding pharmacophores (MBPs) that coordinate to the dinuclear Mn2+ active site. In this study, several metal-binding isosteres (MBIs) were examined where the carboxylic acid moiety of a hydroxypyridinone MBP is replaced with other groups to modulate the physicochemical properties of the compound. MBIs were evaluated for their ability to inhibit PAN using a FRET-based enzymatic assay, and their mode of binding in PAN was determined using X-ray crystallography

Carboxylic Acid Isostere Derivatives of Hydroxypyridinones as Core Scaffolds for Influenza Endonuclease Inhibitors

Among the most important influenza virus targets is the RNA-dependent RNA polymerase acidic N-terminal (PAN) endonuclease, which is a critical component of the viral replication machinery. To inhibit the activity of this metalloenzyme, small-molecule inhibitors employ metal-binding pharmacophores (MBPs) that coordinate to the dinuclear Mn2+ active site. In this study, several metal-binding isosteres (MBIs) were examined where the carboxylic acid moiety of a hydroxypyridinone MBP is replaced with other groups to modulate the physicochemical properties of the compound. MBIs were evaluated for their ability to inhibit PAN using a FRET-based enzymatic assay, and their mode of binding in PAN was determined using X-ray crystallography

Neutron Diffraction Reveals Hydrogen Bonds Critical for cGMP-Selective Activation: Insights for cGMP-Dependent Protein Kinase Agonist Design

High selectivity of cyclic-nucleotide binding (CNB) domains for cAMP and cGMP are required for segregating signaling pathways; however, the mechanism of selectivity remains unclear. To investigate the mechanism of high selectivity in cGMP-dependent protein kinase (PKG), we determined a room-temperature joint X-ray/neutron (XN) structure of PKG Iβ CNB-B, a domain 200-fold selective for cGMP over cAMP, bound to cGMP (2.2 Å), and a low-temperature X-ray structure of CNB-B with cAMP (1.3 Å). The XN structure directly describes the hydrogen bonding interactions that modulate high selectivity for cGMP, while the structure with cAMP reveals that all these contacts are disrupted, explaining its low affinity for cAMP

Implementing Fluorescence Anisotropy Screening and Crystallographic Analysis to Define PKA Isoform-Selective Activation by cAMP Analogs

Cyclic AMP (cAMP) is a ubiquitous second messenger that regulates many proteins, most notably cAMP-dependent protein kinase (PKA). PKA holoenzymes (comprised of two catalytic (C) and two regulatory (R) subunits) regulate a wide variety of cellular processes, and its functional diversity is amplified by the presence of four R-subunit isoforms, RIα, RIβ, RIIα, and RIIβ. Although these isoforms all respond to cAMP, they are functionally nonredundant and exhibit different biochemical properties. In order to understand the functional differences between these isoforms, we screened cAMP derivatives for their ability to selectively activate RI and RII PKA holoenzymes using a fluorescence anisotropy assay. Our results indicate that RIα holoenzymes are selectively activated by C8-substituted analogs and RIIβ holoenzymes by N6-substituted analogs, where HE33 is the most prominent RII activator. We also solved the crystal structures of both RIα and RIIβ bound to HE33. The RIIβ structure shows the bulky aliphatic substituent of HE33 is fully encompassed by a pocket comprising of hydrophobic residues. RIα lacks this hydrophobic lining in Domain A, and the side chains are displaced to accommodate the HE33 dipropyl groups. Comparison between cAMP-bound structures reveals that RIIβ, but not RIα, contains a cavity near the N6 site. This study suggests that the selective activation of RII over RI isoforms by N6 analogs is driven by the spatial and chemical constraints of Domain A and paves the way for the development of potent noncyclic nucleotide activators to specifically target PKA iso-holoenyzmes