Diversified strategy for the synthesis of DNA-encoded oxindole libraries.

DNA-encoded library technology (DELT) employs DNA as a barcode to track the sequence of chemical reactions and enables the design and synthesis of libraries with billions of small molecules through combinatorial expansion. This powerful technology platform has been successfully demonstrated for hit identification and target validation for many types of diseases. As a highly integrated technology platform, DEL is capable of accelerating the translation of synthetic chemistry by using on-DNA compatible reactions or off-DNA scaffold synthesis. Herein, we report the development of a series of novel on-DNA transformations based on oxindole scaffolds for the design and synthesis of diversity-oriented DNA-encoded libraries for screening. Specifically, we have developed 1,3-dipolar cyclizations, cyclopropanations, ring-opening of reactions of aziridines and Claisen-Schmidt condensations to construct diverse oxindole derivatives. The majority of these transformations enable a diversity-oriented synthesis of DNA-encoded oxindole libraries which have been used in the successful hit identification for three protein targets. We have demonstrated that a diversified strategy for DEL synthesis could accelerate the application of synthetic chemistry for drug discovery.

Construction of Thiazole-Fused Dihydropyrans via Formal [4 + 2] Cycloaddition Reaction on DNA.

An efficient and facile formal [4 + 2] cycloaddition reaction was developed to synthesize diverse thiazole-fused dihydropyrans (TFDP) on DNA. Mild reaction conditions, broad substrate scope, and compatibility with subsequent enzymatic ligation demonstrated the utility of this methodology in DNA-encoded library synthesis.

Fragment-Based Computational Method for Designing GPCR Ligands.

G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors, which is arguably the most important family of drug target. With the technology breakthroughs in X-ray crystallography and cryo-electron microscopy, more than 300 GPCR-ligand complex structures have been publicly reported since 2007, covering about 60 unique GPCRs. Such abundant structural information certainly will facilitate the structure-based drug design by targeting GPCRs. In this study, we have developed a fragment-based computational method for designing novel GPCR ligands. We first extracted the characteristic interaction patterns (CIPs) on the binding interfaces between GPCRs and their ligands. The CIPs were used as queries to search the chemical fragments derived from GPCR ligands, which were required to form similar interaction patterns with GPCR. Then, the selected chemical fragments were assembled into complete molecules by using the AutoT&T2 software. In this work, we chose ß-adrenergic receptor (ß-AR) and muscarinic acetylcholine receptor (mAChR) as the targets to validate this method. Based on the designs suggested by our method, samples of 63 compounds were purchased and tested in a cell-based functional assay. A total of 15 and 22 compounds were identified as active antagonists for ß2-AR and mAChR M1, respectively. Molecular dynamics simulations and binding free energy analysis were performed to explore the key interactions (e.g., hydrogen bonds and π-π interactions) between those active compounds and their target GPCRs. In summary, our work presents a useful approach to the de novo design of GPCR ligands based on the relevant 3D structural information.

Palladium-Promoted DNA-Compatible Heck Reaction.

Optimal conditions for palladium-promoted Heck reaction on DNA were developed with good to excellent conversions. Versatility with either DNA-conjugated styrene/acrylamide or aryl iodide and a broad substrate scope of the corresponding coupling partners were established. Furthermore, robustness of the Heck reaction conditions on single-strand DNA and feasibility for DNA-encoded library production were demonstrated.

A potent Lassa virus antiviral targets an arenavirus virulence determinant.

Arenaviruses are a significant cause of hemorrhagic fever, an often-fatal disease for which there is no approved antiviral therapy. Lassa fever in particular generates high morbidity and mortality in West Africa, where the disease is endemic, and a recent outbreak in Nigeria was larger and more geographically diverse than usual. We are developing LHF-535, a small-molecule viral entry inhibitor that targets the arenavirus envelope glycoprotein, as a therapeutic candidate for Lassa fever and other hemorrhagic fevers of arenavirus origin. Using a lentiviral pseudotype infectivity assay, we determined that LHF-535 had sub-nanomolar potency against the viral envelope glycoproteins from all Lassa virus lineages, with the exception of the glycoprotein from the LP strain from lineage I, which was 100-fold less sensitive than that of other strains. This reduced sensitivity was mediated by a unique amino acid substitution, V434I, in the transmembrane domain of the envelope glycoprotein GP2 subunit. This position corresponds to the attenuation determinant of Candid#1, a live-attenuated Junín virus vaccine strain used to prevent Argentine hemorrhagic fever. Using a virus-yield reduction assay, we determined that LHF-535 potently inhibited Junín virus, but not Candid#1, and the Candid#1 attenuation determinant, F427I, regulated this difference in sensitivity. We also demonstrated that a daily oral dose of LHF-535 at 10 mg/kg protected mice from a lethal dose of Tacaribe virus. Serial passage of Tacaribe virus in LHF-535-treated Vero cells yielded viruses that were resistant to LHF-535, and the majority of drug-resistant viruses exhibited attenuated pathogenesis. These findings provide a framework for the clinical development of LHF-535 as a broad-spectrum inhibitor of arenavirus entry and provide an important context for monitoring the emergence of drug-resistant viruses.

Inverse-Electron-Demand Diels-Alder Reactions for the Synthesis of Pyridazines on DNA.

The synthesis of pyridazines on DNA has been developed on the basis of inverse-electron-demand Diels-Alder (IEDDA) reactions of 1,2,4,5-tetrazines. The broad substrate scope is explored. Functionalized pyridazine products are selected for subsequent DNA-compatible Suzuki-Miyaura coupling, acylation, and SNAr substitution reactions, demonstrating the feasibility and versatility of IEDDA reactions for DNA-encoded library synthesis.

Ruthenium-Promoted C-H Activation Reactions between DNA-Conjugated Acrylamide and Aromatic Acids.

The first example of DNA-compatible C-H activation reaction between DNA-conjugated acrylamides and aromatic acids has been developed. This new transformation enables aromatic acid, previously considered as monofunctional building block, to act like a bifunctional building block for the DNA encoded library synthesis. The general scope of aromatic acid was established for this new on-DNA C-H activation, which paved the way for its application in combinatorial library preparation.

Lead optimization of an acylhydrazone scaffold possessing antiviral activity against Lassa virus.

Previously we reported the optimization of antiviral scaffolds containing benzimidazole and related heterocycles possessing activity against a variety of arenaviruses. These series of compounds were discovered through an HTS campaign of a 400,000 small molecule library using lentivirus-based pseudotypes incorporated with the Lassa virus envelope glycoprotein (LASV GP). This screening also uncovered an alternate series of very potent arenavirus inhibitors based upon an acylhydrazone scaffold. Subsequent SAR analysis of this chemical series involved various substitutions throughout the chemical framework along with assessment of the preferred stereochemistry. These studies led to an optimized analog (ST-161) possessing subnanomolar activity against LASV and submicromolar activity against a number of other viruses in the Arenaviridae family.

Novel benzoxazole inhibitor of dengue virus replication that targets the NS3 helicase.

Dengue virus (DENV) is the predominant mosquito-borne viral pathogen that infects humans with an estimated 50 to 100 million infections per year worldwide. Over the past 50 years, the incidence of dengue disease has increased dramatically and the virus is now endemic in more than 100 countries. Moreover, multiple serotypes of DENV are now found in the same geographic region, increasing the likelihood of more severe forms of disease. Despite extensive research, there are still no approved vaccines or therapeutics commercially available to treat DENV infection. Here we report the results of a high-throughput screen of a chemical compound library using a whole-virus assay that identified a novel small-molecule inhibitor of DENV, ST-610, that potently and selectively inhibits all four serotypes of DENV replication in vitro. Sequence analysis of drug-resistant virus isolates has identified a single point mutation, A263T, in the NS3 helicase domain that confers resistance to this compound. ST-610 inhibits DENV NS3 helicase RNA unwinding activity in a molecular-beacon-based helicase assay but does not inhibit nucleoside triphosphatase activity based on a malachite green ATPase assay. ST-610 is nonmutagenic, is well tolerated (nontoxic) in mice, and has shown efficacy in a sublethal murine model of DENV infection with the ability to significantly reduce viremia and viral load compared to vehicle controls.

A novel inhibitor of dengue virus replication that targets the capsid protein.

Dengue viruses (DENV) infect 50 to 100 million people worldwide per year, of which 500,000 develop severe life-threatening disease. This mosquito-borne illness is endemic in most tropical and subtropical countries and has spread significantly over the last decade. While there are several promising vaccine candidates in clinical trials, there are currently no approved vaccines or therapeutics available for treatment of dengue infection. Here, we describe a novel small-molecule compound, ST-148, that is a potent inhibitor of all four serotypes of DENV in vitro. ST-148 significantly reduced viremia and viral load in vital organs and tended to lower cytokine levels in the plasma in a nonlethal model of DENV infection in AG129 mice. Compound resistance mapped to the DENV capsid (C) gene, and a direct interaction of ST-148 with C protein is suggested by alterations of the intrinsic fluorescence of the protein in the presence of compound. Thus, ST-148 appears to interact with the DENV C protein and inhibits a distinct step(s) of the viral replication cycle.

Discovery and optimization of potent broad-spectrum arenavirus inhibitors derived from benzimidazole.

A chemically diverse library of about 400,000 small molecules was screened for antiviral activity against lentiviral pseudotypes with the Lassa virus envelope glycoprotein (LASV GP) gene incorporated. High-throughput screening resulted in discovery of a hit compound (ST-37) possessing a benzimidazole core which led to a potent compound series. Herein, we report SAR studies which involved structural modifications to the phenyl rings and methylamino linker portion attached to the benzimidazole core. Many analogs in this study possessed single digit nanomolar potency against LASV pseudotypes. Compounds in this benzimidazole series also exhibited nanomolar antiviral activity against pseudotypes generated from other arenavirus envelopes indicating the potential for development of a broad-spectrum inhibitor. Ultimately, lead compound ST-193 was identified and later found to be efficacious in a lethal LASV guinea pig model showing superior protection compared to ribavirin treatment.

Discovery and optimization of potent broad-spectrum arenavirus inhibitors derived from benzimidazole and related heterocycles.

A series of potent arenavirus inhibitors sharing a benzimidazole core were previously reported by our group. SAR studies were expanded beyond the previous analysis, which involved the attached phenyl rings and methylamino linker portion, to include modifications focused on the benzimidazole core. These changes included the introduction of various substituents to the bicyclic benzimidazole ring system along with alternate core heterocycles. Many of the analogs containing alternate nitrogen-based bicyclic ring systems were found to retain antiviral potency compared to the benzimidazole series from which we derived our lead compound, ST-193. In fact, 21 h, built on an imidazopyridine core, possessed a near tenfold increase in potency against Lassa virus pseudotypes compared to ST-193. As found with the benzimidazole series, broad-spectrum arenavirus activity was also observed for a number of the analogs discovered during this study.

SAR analysis of a series of acylthiourea derivatives possessing broad-spectrum antiviral activity.

A series of acylthiourea derivatives were designed, synthesized, and evaluated for broad-spectrum antiviral activity with selected viruses from Poxviridae (vaccinia virus) and two different genera of the family Bunyaviridae (Rift Valley fever and La Crosse viruses). A compound selected from a library screen, compound 1, displayed submicromolar antiviral activity against both vaccinia virus (EC(50)=0.25 µM) and La Crosse virus (EC(50)=0.27 µM) in cytopathic effect (CPE) assays. SAR analysis was performed to further improve antiviral potency and to optimize drug-like properties of the initial hits. During our analysis, we identified 26, which was found to be nearly fourfold more potent than 1 against both vaccinia and La Crosse viruses. Selected compounds were further tested to more fully characterize the spectrum of antiviral activity. Many of these possessed single digit micromolar and sub-micromolar antiviral activity against a diverse array of targets, including influenza virus (Orthomyxoviridae), Tacaribe virus (Arenaviridae), and dengue virus (Flaviviridae).

Evaluation of Lassa antiviral compound ST-193 in a guinea pig model.

Lassa virus (LASV), a member of the Arenaviridae family, causes a viral hemorrhagic fever endemic to West Africa, where as many as 300,000 infections occur per year. Presently, there are no FDA-approved LASV-specific vaccines or antiviral agents, although the antiviral drug ribavirin has shown some efficacy. A recently identified small-molecule inhibitor of arenavirus entry, ST-193, exhibits submicromolar antiviral activity in vitro. To determine the antiviral utility of ST-193 in vivo, we tested the efficacy of this compound in the LASV guinea pig model. Four groups of strain 13 guinea pigs were administered 25 or 80 mg/kg ST-193, 25 mg/kg of ribavirin, or the vehicle by the intraperitoneal (i.p.) route before infection with a lethal dose of LASV, strain Josiah, and continuing once daily for 14 days. Control animals exhibited severe disease, becoming moribund between days 10 and 15 postinfection. ST-193-treated animals exhibited fewer signs of disease and enhanced survival when compared to the ribavirin or vehicle groups. Body temperatures in all groups were elevated by day 9, but returned to normal by day 19 postinfection in the majority of ST-193-treated animals. ST-193 treatment mediated a 2-3-log reduction in viremia relative to vehicle-treated controls. The overall survival rate for the ST-193-treated guinea pigs was 62.5% (10/16) compared with 0% in the ribavirin (0/8) and vehicle (0/7) groups. These data suggest that ST-193 may serve as an improved candidate for the treatment of Lassa fever.

A specific interaction of small molecule entry inhibitors with the envelope glycoprotein complex of the Junín hemorrhagic fever arenavirus.

Arenaviruses are responsible for acute hemorrhagic fevers worldwide and are recognized to pose significant threats to public health and biodefense. Small molecule compounds have recently been discovered that inhibit arenavirus entry and protect against lethal infection in animal models. These chemically distinct inhibitors act on the tripartite envelope glycoprotein (GPC) through its unusual stable signal peptide subunit to stabilize the complex against pH-induced activation of membrane fusion in the endosome. Here, we report the production and characterization of the intact transmembrane GPC complex of Junín arenavirus and its interaction with these inhibitors. The solubilized GPC is antigenically indistinguishable from the native protein and forms a homogeneous trimer in solution. When reconstituted into a lipid bilayer, the purified complex interacts specifically with its cell-surface receptor transferrin receptor-1. We show that small molecule entry inhibitors specific to New World or Old World arenaviruses bind to the membrane-associated GPC complex in accordance with their respective species selectivities and with dissociation constants comparable with concentrations that inhibit GPC-mediated membrane fusion. Furthermore, competitive binding studies reveal that these chemically distinct inhibitors share a common binding pocket on GPC. In conjunction with previous genetic studies, these findings identify the pH-sensing interface of GPC as a highly vulnerable target for antiviral intervention. This work expands our mechanistic understanding of arenavirus entry and provides a foundation to guide the development of small molecule compounds for the treatment of arenavirus hemorrhagic fevers.

pH-induced activation of arenavirus membrane fusion is antagonized by small-molecule inhibitors.

The arenavirus envelope glycoprotein (GPC) mediates viral entry through pH-induced membrane fusion in the endosome. This crucial process in the viral life cycle can be specifically inhibited in the New World arenaviruses by the small-molecule compound ST-294. Here, we show that ST-294 interferes with GPC-mediated membrane fusion by targeting the interaction of the G2 fusion subunit with the stable signal peptide (SSP). We demonstrate that amino acid substitutions at lysine-33 of the Junín virus SSP confer resistance to ST-294 and engender de novo sensitivity to ST-161, a chemically distinct inhibitor of the Old World Lassa fever virus. These compounds, as well as a broadly active inhibitor, ST-193, likely share a molecular target at the SSP-G2 interface. We also show that both ST-294 and ST-193 inhibit pH-induced dissociation of the G1 receptor-binding subunit from GPC, a process concomitant with fusion activation. Interestingly, the inhibitory activity of these molecules can in some cases be overcome by further lowering the pH used for activation. Our results suggest that these small molecules act to stabilize the prefusion GPC complex against acidic pH. The pH-sensitive interaction between SSP and G2 in GPC represents a robust molecular target for the development of antiviral compounds for the treatment of arenavirus hemorrhagic fevers.

Identification of a broad-spectrum arenavirus entry inhibitor.

Several arenaviruses, including Lassa virus (LASV), are causative agents of hemorrhagic fever, for which effective therapeutic options are lacking. The LASV envelope glycoprotein (GP) gene was used to generate lentiviral pseudotypes to identify small-molecule inhibitors of viral entry. A benzimidazole derivative with potent antiviral activity was identified from a high-throughput screen utilizing this strategy. Subsequent lead optimization for antiviral activity identified a modified structure, ST-193, with a 50% inhibitory concentration (IC(50)) of 1.6 nM against LASV pseudotypes. ST-193 inhibited pseudotypes generated with other arenavirus envelopes as well, including the remaining four commonly associated with hemorrhagic fever (IC(50)s for Junín, Machupo, Guanarito, and Sabiá were in the 0.2 to 12 nM range) but exhibited no antiviral activity against pseudotypes incorporating either the GP from the LASV-related arenavirus lymphocytic choriomeningitis virus (LCMV) or the unrelated G protein from vesicular stomatitis virus, at concentrations of up to 10 microM. Determinants of ST-193 sensitivity were mapped through a combination of LASV-LCMV domain-swapping experiments, genetic selection of viral variants, and site-directed mutagenesis. Taken together, these studies demonstrate that sensitivity to ST-193 is dictated by a segment of about 30 amino acids within the GP2 subunit. This region includes the carboxy-terminal region of the ectodomain and the predicted transmembrane domain of the envelope protein, revealing a novel antiviral target within the arenavirus envelope GP.

Discovery of small molecule inhibitors of ubiquitin-like poxvirus proteinase I7L using homology modeling and covalent docking approaches.

Essential for viral replication and highly conserved among poxviridae, the vaccinia virus I7L ubiquitin-like proteinase (ULP) is an attractive target for development of smallpox antiviral drugs. At the same time, the I7L proteinase exemplifies several interesting challenges from the rational drug design perspective. In the absence of a published I7L X-ray structure, we have built a detailed 3D model of the I7L ligand binding site (S2-S2' pocket) based on exceptionally high structural conservation of this site in proteases of the ULP family. The accuracy and limitations of this model were assessed through comparative analysis of available X-ray structures of ULPs, as well as energy based conformational modeling. The 3D model of the I7L ligand binding site was used to perform covalent docking and VLS of a comprehensive library of about 230,000 available ketone and aldehyde compounds. Out of 456 predicted ligands, 97 inhibitors of I7L proteinase activity were confirmed in biochemical assays ( approximately 20% overall hit rate). These experimental results both validate our I7L ligand binding model and provide initial leads for rational optimization of poxvirus I7L proteinase inhibitors. Thus, fragments predicted to bind in the prime portion of the active site can be combined with fragments on non-prime side to yield compounds with improved activity and specificity.

N-(3,3a,4,4a,5,5a,6,6a-Octahydro-1,3-dioxo-4,6- ethenocycloprop[f]isoindol-2-(1H)-yl)carboxamides: Identification of novel orthopoxvirus egress inhibitors.

A series of novel, potent orthopoxvirus egress inhibitors was identified during high-throughput screening of the ViroPharma small molecule collection. Using structure--activity relationship information inferred from early hits, several compounds were synthesized, and compound 14 was identified as a potent, orally bioavailable first-in-class inhibitor of orthopoxvirus egress from infected cells. Compound 14 has shown comparable efficaciousness in three murine orthopoxvirus models and has entered Phase I clinical trials.