Inhibition mechanisms of AcrF9, AcrF8, and AcrF6 against type I-F CRISPR-Cas complex revealed by cryo-EM.

Prokaryotes and viruses have fought a long battle against each other. Prokaryotes use CRISPR-Cas-mediated adaptive immunity, while conversely, viruses evolve multiple anti-CRISPR (Acr) proteins to defeat these CRISPR-Cas systems. The type I-F CRISPR-Cas system in Pseudomonas aeruginosa requires the crRNA-guided surveillance complex (Csy complex) to recognize the invading DNA. Although some Acr proteins against the Csy complex have been reported, other relevant Acr proteins still need studies to understand their mechanisms. Here, we obtain three structures of previously unresolved Acr proteins (AcrF9, AcrF8, and AcrF6) bound to the Csy complex using electron cryo-microscopy (cryo-EM), with resolution at 2.57 Å, 3.42 Å, and 3.15 Å, respectively. The 2.57-Å structure reveals fine details for each molecular component within the Csy complex as well as the direct and water-mediated interactions between proteins and CRISPR RNA (crRNA). Our structures also show unambiguously how these Acr proteins bind differently to the Csy complex. AcrF9 binds to key DNA-binding sites on the Csy spiral backbone. AcrF6 binds at the junction between Cas7.6f and Cas8f, which is critical for DNA duplex splitting. AcrF8 binds to a distinct position on the Csy spiral backbone and forms interactions with crRNA, which has not been seen in other Acr proteins against the Csy complex. Our structure-guided mutagenesis and biochemistry experiments further support the anti-CRISPR mechanisms of these Acr proteins. Our findings support the convergent consequence of inhibiting degradation of invading DNA by these Acr proteins, albeit with different modes of interactions with the type I-F CRISPR-Cas system.

Cryo-EM structures of Helicobacter pylori vacuolating cytotoxin A oligomeric assemblies at near-atomic resolution.

Human gastric pathogen Helicobacter pylori (H. pylori) is the primary risk factor for gastric cancer and is one of the most prevalent carcinogenic infectious agents. Vacuolating cytotoxin A (VacA) is a key virulence factor secreted by H. pylori and induces multiple cellular responses. Although structural and functional studies of VacA have been extensively performed, the high-resolution structure of a full-length VacA protomer and the molecular basis of its oligomerization are still unknown. Here, we use cryoelectron microscopy to resolve 10 structures of VacA assemblies, including monolayer (hexamer and heptamer) and bilayer (dodecamer, tridecamer, and tetradecamer) oligomers. The models of the 88-kDa full-length VacA protomer derived from the near-atomic resolution maps are highly conserved among different oligomers and show a continuous right-handed ß-helix made up of two domains with extensive domain-domain interactions. The specific interactions between adjacent protomers in the same layer stabilizing the oligomers are well resolved. For double-layer oligomers, we found short- and/or long-range hydrophobic interactions between protomers across the two layers. Our structures and other previous observations lead to a mechanistic model wherein VacA hexamer would correspond to the prepore-forming state, and the N-terminal region of VacA responsible for the membrane insertion would undergo a large conformational change to bring the hydrophobic transmembrane region to the center of the oligomer for the membrane channel formation.

Structural basis of subunit selectivity for competitive NMDA receptor antagonists with preference for GluN2A over GluN2B subunits.

NMDA-type glutamate receptors are ligand-gated ion channels that contribute to excitatory neurotransmission in the central nervous system (CNS). Most NMDA receptors comprise two glycine-binding GluN1 and two glutamate-binding GluN2 subunits (GluN2A-D). We describe highly potent (S)-5-[(R)-2-amino-2-carboxyethyl]-4,5-dihydro-1H-pyrazole-3-carboxylic acid (ACEPC) competitive GluN2 antagonists, of which ST3 has a binding affinity of 52 nM at GluN1/2A and 782 nM at GluN1/2B receptors. This 15-fold preference of ST3 for GluN1/2A over GluN1/2B is improved compared with NVP-AAM077, a widely used GluN2A-selective antagonist, which we show has 11-fold preference for GluN1/2A over GluN1/2B. Crystal structures of the GluN1/2A agonist binding domain (ABD) heterodimer with bound ACEPC antagonists reveal a binding mode in which the ligands occupy a cavity that extends toward the subunit interface between GluN1 and GluN2A ABDs. Mutational analyses show that the GluN2A preference of ST3 is primarily mediated by four nonconserved residues that are not directly contacting the ligand, but positioned within 12 Å of the glutamate binding site. Two of these residues influence the cavity occupied by ST3 in a manner that results in favorable binding to GluN2A, but occludes binding to GluN2B. Thus, we reveal opportunities for the design of subunit-selective competitive NMDA receptor antagonists by identifying a cavity for ligand binding in which variations exist between GluN2A and GluN2B subunits. This structural insight suggests that subunit selectivity of glutamate-site antagonists can be mediated by mechanisms in addition to direct contributions of contact residues to binding affinity.

Effect of a K72A Mutation on the Structure, Stability, Dynamics, and Peroxidase Activity of Human Cytochrome c.

We test the hypothesis that Lys72 suppresses the intrinsic peroxidase activity of human cytochrome c, as observed previously for yeast iso-1-cytochrome c [McClelland, L. J., et al. (2014) Proc. Natl. Acad. Sci. U. S. A. 111, 6648-6653]. A 1.25 Å X-ray structure of K72A human cytochrome c shows that the mutation minimally affects structure. Guanidine hydrochloride denaturation demonstrates that the K72A mutation increases global stability by 0.5 kcal/mol. The K72A mutation also increases the apparent pKa of the alkaline transition, a measure of the stability of the heme crevice, by 0.5 unit. Consistent with the increase in the apparent pKa, the rate of formation of the dominant alkaline conformer decreases, and this conformer is no longer stabilized by proline isomerization. Peroxidase activity measurements show that the K72A mutation increases kcat by 1.6-4-fold at pH 7-10, an effect larger than that seen for the yeast protein. X-ray structures of wild type and K72A human cytochrome c indicate that direct interactions of Lys72 with the far side of Ω-loop D, which are seen in X-ray structures of horse and yeast cytochrome c and could suppress peroxidase activity, are lacking. Instead, we propose that the stronger effect of the K72A mutation on the peroxidase activity of human versus yeast cytochrome c results from relief of steric interactions between the side chains at positions 72 and 81 (Ile in human vs Ala in yeast), which suppress the dynamics of Ω-loop D necessary for the intrinsic peroxidase activity of cytochrome c.

Structure of a mitochondrial cytochrome c conformer competent for peroxidase activity.

At the onset of apoptosis, the peroxidation of cardiolipin at the inner mitochondrial membrane by cytochrome c requires an open coordination site on the heme. We report a 1.45-Å resolution structure of yeast iso-1-cytochrome c with the Met80 heme ligand swung out of the heme crevice and replaced by a water molecule. This conformational change requires modest adjustments to the main chain of the heme crevice loop and is facilitated by a trimethyllysine 72-to-alanine mutation. This mutation also enhances the peroxidase activity of iso-1-cytochrome c. The structure shows a buried water channel capable of facilitating peroxide access to the active site and of moving protons produced during peroxidase activity to the protein surface. Alternate positions of the side chain of Arg38 appear to mediate opening and closing of the buried water channel. In addition, two buried water molecules can adopt alternate positions that change the network of hydrogen bonds in the buried water channel. Taken together, these observations suggest that low and high proton conductivity states may mediate peroxidase function. Comparison of yeast and mammalian cytochrome c sequences, in the context of the steric factors that permit opening of the heme crevice, suggests that higher organisms have evolved to inhibit peroxidase activity, providing a more stringent barrier to the onset of apoptosis.

Cytochrome c Can Form a Well-Defined Binding Pocket for Hydrocarbons.

Cytochrome c can acquire peroxidase activity when it binds to cardiolipin in mitochondrial membranes. The resulting oxygenation of cardiolipin by cytochrome c provides an early signal for the onset of apoptosis. The structure of this enzyme-substrate complex is a matter of considerable debate. We present three structures at 1.7-2.0 Å resolution of a domain-swapped dimer of yeast iso-1-cytochrome c with the detergents, CYMAL-5, CYMAL-6, and ω-undecylenyl-ß-d-maltopyranoside, bound in a channel that places the hydrocarbon moieties of these detergents next to the heme. The heme is poised for peroxidase activity with water bound in place of Met80, which serves as the axial heme ligand when cytochrome c functions as an electron carrier. The hydroxyl group of Tyr67 sits 3.6-4.0 Å from the nearest carbon of the detergents, positioned to act as a relay in radical abstraction during peroxidase activity. Docking studies with linoleic acid, the most common fatty acid component of cardiolipin, show that C11 of linoleic acid can sit adjacent to Tyr67 and the heme, consistent with the oxygenation pattern observed in lipidomics studies. The well-defined hydrocarbon binding pocket provides atomic resolution evidence for the extended lipid anchorage model for cytochrome c/cardiolipin binding. Dimer dissociation/association kinetics for yeast versus equine cytochrome c indicate that formation of mammalian cytochrome c dimers in vivo would require catalysis. However, the dimer structure shows that only a modest deformation of monomeric cytochrome c would suffice to form the hydrocarbon binding site occupied by these detergents.

Discovery of Potent and Selective Covalent Inhibitors of HER2WT and HER2YVMA.

HER2 overexpression and amplification have been identified as oncogenic drivers, and the development of therapies to treat tumors harboring these markers has received considerable attention. Activation of HER2 signaling and subsequent cell growth can also be induced by HER2 mutations, including the common YVMA insertion in exon 20 within the kinase domain. Enhertu is currently the only approved treatment for HER2 mutant tumors in NSCLC. TKIs tested in this space have suffered from off-target activity, primarily due to EGFRWT inhibition or attenuated activity against HER2 mutants. The goal of this work was to identify a TKI that would provide robust inhibition of oncogenic HER2WT and HER2 mutants while sparing EGFRWT activity. Herein, we describe the development of a potent, covalent inhibitor of HER2WT and the YVMA insertion mutant while providing oral bioavailability and avoiding the inhibition of EGFRWT.

A next-generation BRAF inhibitor overcomes resistance to BRAF inhibition in patients with BRAF-mutant cancers using pharmacokinetics-informed dose escalation.

RAF inhibitors have transformed treatment for BRAF V600-mutant cancer patients, but clinical benefit is limited by adaptive induction of ERK signaling, genetic alterations that induce BRAF V600 dimerization, and poor brain penetration. Next-generation pan-RAF dimer inhibitors are limited by narrow therapeutic index. PF-07799933 (ARRY-440) is a brain-penetrant, selective, pan-mutant BRAF inhibitor. PF-07799933 inhibited signaling in vitro, disrupted endogenous mutant-BRAF:wild-type-CRAF dimers, and spared wild-type ERK signaling. PF-07799933 ± binimetinib inhibited growth of mouse xenograft tumors driven by mutant BRAF that functions as dimers and by BRAF V600E with acquired resistance to current RAF inhibitors. We treated patients with treatment-refractory BRAF-mutant solid tumors in a first-in-human clinical trial (NCT05355701) that utilized a novel, flexible, pharmacokinetics-informed dose escalation design that allowed rapid achievement of PF-07799933 efficacious concentrations. PF-07799933 ± binimetinib was well-tolerated and resulted in multiple confirmed responses, systemically and in the brain, in BRAF-mutant cancer patients refractory to approved RAF inhibitors.

Application of sulfur SAD to small crystals with a large asymmetric unit and anomalous substructure.

The application of sulfur single-wavelength anomalous dispersion (S-SAD) to determine the crystal structures of macromolecules can be challenging if the asymmetric unit is large, the crystals are small, the size of the anomalously scattering sulfur structure is large and the resolution at which the anomalous signals can be accurately measured is modest. Here, as a study of such a case, approaches to the SAD phasing of orthorhombic Ric-8A crystals are described. The structure of Ric-8A was published with only a brief description of the phasing process [Zeng et al. (2019), Structure, 27, 1137-1141]. Here, alternative approaches to determining the 40-atom sulfur substructure of the 103 kDa Ric-8A dimer that composes the asymmetric unit are explored. At the data-collection wavelength of 1.77 Šmeasured at the Frontier micro-focusing Macromolecular Crystallography (FMX) beamline at National Synchrotron Light Source II, the sulfur anomalous signal strength, |Δano|/σΔano (d''/sig), approaches 1.4 at 3.4 Šresolution. The highly redundant, 11 000 000-reflection data set measured from 18 crystals was segmented into isomorphous clusters using BLEND in the CCP4 program suite. Data sets within clusters or sets of clusters were scaled and merged using AIMLESS from CCP4 or, alternatively, the phenix.scale_and_merge tool from the Phenix suite. The latter proved to be the more effective in extracting anomalous signals. The HySS tool in Phenix, SHELXC/D and PRASA as implemented in the CRANK2 program suite were each employed to determine the sulfur substructure. All of these approaches were effective, although HySS, as a component of the phenix.autosol tool, required data from all crystals to find the positions of the sulfur atoms. Critical contributors in this case study to successful phase determination by SAD included (i) the high-flux FMX beamline, featuring helical-mode data collection and a helium-filled beam path, (ii) as recognized by many authors, a very highly redundant, multiple-crystal data set and (iii) the inclusion within that data set of data from crystals that were scanned over large ω ranges, yielding highly isomorphous and highly redundant intensity measurements.

Structural basis for the high-affinity inhibition of mammalian membranous adenylyl cyclase by 2',3'-o-(N-methylanthraniloyl)-inosine 5'-triphosphate.

2',3'-O-(N-Methylanthraniloyl)-ITP (MANT-ITP) is the most potent inhibitor of mammalian membranous adenylyl cyclase (mAC) 5 (AC5, K(i), 1 nM) yet discovered and surpasses the potency of MANT-GTP by 55-fold (J Pharmacol Exp Ther 329:1156-1165, 2009). AC5 inhibitors may be valuable drugs for treatment of heart failure. The aim of this study was to elucidate the structural basis for the high-affinity inhibition of mAC by MANT-ITP. MANT-ITP was a considerably more potent inhibitor of the purified catalytic domains VC1 and IIC2 of mAC than MANT-GTP (K(i), 0.7 versus 18 nM). Moreover, there was considerably more efficient fluorescence resonance energy transfer between Trp1020 of IIC2 and the MANT group of MANT-ITP compared with MANT-GTP, indicating optimal interaction of the MANT group of MANT-ITP with the hydrophobic pocket. The crystal structure of MANT-ITP in complex with the G(s)α- and forskolin-activated catalytic domains VC1:IIC2 compared with the existing MANT-GTP crystal structure revealed only subtle differences in binding mode. The higher affinity of MANT-ITP to mAC compared with MANT-GTP is probably due to fewer stereochemical constraints upon the nucleotide base in the purine binding pocket, allowing a stronger interaction with the hydrophobic regions of IIC2 domain, as assessed by fluorescence spectroscopy. Stronger interaction is also achieved in the phosphate-binding site. The triphosphate group of MANT-ITP exhibits better metal coordination than the triphosphate group of MANT-GTP, as confirmed by molecular dynamics simulations. Collectively, the subtle differences in ligand structure have profound effects on affinity for mAC.

Stereoselective synthesis of novel 2'-(S)-CCG-IV analogues as potent NMDA receptor agonists.

We developed a versatile stereoselective route for the synthesis of new 2'-(S)-CCG-IV analogues. The route allows for late stage diversification and thereby provides access to a great variety of conformationally restricted cyclopropyl glutamate analogues. A selection of the 2'-(S)-CCG-IV analogues were evaluated using two-electrode voltage-clamp electrophysiology at recombinant GluN1/GluN2A-D receptors, demonstrating that agonists can be developed with GluN2 subunit-dependent potency and agonist efficacy. We also describe a crystal structure of the GluN2A agonist binding domain in complex with 2'-butyl-(S)-CCG-IV that determines the position of 2'-substituents in (S)-CCG-IV agonists in the glutamate binding site and provides further insight to the structural determinants of their agonist efficacy. The stereoselective synthesis described here enables versatile and straight-forward modifications to diverse analogues of interest for the development of potent subtype-specific NMDA receptor agonists and other applications.

The structure of the cysteine-rich region from human histone-lysine N-methyltransferase EHMT2 (G9a).

Euchromatic histone-lysine N-methyltransferase 1 (EHMT1; G9a-like protein; GLP) and euchromatic histone-lysine N-methyltransferase 2 (EHMT2; G9a) are protein lysine methyltransferases that regulate gene expression and are essential for development and the ability of organisms to change and adapt. In addition to ankyrin repeats and the catalytic SET domain, the EHMT proteins contain a unique cysteine-rich region (CRR) that mediates protein-protein interactions and recruitment of the methyltransferases to specific sites in chromatin. We have determined the structure of the CRR from human EHMT2 by X-ray crystallography and show that the CRR adopts an unusual compact fold with four bound zinc atoms. The structure consists of a RING domain preceded by a smaller zinc-binding motif and an N-terminal segment. The smaller zinc-binding motif straddles the N-terminal end of the RING domain, and the N-terminal segment runs in an extended conformation along one side of the structure and interacts with both the smaller zinc-binding motif and the RING domain. The interface between the N-terminal segment and the RING domain includes one of the zinc atoms. The RING domain is partially sequestered within the CRR and unlikely to function as a ubiquitin ligase.

Structure of the G protein chaperone and guanine nucleotide exchange factor Ric-8A bound to Gαi1.

Ric-8A is a cytosolic Guanine Nucleotide exchange Factor (GEF) that activates heterotrimeric G protein alpha subunits (Gα) and serves as an essential Gα chaperone. Mechanisms by which Ric-8A catalyzes these activities, which are stimulated by Casein Kinase II phosphorylation, are unknown. We report the structure of the nanobody-stabilized complex of nucleotide-free Gα bound to phosphorylated Ric-8A at near atomic resolution by cryo-electron microscopy and X-ray crystallography. The mechanism of Ric-8A GEF activity differs considerably from that employed by G protein-coupled receptors at the plasma membrane. Ric-8A engages a specific conformation of Gα at multiple interfaces to form a complex that is stabilized by phosphorylation within a Ric-8A segment that connects two Gα binding sites. The C-terminus of Gα is ejected from its beta sheet core, thereby dismantling the GDP binding site. Ric-8A binds to the exposed Gα beta sheet and switch II to stabilize the nucleotide-free state of Gα.

Structural basis for inhibition of mammalian adenylyl cyclase by calcium.

Type V and VI mammalian adenylyl cyclases (AC5, AC6) are inhibited by Ca(2+) at both sub- and supramicromolar concentration. This inhibition may provide feedback in situations where cAMP promotes opening of Ca(2+) channels, allowing fine control of cardiac contraction and rhythmicity in cardiac tissue where AC5 and AC6 predominate. Ca(2+) inhibits the soluble AC core composed of the C1 domain of AC5 (VC1) and the C2 domain of AC2 (IIC2). As observed for holo-AC5, inhibition is biphasic, showing "high-affinity" (K(i) = approximately 0.4 microM) and "low-affinity" (K(i) = approximately 100 microM) modes of inhibition. At micromolar concentration, Ca(2+) inhibition is nonexclusive with respect to pyrophosphate (PP(i)), a noncompetitive inhibitor with respect to ATP, but at >100 microM Ca(2+), inhibition appears to be exclusive with respect to PP(i). The 3.0 A resolution structure of Galphas.GTPgammaS/forskolin-activated VC1:IIC2 crystals soaked in the presence of ATPalphaS and 8 microM free Ca(2+) contains a single, loosely coordinated metal ion. ATP soaked into VC1:IIC2 crystals in the presence of 1.5 mM Ca(2+) is not cyclized, and two calcium ions are observed in the 2.9 A resolution structure of the complex. In both of the latter complexes VC1:IIC2 adopts the "open", catalytically inactive conformation characteristic of the apoenzyme, in contrast to the "closed", active conformation seen in the presence of ATP analogues and Mg(2+) or Mn(2+). Structures of the pyrophosphate (PP(i)) complex with 10 mM Mg(2+) (2.8 A) or 2 mM Ca(2+) (2.7 A) also adopt the open conformation, indicating that the closed to open transition occurs after cAMP release. In the latter complexes, Ca(2+) and Mg(2+) bind only to the high-affinity "B" metal site associated with substrate/product stabilization. Ca(2+) thus stabilizes the inactive conformation in both ATP- and PP(i)-bound states.

Differential inhibition of various adenylyl cyclase isoforms and soluble guanylyl cyclase by 2',3'-O-(2,4,6-trinitrophenyl)-substituted nucleoside 5'-triphosphates.

Adenylyl cyclases (ACs) catalyze the conversion of ATP into the second messenger cAMP and play a key role in signal transduction. In a recent study (Mol Pharmacol 70:878-886, 2006), we reported that 2',3'-O-(2,4,6-trinitrophenyl)-substituted nucleoside 5'-triphosphates (TNP-NTPs) are potent inhibitors (K(i) values in the 10 nM range) of the purified catalytic subunits VC1 and IIC2 of membranous AC (mAC). The crystal structure of VC1:IIC2 in complex with TNP-ATP revealed that the nucleotide binds to the catalytic site with the TNP-group projecting into a hydrophobic pocket. The aims of this study were to analyze the interaction of TNP-nucleotides with VC1:IIC2 by fluorescence spectroscopy and to analyze inhibition of mAC isoforms, soluble AC (sAC), soluble guanylyl cyclase (sGC), and G-proteins by TNP-nucleotides. Interaction of VC1:IIC2 with TNP-NDPs and TNP-NTPs resulted in large fluorescence increases that were differentially reduced by a water-soluble forskolin analog. TNP-ATP turned out to be the most potent inhibitor for ACV (K(i), 3.7 nM) and sGC (K(i), 7.3 nM). TNP-UTP was identified as the most potent inhibitor for ACI (K(i), 7.1 nM) and ACII (K(i), 24 nM). TNP-NTPs inhibited sAC and GTP hydrolysis by G(s)- and G(i)-proteins only with low potencies. Molecular modeling revealed that TNP-GTP and TNP-ATP interact very similarly, but not identically, with VC1:IIC2. Collectively, our data show that TNP-nucleotides are useful fluorescent probes to monitor conformational changes in VC1:IIC2 and that TNP-NTPs are a promising starting point to develop isoform-selective AC and sGC inhibitors. TNP-ATP is the most potent sGC inhibitor known so far.

Cryo-EM structure of a 40 kDa SAM-IV riboswitch RNA at 3.7 Å resolution.

Specimens below 50 kDa have generally been considered too small to be analyzed by single-particle cryo-electron microscopy (cryo-EM). The high flexibility of pure RNAs makes it difficult to obtain high-resolution structures by cryo-EM. In bacteria, riboswitches regulate sulfur metabolism through binding to the S-adenosylmethionine (SAM) ligand and offer compelling targets for new antibiotics. SAM-I, SAM-I/IV, and SAM-IV are the three most commonly found SAM riboswitches, but the structure of SAM-IV is still unknown. Here, we report the structures of apo and SAM-bound SAM-IV riboswitches (119-nt, ~40 kDa) to 3.7 Å and 4.1 Å resolution, respectively, using cryo-EM. The structures illustrate homologies in the ligand-binding core but distinct peripheral tertiary contacts in SAM-IV compared to SAM-I and SAM-I/IV. Our results demonstrate the feasibility of resolving small RNAs with enough detail to enable detection of their ligand-binding pockets and suggest that cryo-EM could play a role in structure-assisted drug design for RNA.

Structure, Function, and Dynamics of the Gα Binding Domain of Ric-8A.

Ric-8A is a 530-amino acid cytoplasmic molecular chaperone and guanine nucleotide exchange factor (GEF) for i, q, and 12/13 classes of heterortrimeric G protein alpha subunits (Gα). We report the 2.2-Å crystal structure of the Ric-8A Gα-binding domain with GEF activity, residues 1-452, and is phosphorylated at Ser435 and Thr440. Residues 1-429 adopt a superhelical fold comprised of Armadillo (ARM) and HEAT repeats, and the C terminus is disordered. One of the phosphorylated residues potentially binds to a basic cluster in an ARM motif. Amino acid sequence conservation and published hydrogen-deuterium exchange data indicate repeats 3 through 6 to be a putative Gα-binding surface. Normal mode modeling of small-angle X-ray scattering data indicates that phosphorylation induces relative rotation between repeats 1-4, 5-6, and 7-9. 2D 1H-15N-TROSY spectra of [2H,15N]-labeled Gαi1 in the presence of R452 reveals chemical shift perturbations of the C terminus and Gαi1 residues involved in nucleotide binding.

Activation and inhibition of adenylyl cyclase isoforms by forskolin analogs.

Adenylyl cyclase (AC) isoforms 1 to 9 are differentially expressed in tissues and constitute an interesting drug target. ACs 1 to 8 are activated by the diterpene, forskolin (FS). It is unfortunate that there is a paucity of AC isoform-selective activators. To develop such compounds, an understanding of the structure/activity relationships of diterpenes is necessary. Therefore, we examined the effects of FS and nine FS analogs on ACs 1, 2, and 5 expressed in Spodoptera frugiperda insect cells. Diterpenes showed the highest potencies at AC1 and the lowest potencies at AC2. We identified full agonists, partial agonists, antagonists, and inverse agonists, i.e., diterpenes that reduced basal AC activity. Each AC isoform exhibited a distinct pharmacological profile. AC2 showed the highest basal activity of all AC isoforms and highest sensitivity to inverse agonistic effects of 1-deoxy-forskolin, 7-deacetyl-1,9-dideoxy-forskolin, and, particularly, BODIPY-forskolin. In contrast, BODIPY-forskolin acted as partial agonist at the other ACs. 1-Deoxy-forskolin analogs were devoid of agonistic activity at ACs but antagonized the effects of FS in a mixed competitive/noncompetitive manner. At purified catalytic AC subunits, BODIPY-forskolin acted as weak partial agonist/strong partial antagonist. Molecular modeling revealed that the BODIPY group rotates promiscuously outside of the FS-binding site. Collectively, ACs are not uniformly activated and inhibited by FS and FS analogs, demonstrating the feasibility to design isoform-selective FS analogs. The two- and multiple-state models, originally developed to conceptualize ligand effects at G-protein-coupled receptors, can be applied to ACs to explain certain experimental data.

Crystal structure of the major quadruplex formed in the promoter region of the human c-MYC oncogene.

The c-MYC oncogene mediates multiple tumor cell survival pathways and is dysregulated or overexpressed in the majority of human cancers. The NHE III1 region of the c-MYC promoter forms a DNA quadruplex. Stabilization of this structure with small molecules has been shown to reduce expression of c-MYC, and targeting the c-MYC quadruplex has become an emerging strategy for development of antitumor compounds. Previous solution NMR studies of the c-MYC quadruplex have assigned the major conformer and topology of this important target, however, regions outside the G-quartet core were not as well-defined. Here, we report a high-resolution crystal structure (2.35 Å) of the major quadruplex formed in the NHE III1 region of the c-MYC promoter. The crystal structure is in general agreement with the solution NMR structure, however, key differences are observed in the position of nucleotides outside the G-quartet core. The crystal structure provides an alternative model that, along with comparisons to other reported quadruplex crystal structures, will be important to the rational design of selective compounds. This work will aid in development of ligands to target the c-MYC promoter quadruplex with the goal of creating novel anticancer therapies.

The high binding affinity of phosphorothioate-modified oligomers for Ff gene 5 protein is moderated by the addition of C-5 propyne or 2'-O-methyl modifications.

One of the problems that hamper the use of antisense DNAs as effective drugs is the non-specific binding of chemically-modified oligonucleotides to cellular proteins. We previously showed that the affinity of a model ssDNA-binding protein, the Ff gene 5 protein (g5p), was >300-fold higher for phosphorothioate-modified DNA (S-DNA) than for unmodified dA(36), consistent with the propensity of S-DNA to bind indiscriminately to proteins. The current work shows that g5p binding is also sensitive to sugar and pyrimidine modifications used in antisense oligomers. Binding affinities of g5p for 10 36mer oligomers were quantitated using solution circular dichroism measurements. The oligomers contained C-5-propyne (prC), 2'-O-methyl (2'-O-Me) or 2'-OH (RNA) groups, alone or combined with the phosphorothioate modification. In agreement with reported increases in antisense activity, the addition of prC or 2'-O-Me modifications substantially reduced the affinity of oligomers for g5p by approximately 2-fold compared with the same DNA oligomer sequences containing only phosphorothioate linkages. That is, such modifications moderated the propensity of the phosphorothioate group to bind tightly to the g5p. The Ff g5p could be a useful model protein for assessing non-specific binding effects of antisense oligomer modifications.