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.

A "Double-Edged" Scaffold: Antitumor Power within the Antibacterial Quinolone.

In the late 1980s, reports emerged describing experimental antibacterial quinolones having significant potency against eukaryotic Type II topoisomerases (topo II) and showing cytotoxic activity against tumor cell lines. As a result, several pharmaceutical companies initiated quinolone anticancer programs to explore the potential of this class in comparison to conventional human topo II inhibiting antitumor drugs such as doxorubicin and etoposide. In this review, we present a modern re-evaluation of the anticancer potential of the quinolone class in the context of today's predominantly pathway-based (rather than cytotoxicity-based) oncology drug R&D environment. The quinolone eukaryotic SAR is comprehensively discussed, contrasted with the corresponding prokaryotic data, and merged with recent structural biology information which is now beginning to help explain the basis for that SAR. Quinolone topo II inhibitors appear to be much less susceptible to efflux-mediated resistance, a current limitation of therapy with conventional agents. Recent advances in the biological understanding of human topo II isoforms suggest that significant progress might now be made in overcoming two other treatment-limiting disadvantages of conventional topo II inhibitors, namely cardiotoxicity and drug-induced secondary leukemias. We propose that quinolone class topo II inhibitors could have a useful future therapeutic role due to the continued need for effective topo II drugs in many cancer treatment settings, and due to the recent biological and structural advances which can now provide, for the first time, specific guidance for the design of a new class of inhibitors potentially superior to existing agents.

Origins of the Quinolone Class of Antibacterials: An Expanded "Discovery Story".

Published descriptions of the specific lines of research leading to the discovery of therapeutically important medicines, especially major new class medicines, have long provided value to the biopharmaceutical community as models of success, often influencing the strategies and methods of subsequent drug research. Quinolone antibacterials represent one of medicine's most important classes of anti-infective agents; yet in contrast to many other classes of anti-infectives, astonishingly few details concerning the origin of the class or the rationale leading to the selection of the first clinical agent, nalidixic acid, were ever published by the discoverers. Moreover, earlier disclosures of an independent discovery of the quinolone class of antibacterials have been almost entirely overlooked by the scientific literature. This review brings together all the available information from primary literature sources relating to both discoveries and provides for the first time a much fuller, if still partially speculative, story of the earliest years of this important class of drugs.

A New-Class Antibacterial-Almost. Lessons in Drug Discovery and Development: A Critical Analysis of More than 50 Years of Effort toward ATPase Inhibitors of DNA Gyrase and Topoisomerase IV.

The introduction into clinical practice of an ATPase inhibitor of bacterial DNA gyrase and topoisomerase IV (topo IV) would represent a new-class agent for the treatment of resistant bacterial infections. Novobiocin, the only historical member of this class, established the clinical proof of concept for this novel mechanism during the late 1950s, but its use declined rapidly and it was eventually withdrawn from the market. Despite significant and prolonged effort across the biopharmaceutical industry to develop other agents of this class, novobiocin remains the only ATPase inhibitor of gyrase and topo IV ever to progress beyond Phase I. In this review, we analyze the historical attempts to discover and develop agents within this class and highlight factors that might have hindered those efforts. Within the last 15 years, however, our technical understanding of the molecular details of the inhibition of the gyrase and topo IV ATPases, the factors governing resistance development to such inhibitors, and our knowledge of the physical properties required for robust clinical drug candidates have all matured to the point wherein the industry may now address this mechanism of action with greater confidence. The antibacterial spectrum within this class has recently been extended to begin to include serious Gram negative pathogens such as Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae. In spite of this recent technical progress, adverse economics associated with antibacterial R&D over the last 20 years has diminished industry's ability to commit the resources and perseverance needed to bring new-class agents to launch. Consequently, a number of recent efforts in the ATPase class have been derailed by organizational rather than scientific factors. Nevertheless, within this context we discuss the unique opportunity for the development of ATPase inhibitors of gyrase and topo IV as new-class antibacterial agents with broad spectrum potential.

Phenylimidazoles as potent and selective inhibitors of coagulation factor XIa with in vivo antithrombotic activity.

Novel inhibitors of FXIa containing an (S)-2-phenyl-1-(4-phenyl-1H-imidazol-2-yl)ethanamine core have been optimized to provide compound 16b, a potent, reversible inhibitor of FXIa (Ki = 0.3 nM) having in vivo antithrombotic efficacy in the rabbit AV-shunt thrombosis model (ID50 = 0.6 mg/kg + 1 mg kg(-1) h(-1)). Initial analog selection was informed by molecular modeling using compounds 11a and 11h overlaid onto the X-ray crystal structure of tetrahydroquinoline 3 complexed to FXIa. Further optimization was achieved by specific modifications derived from careful analysis of the X-ray crystal structure of the FXIa/11h complex. Compound 16b was well tolerated and enabled extensive pharmacologic evaluation of the FXIa mechanism up to the ID90 for thrombus inhibition.

Discovery of nonbenzamidine factor VIIa inhibitors using a biaryl acid scaffold.

In this Letter, we describe the synthesis of several nonamidine analogs of biaryl acid factor VIIa inhibitor 1 containing weakly basic or nonbasic P1 groups. 2-Aminoisoquinoline was found to be an excellent surrogate for the benzamidine group (compound 2) wherein potent inhibition of factor VIIa is maintained relative to most other related serine proteases. In an unanticipated result, the m-benzamide P1 (compounds 21a and 21b) proved to be a viable benzamidine replacement, albeit with a 20-40 fold loss in potency against factor VIIa.

Structure of the complex of trypsin with a highly potent synthetic inhibitor at 0.97 A resolution.

The structure of the complex formed between bovine beta-trypsin and the highly potent synthetic inhibitor 2-{3'-[5'-methoxy-2'-(N-p-diaminomethylphenyl)amido]-1'-pyrido}-5-(N-2''-t-butylethanol)amidobenzoic acid (C(28)H(32)N(5)O(6)) has been determined at 0.97 A resolution. X-ray intensity data were collected to 0.97 A under cryocooled conditions. The structure was refined anisotropically using REFMAC5 and SHELX-97 to R(cryst) factors of 13.4 and 12.6% and R(free) factors of 15.7 and 16.3%, respectively. Several regions of the main chain and side chains that have not been previously observed were clearly defined in the present structure. H atoms are indicated as significant peaks in an |F(o) - F(c)| difference map, which accounts for an estimated 35% of all H atoms at the 2.5sigma level. The C, N and O atoms are definitively differentiated in the electron-density maps. The amido part of the inhibitor occupies the specificity pocket and the remainder fills the remaining part of the ligand-binding cleft and interacts with the enzyme through an extensive network of hydrogen bonds. The inhibitor distorts the stereochemistry of the catalytic triad, Ser195-His57-Asp102, thereby blocking the proton-relay process of the active site by preventing the formation of the crucial hydrogen bond between His57 N(delta1) and Asp102 O(delta1).

Ketene aminal-based lactam derivatives as a novel class of orally active FXa inhibitors.

N,N'-Disubstituted ketene aminals are good bioisosteres of thiourea functional groups. We report the design and synthesis of a novel class of ketene aminal-based lactam derivatives as potent and orally active FXa inhibitors.

Synthesis of potent and highly selective nonguanidine azetidinone inhibitors of human tryptase.

Azetidinones such as BMS-363131 (2) and BMS-363130 (3), which contain a guanidine group in the C-3 side chain were previously shown to be very potent inhibitors of human tryptase with high selectivity versus other serine proteases, including trypsin. In this letter, we describe the discovery of a number of potent azetidinone tryptase inhibitors in which the guanidine moiety at the ring C-3 position is replaced with primary or secondary amine or aminopyridine functionality. In particular, BMS-354326 (4) is a highly potent tryptase inhibitor (IC(50)=1.8 nM), which has excellent selectivity against trypsin and most other related serine proteases.

Solid-phase synthesis and SAR of 4-carboxy-2-azetidinone mechanism-based tryptase inhibitors.

A series of nonguanidine N1-activated C4-carboxy azetidinone tryptase inhibitors was prepared by solid-phase methodology to quickly assess the SAR associated with distal functionality on the N1-activating group. From these studies, potent inhibitors with improved specificity were discovered.

Synthesis of potent and selective 2-azepanone inhibitors of human tryptase.

The serine protease tryptase has been associated with a broad range of allergic and inflammatory diseases and, in particular, has been implicated as a critical mediator of asthma. The inhibition of tryptase therefore has the potential to be a valuable therapy for asthma. The synthesis, employing solution phase parallel methods, and SAR of a series of novel 2-azepanone tryptase inhibitors are presented. A member of this series, 8t, was identified as a potent inhibitor of human tryptase (IC(50)=38 nM) with selectivity >/=330-fold versus related serine proteases (trypsin, plasmin, uPA, tPA, APC, alpha-thrombin, and FXa) [corrected].

Synthesis and SAR of 4-carboxy-2-azetidinone mechanism-based tryptase inhibitors.

A series of N1-activated C4-carboxy azetidinones was prepared and tested as inhibitors of human tryptase. The key stereochemical and functional features required for potency, serine protease specificity and aqueous stability were determined. From these studies compound 2, BMS-262084, was identified as a potent and selective tryptase inhibitor which, when dosed intratracheally in ovalbumin-sensitized guinea pigs, reduced allergen-induced bronchoconstriction and inflammatory cell infiltration into the lung.

Synthesis of potent and highly selective inhibitors of human tryptase.

The serine protease tryptase has been implicated in allergic and inflammatory diseases and associated with asthma. The synthesis and SAR of a series of N1-activated-4-carboxy azetidinones are described, resulting in identification of BMS-363131 (2) as a potent inhibitor of human tryptase (IC(50)<1.7 nM) with high selectivity (>3000-fold) for tryptase versus related serine proteases including trypsin.