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.

Discovery of a Potent and Selective Tyrosine Kinase 2 Inhibitor: TAK-279.

TYK2 is a key mediator of IL12, IL23, and type I interferon signaling, and these cytokines have been implicated in the pathogenesis of multiple inflammatory and autoimmune diseases such as psoriasis, rheumatoid arthritis, lupus, and inflammatory bowel diseases. Supported by compelling data from human genome-wide association studies and clinical results, TYK2 inhibition through small molecules is an attractive therapeutic strategy to treat these diseases. Herein, we report the discovery of a series of highly selective pseudokinase (Janus homology 2, JH2) domain inhibitors of TYK2 enzymatic activity. A computationally enabled design strategy, including the use of FEP+, was instrumental in identifying a pyrazolo-pyrimidine core. We highlight the utility of computational physics-based predictions used to optimize this series of molecules to identify the development candidate 30, a potent, exquisitely selective cellular TYK2 inhibitor that is currently in Phase 2 clinical trials for the treatment of psoriasis and psoriatic arthritis.

Potent and selective TYK2-JH1 inhibitors highly efficacious in rodent model of psoriasis.

TYK2 is a member of the JAK family of kinases and a key mediator of IL-12, IL-23, and type I interferon signaling. These cytokines have been implicated in the pathogenesis of multiple inflammatory and autoimmune diseases such as psoriasis, rheumatoid arthritis, lupus, and inflammatory bowel diseases. Supported by compelling data from human genetic association studies, TYK2 inhibition is an attractive therapeutic strategy for these diseases. Herein, we report the discovery of a series of highly selective catalytic site TYK2 inhibitors designed using FEP+ and structurally enabled design starting from a virtual screen hit. We highlight the structure-based optimization to identify a lead candidate 30, a potent cellular TYK2 inhibitor with excellent selectivity, pharmacokinetic properties, and in vivo efficacy in a mouse psoriasis model.

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.

Design and Synthesis of Selective Phosphodiesterase 4D (PDE4D) Allosteric Inhibitors for the Treatment of Fragile X Syndrome and Other Brain Disorders.

Novel pyridine- and pyrimidine-based allosteric inhibitors are reported that achieve PDE4D subtype selectivity through recognition of a single amino acid difference on a key regulatory domain, known as UCR2, that opens and closes over the catalytic site for cAMP hydrolysis. The design and optimization of lead compounds was based on iterative analysis of X-ray crystal structures combined with metabolite identification. Selectivity for the activated, dimeric form of PDE4D provided potent memory enhancing effects in a mouse model of novel object recognition with improved tolerability and reduced vascular toxicity over earlier PDE4 inhibitors that lack subtype selectivity. The lead compound, 28 (BPN14770), has entered midstage, human phase 2 clinical trials for the treatment of Fragile X Syndrome.

Selective interleukin-1 receptor-associated kinase 4 inhibitors for the treatment of autoimmune disorders and lymphoid malignancy.

Pathological activation of the Toll-like receptor signaling adaptor protein MYD88 underlies many autoimmune and inflammatory disease states. In the activated B cell-like (ABC) subtype of diffuse large B cell lymphoma (DLBCL), the oncogenic MYD88 L265P mutation occurs in 29% of cases, making it the most prevalent activating mutation in this malignancy. IRAK4 kinase accounts for almost all of the biological functions of MYD88, highlighting IRAK4 as a therapeutic target for diseases driven by aberrant MYD88 signaling. Using innovative structure-based drug design methodologies, we report the development of highly selective and bioavailable small molecule IRAK4 inhibitors, ND-2158 and ND-2110. These small molecules suppressed LPS-induced TNF production, alleviated collagen-induced arthritis, and blocked gout formation in mouse models. IRAK4 inhibition promoted killing of ABC DLBCL lines harboring MYD88 L265P, by down-modulating survival signals, including NF-κB and autocrine IL-6/IL-10 engagement of the JAK-STAT3 pathway. In ABC DLBCL xenograft models, IRAK4 inhibition suppressed tumor growth as a single agent, and in combination with the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib or the Bcl-2 inhibitor ABT-199. Our findings support pharmacological inhibition of IRAK4 as a therapeutic strategy in autoimmune disorders, in a genetically defined population of ABC DLBCL, and possibly other malignancies dependent on aberrant MYD88 signaling.

Accurate and reliable prediction of relative ligand binding potency in prospective drug discovery by way of a modern free-energy calculation protocol and force field.

Designing tight-binding ligands is a primary objective of small-molecule drug discovery. Over the past few decades, free-energy calculations have benefited from improved force fields and sampling algorithms, as well as the advent of low-cost parallel computing. However, it has proven to be challenging to reliably achieve the level of accuracy that would be needed to guide lead optimization (∼5× in binding affinity) for a wide range of ligands and protein targets. Not surprisingly, widespread commercial application of free-energy simulations has been limited due to the lack of large-scale validation coupled with the technical challenges traditionally associated with running these types of calculations. Here, we report an approach that achieves an unprecedented level of accuracy across a broad range of target classes and ligands, with retrospective results encompassing 200 ligands and a wide variety of chemical perturbations, many of which involve significant changes in ligand chemical structures. In addition, we have applied the method in prospective drug discovery projects and found a significant improvement in the quality of the compounds synthesized that have been predicted to be potent. Compounds predicted to be potent by this approach have a substantial reduction in false positives relative to compounds synthesized on the basis of other computational or medicinal chemistry approaches. Furthermore, the results are consistent with those obtained from our retrospective studies, demonstrating the robustness and broad range of applicability of this approach, which can be used to drive decisions in lead optimization.

Recent advances in the discovery of small molecule inhibitors of interleukin-1 receptor-associated kinase 4 (IRAK4) as a therapeutic target for inflammation and oncology disorders.

IRAK4, a serine/threonine kinase, plays a key role in both inflammation and oncology diseases. Herein, we summarize the compelling biology surrounding the IRAK4 signaling node in disease, review key structural features of IRAK4 including selectivity challenges, and describe efforts to discover clinically viable IRAK4 inhibitors. Finally, a view of knowledge gained and remaining challenges is provided.

Current HIV therapeutics: mechanistic and chemical determinants of toxicity.

This review discusses current recommendations for initial therapy in HIV-infected individuals and the toxicity of certain components of these therapeutic regimens. In developed regions, therapy is headed in a direction of individualized treatment where AIDS can be converted into a treatable, chronic disease that is characterized by viral loads below assay detection levels. In developing nations, where sophisticated diagnostics are less applicable, treatment continues on a population basis. Because nucleoside reverse transcriptase inhibitors remain a cornerstone of current recommended regimens both in developed and developing regions, the mechanistic and chemical determinants of mitochondrial toxicity are highlighted and discussed in detail.

Bacterial translation inhibitors, 1-acylindazol-3-ols as anthranilic acid mimics.

The discovery and initial optimization of a novel anthranilic acid derived class of antibacterial agents has been described in a recent series of papers. This paper describes the discovery of 1-acylindazol-3-ols as a novel bioisostere of an anthranilic acid. The synthesis and structure-activity relationships of the indazol bioisosteres are described herein.

Resistance mapping and mode of action of a novel class of antibacterial anthranilic acids: evidence for disruption of cell wall biosynthesis.

OBJECTIVES: The aim of this study was to characterize the mechanism of action of a novel class of bacterial protein synthesis inhibitors identified in a high-throughput coupled transcription-translation assay. METHODS: Evaluation of the cross-resistance to antibiotics with known mechanisms of action, resistance mapping and biochemical characterization of a novel class of antibacterial anthranilic acids was performed. RESULTS: No cross-resistance to established classes of antibiotics was found. Resistance was mapped to SA1575, an essential, integral membrane protein predicted to be involved in polysaccharide biosynthesis. Biochemical analysis demonstrated the inhibition of cell wall biosynthesis. CONCLUSIONS: This novel class of antibacterial anthranilic acids inhibits cell wall biosynthesis. Resistance mapped to SA1575, which may represent a novel target for antibacterial drug discovery.

Correlation of carboxylic acid pKa to protein binding and antibacterial activity of a novel class of bacterial translation inhibitors.

Previously we reported the discovery and initial optimization of a novel anthranilic acid derived class of antibacterial agents which suffered from extensive protein binding. This report describes efforts directed toward understanding the relationship of the acidity of the carboxylic acid with the extent of protein binding. The pK(a) of the acid was modified via the synthesis of a number of anthranilic acid analogs which vary the aromatic ring substituent at the 4-position. The pK(a) and HSA binding constants have been determined for each of the analogs. Our results indicate a correlation between pK(a) and HSA K(d). The physical properties and antibacterial activities will be discussed as well as how these results help address the protein binding issue with this series of compounds.

Structure-activity relationships of bioisosteres of a carboxylic acid in a novel class of bacterial translation inhibitors.

The discovery and initial optimization of a novel anthranilic acid derived class of antibacterial agents which suffered from extensive protein binding has been previously reported. The structure-activity relationships around the carboxylic acid substituent are described herein. This acid was replaced by several alternative functional groups in attempts to retain bioactivity while reducing protein binding. Only groups with an acidic proton retained activity, and analogs containing those groups maintained the protein binding inherent to this class of antibacterial agents.

Human serum albumin binding assay based on displacement of a non selective fluorescent inhibitor.

In this paper, we describe a fluorescent antibacterial analog, 6, with utility as a competition probe to determine affinities of other antibacterial analogs for human serum albumin (HSA). Analog 6 bound to HSA with an affinity of 400+/-100 nM and the fluorescence was environmentally sensitive. With 370 nm excitation, environmental sensitivity was indicated by a quenching of the 530 nm emission when the probe bound to HSA. Displacement of dansylsarcosine from HSA by 6 indicated it competed with compounds that bound at site II (ibuprofen binding site) on HSA. Analog 6 also shifted the NMR peaks of an HSA bound oleic acid molecule that itself was affected by compounds that bound at site II. In addition to binding at site II, 6 interacted at site I (warfarin binding site) as indicated by displacement of dansylamide and the shifting of NMR peaks of an HSA bound oleic acid molecule affected by warfarin site binding. Additional evidence for multiple site interaction was discovered when a percentage of 6 could be displaced by either ibuprofen or phenylbutazone. A competition assay was established using 6 to determine relative affinities of other antibacterial inhibitors for HSA.

Preparation of novel anthranilic acids as antibacterial agents: extensive evaluation of structural and physical properties on antibacterial activity and human serum albumin affinity.

In the past few years a significant effort has been devoted by Pharmacia toward the discovery of novel antibiotics. We have recently described the identification of an anthranilic acid lead 1 and the optimization resulting in the advanced lead 2. In this report, we describe the preparation of several selected analogs to probe the dependency of this template for antibacterial activity and the affinity these compounds have for human serum albumin (HSA). These analogs illustrate that decreased affinity for HSA can be achieved while retaining relevant antibacterial activity. The most important factor for reduced HSA affinity is decrease in logP rather than a structural change.

Preparation of novel antibacterial agents. Replacement of the central aromatic ring with heterocycles.

Discovery of novel antibacterial agents is a significant challenge. We have recently reported on our discovery of novel antibacterial agents in which we have rapidly optimized potency utilizing a parallel chemistry approach. These advanced leads suffer from high affinity for human serum albumin (HSA). In an effort to decrease the affinity for HSA we have prepared a series of heterocyclic analogs, which retained antibacterial activity and demonstrated reduced affinity for HSA.

Preparation of novel anthranilic acids as antibacterial agents. Extensive evaluation of alternative amide bioisosteres connecting the A- and the B-rings.

In the past few years, a significant effort has been devoted by Pharmacia toward the discovery of novel antibiotics. We have recently described the identification of an anthranilic acid lead 1 and the optimization resulting in the advanced lead 2. In this report, we describe the preparation of several selected amide bioisosteres connecting the A- and the B-rings. The E-alkene provided a rigid analog with equal potency to the corresponding amide. This indicates that the amide is not a recognition element rather acts as an appropriate spatial linker of the two important aryl A and B rings. The work here clearly demonstrates that the amide linker can be replaced with several functionalities without significant deterioration in the MIC activity.

Discovery and initial development of a novel class of antibacterials: inhibitors of Staphylococcus aureus transcription/translation.

The novel bacterial transcription/translation (TT) inhibitor 1 was identified through a combination of high throughput screening and exploratory medicinal chemistry. Initial optimization of the anthranilic acid moiety and sulfonamide amine diversity was accomplished via 1- and two-dimensional solution phase libraries, resulting in an improvement in the MIC of the lead from 64 to 8mug/mL (compound 4l). Subsequent modification of the central aromatic ring and further refinement of the sulfonamide amines required the development of a solid phase route on Wang resin. The resulting libraries generated a number of potent antibacterials with MICs of 1mug/mL (e.g., 10b, 12, and 13). During the course of this work, it became apparent that the antibacterial activity of the series is not fully correlated with TT inhibition, suggesting that at least one additional mechanism of action is operative.

A stereoselective intramolecular 1,3-dipolar nitrone cycloaddition for the synthesis of substituted chromanes.

A stereoselective intramolecular 1,3-dipolar nitrone cycloaddition useful in the synthesis of chromanes is described. The reaction relies on the use of a chiral auxiliary on the nitrone partner. Key to the success of the reaction is the choice of auxiliary and the choice of Lewis acid catalyst. Utilizing an auxiliary with a pendant coordinating group, and Zn(OTf)(2) as the Lewis acid, diastereoselectivities up to 22:1 could be achieved.