Structures, functions and adaptations of the human LINE-1 ORF2 protein.

The LINE-1 (L1) retrotransposon is an ancient genetic parasite that has written around one-third of the human genome through a 'copy and paste' mechanism catalysed by its multifunctional enzyme, open reading frame 2 protein (ORF2p)1. ORF2p reverse transcriptase (RT) and endonuclease activities have been implicated in the pathophysiology of cancer2,3, autoimmunity4,5 and ageing6,7, making ORF2p a potential therapeutic target. However, a lack of structural and mechanistic knowledge has hampered efforts to rationally exploit it. We report structures of the human ORF2p 'core' (residues 238-1061, including the RT domain) by X-ray crystallography and cryo-electron microscopy in several conformational states. Our analyses identified two previously undescribed folded domains, extensive contacts to RNA templates and associated adaptations that contribute to unique aspects of the L1 replication cycle. Computed integrative structural models of full-length ORF2p show a dynamic closed-ring conformation that appears to open during retrotransposition. We characterize ORF2p RT inhibition and reveal its underlying structural basis. Imaging and biochemistry show that non-canonical cytosolic ORF2p RT activity can produce RNA:DNA hybrids, activating innate immune signalling through cGAS/STING and resulting in interferon production6-8. In contrast to retroviral RTs, L1 RT is efficiently primed by short RNAs and hairpins, which probably explains cytosolic priming. Other biochemical activities including processivity, DNA-directed polymerization, non-templated base addition and template switching together allow us to propose a revised L1 insertion model. Finally, our evolutionary analysis demonstrates structural conservation between ORF2p and other RNA- and DNA-dependent polymerases. We therefore provide key mechanistic insights into L1 polymerization and insertion, shed light on the evolutionary history of L1 and enable rational drug development targeting L1.

Human endogenous retrovirus-K (HERV-K) reverse transcriptase (RT) structure and biochemistry reveals remarkable similarities to HIV-1 RT and opportunities for HERV-K-specific inhibition.

Human endogenous retroviruses (HERVs) comprise nearly 8% of the human genome and are derived from ancient integrations of retroviruses into the germline. The biology of HERVs is poorly defined, but there is accumulating evidence supporting pathological roles in diverse diseases, such as cancer, autoimmune, and neurodegenerative diseases. Functional proteins are produced by HERV-encoded genes, including reverse transcriptases (RTs), which could be a contributor to the pathology attributed to aberrant HERV-K expression. To facilitate the discovery and development of HERV-K RT potent and selective inhibitors, we expressed active HERV-K RT and determined the crystal structure of a ternary complex of this enzyme with a double-stranded DNA substrate. We demonstrate a range of RT inhibition with antiretroviral nucleotide analogs, while classic nonnucleoside analogs do not inhibit HERV-K RT. Detailed comparisons of HERV-K RT with other known RTs demonstrate similarities to diverse RT families and a striking similarity to the HIV-1 RT asymmetric heterodimer. Our analysis further reveals opportunities for selective HERV-K RT inhibition.

Discovery of new acylaminopyridines as GSK-3 inhibitors by a structure guided in-depth exploration of chemical space around a pyrrolopyridinone core.

Glycogen synthase kinase-3 (GSK-3) has been proposed to play a crucial role in the pathogenesis of many diseases including cancer, stroke, bipolar disorders, diabetes and neurodegenerative diseases. GSK-3 inhibition has been a major area of pharmaceutical interest over the last two decades. A plethora of reports appeared recently on selective inhibitors and their co-crystal structures in GSK-3ß. We identified several series of promising new GSK-3ß inhibitors from a coherent design around a pyrrolopyridinone core structure. A systematic exploration of the chemical space around the central spacer led to potent single digit and sub-nanomolar GSK-3ß inhibitors. When dosed orally in a transgenic mouse model of Alzheimer's disease (AD), an exemplary compound showed significant lowering of Tau phosphorylation at one of the GSK-3 phosphorylating sites, Ser396. X-ray crystallography greatly aided in validating the binding hypotheses.

Crystal structure of the herpes simplex virus 1 DNA polymerase.

Herpesviruses are the second leading cause of human viral diseases. Herpes Simplex Virus types 1 and 2 and Varicella-zoster virus produce neurotropic infections such as cutaneous and genital herpes, chickenpox, and shingles. Infections of a lymphotropic nature are caused by cytomegalovirus, HSV-6, HSV-7, and Epstein-Barr virus producing lymphoma, carcinoma, and congenital abnormalities. Yet another series of serious health problems are posed by infections in immunocompromised individuals. Common therapies for herpes viral infections employ nucleoside analogs, such as Acyclovir, and target the viral DNA polymerase, essential for viral DNA replication. Although clinically useful, this class of drugs exhibits a narrow antiviral spectrum, and resistance to these agents is an emerging problem for disease management. A better understanding of herpes virus replication will help the development of new safe and effective broad spectrum anti-herpetic drugs that fill an unmet need. Here, we present the first crystal structure of a herpesvirus polymerase, the Herpes Simplex Virus type 1 DNA polymerase, at 2.7 A resolution. The structural similarity of this polymerase to other alpha polymerases has allowed us to construct high confidence models of a replication complex of the polymerase and of Acyclovir as a DNA chain terminator. We propose a novel inhibition mechanism in which a representative of a series of non-nucleosidic viral polymerase inhibitors, the 4-oxo-dihydroquinolines, binds at the polymerase active site interacting non-covalently with both the polymerase and the DNA duplex.

Co-crystallization of Staphylococcus aureus peptide deformylase (PDF) with potent inhibitors.

In bacteria the biosynthesis of all nascent polypeptides begins with N-formylmethionine. The post-translational removal of the N-formyl group is carried out by peptide deformylase (PDF). Processing of the N-formyl group from critical bacterial proteins is required for cell survival. This formylation/deformylation cycle is unique to eubacteria and is not utilized in eucaryotic cytosolic protein biosynthesis. Thus, inhibition of PDF would halt bacterial growth, spare host cell-function, and would be a novel mechanism for a new class of antibiotic. Diffraction-quality Se-met crystals of S. aureus PDF were prepared that belong to space group C222(1) with unit cell parameters of a = 94.1 b = 121.9 c = 47.6 A. Multiple anomalous dispersion data were collected at the Advanced Photon Source 17-ID beamline and used to solve the PDF structure to 1.9 A resolution. Crystals were also prepared with three PDF inhibitors: thiorphan, actinonin and PNU-172550. The thiorphan and actinonin co-crystals belong to space group C222(1) with similar unit-cell dimensions. Repeated attempts to generate a complex structure of PDF with PNU-172550 from the orthorhombic space group were unsuccessful. Crystallization screening identified an alternate C2 crystal form with unit-cell dimensions of a = 93.4 b = 42.5 c = 104.1 A, beta = 93 degrees.

Physical methods to determine the binding mode of putative ligands for hepatitis C virus NS3 helicase.

Several small molecules identified by high-throughput screening (HTS) were evaluated for their ability to bind to a nonstructural protein 3 (NS3) helicase from hepatitis C virus (HCV). Equilibrium dissociation constants (K(d)'s) of the compounds for this helicase were determined using several techniques including an assay measuring the kinetics of isothermal enzyme denaturation at several concentrations of the test molecule. Effects of two nonhydrolyzable ATP analogs on helicase denaturation were measured as controls using the isothermal denaturation (ITD) assay. Two compounds, 4-(2,4-dimethylphenyl)-2,7,8-trimethyl-4,5-quinolinediamine and 2-phenyl-N-(5-piperazin-1-ylpentyl)quinazolin-4-amine, were identified from screening that inhibited the enzyme and had low micromolar dissociation constants for NS3 helicase in the ITD assay. Low micromolar affinity of the quinolinediamine to helicase was also confirmed by nuclear magnetic resonance experiments. Unfortunately, isothermal titration calorimetry (ITC) experiments indicated that a more water-soluble analog bound to the 47/23-mer oligonucleotide helicase substrate with low micromolar affinity as did the substituted quinazolinamine. There was no further interest in these templates as helicase inhibitors due to the nonspecific binding to enzyme and substrate. A combination of physical methods was required to discern the mode of action of compounds identified by HTS and remove undesirable lead templates from further consideration.