Cyanotriazoles are selective topoisomerase II poisons that rapidly cure trypanosome infections.

Millions who live in Latin America and sub-Saharan Africa are at risk of trypanosomatid infections, which cause Chagas disease and human African trypanosomiasis (HAT). Improved HAT treatments are available, but Chagas disease therapies rely on two nitroheterocycles, which suffer from lengthy drug regimens and safety concerns that cause frequent treatment discontinuation. We performed phenotypic screening against trypanosomes and identified a class of cyanotriazoles (CTs) with potent trypanocidal activity both in vitro and in mouse models of Chagas disease and HAT.

Biochemical Characterization of Respiratory Syncytial Virus RNA-Dependent RNA Polymerase Complex.

RNA-dependent RNA polymerases (RdRPs) from nonsegmented negative strand (NNS) RNA viruses perform both mRNA transcription and genome replication, and these activities are regulated by their interactions with RNA and other accessory proteins within the ribonucleoprotein (RNP) complex. Detailed biochemical characterization of these enzymatic activities and their regulation is essential for understanding the life cycles of many pathogenic RNA viruses and for antiviral drug discovery. We developed biochemical and biophysical kinetic methods to study the RNA synthesis and RNA binding activities of respiratory syncytial virus (RSV) L/P RdRP.

Inhibition of IRF5 cellular activity with cell-penetrating peptides that target homodimerization.

The transcription factor interferon regulatory factor 5 (IRF5) plays essential roles in pathogen-induced immunity downstream of Toll-, nucleotide-binding oligomerization domain-, and retinoic acid-inducible gene I-like receptors and is an autoimmune susceptibility gene. Normally, inactive in the cytoplasm, upon stimulation, IRF5 undergoes posttranslational modification(s), homodimerization, and nuclear translocation, where dimers mediate proinflammatory gene transcription. Here, we report the rational design of cell-penetrating peptides (CPPs) that disrupt IRF5 homodimerization.

Two Distinct Mechanisms of Inhibition of LpxA Acyltransferase Essential for Lipopolysaccharide Biosynthesis.

The lipopolysaccharide biosynthesis pathway is considered an attractive drug target against the rising threat of multi-drug-resistant Gram-negative bacteria. Here, we report two novel small-molecule inhibitors (compounds and ) of the acyltransferase LpxA, the first enzyme in the lipopolysaccharide biosynthesis pathway. We show genetically that the antibacterial activities of the compounds against efflux-deficient are mediated by LpxA inhibition.

A polyomavirus peptide binds to the capsid VP1 pore and has potent antiviral activity against BK and JC polyomaviruses.

In pursuit of therapeutics for human polyomaviruses, we identified a peptide derived from the BK polyomavirus (BKV) minor structural proteins VP2/3 that is a potent inhibitor of BKV infection with no observable cellular toxicity. The thirteen-residue peptide binds to major structural protein VP1 with single-digit nanomolar affinity. Alanine-scanning of the peptide identified three key residues, substitution of each of which results in ~1000 fold loss of binding affinity with a concomitant reduction in antiviral activity.

Structural and Biological Basis of Small Molecule Inhibition of LpxD Acyltransferase Essential for Lipopolysaccharide Biosynthesis.

LpxD, acyl-ACP-dependent -acyltransferase, is the third enzyme of lipid A biosynthesis in Gram-negative bacteria. A recent probe-based screen identified several compounds, including 6359-0284 (compound ), that inhibit the enzymatic activity of () LpxD. Here, we use these inhibitors to chemically validate LpxD as an attractive antibacterial target.

Biased Complement Diversity Selection for Effective Exploration of Chemical Space in Hit-Finding Campaigns.

The success of hit-finding campaigns relies on many factors, including the quality and diversity of the set of compounds that is selected for screening. This paper presents a generalized workflow that guides compound selections from large compound archives with opportunities to bias the selections with available knowledge in order to improve hit quality while still effectively sampling the accessible chemical space. An optional flag in the workflow supports an explicit complement design function where diversity selections complement a given core set of compounds.

Structural basis for therapeutic inhibition of influenza A polymerase PB2 subunit.

Influenza virus uses a unique mechanism to initiate viral transcription named cap-snatching. The PB2 subunit of the viral heterotrimeric RNA polymerase binds the cap structure of cellular pre-mRNA to promote its cleavage by the PA subunit. The resulting 11-13 capped oligomer is used by the PB1 polymerase subunit to initiate transcription of viral proteins.

Inhibition of prenylated KRAS in a lipid environment.

RAS mutations lead to a constitutively active oncogenic protein that signals through multiple effector pathways. In this chemical biology study, we describe a novel coupled biochemical assay that measures activation of the effector BRAF by prenylated KRASG12V in a lipid-dependent manner. Using this assay, we discovered compounds that block biochemical and cellular functions of KRASG12V with low single-digit micromolar potency.

Molecular Basis of mRNA Cap Recognition by Influenza B Polymerase PB2 Subunit.

Influenza virus polymerase catalyzes the transcription of viral mRNAs by a process known as "cap-snatching," where the 5'-cap of cellular pre-mRNA is recognized by the PB2 subunit and cleaved 10-13 nucleotides downstream of the cap by the endonuclease PA subunit. Although this mechanism is common to both influenza A (FluA) and influenza B (FluB) viruses, FluB PB2 recognizes a wider range of cap structures including m(7)GpppGm-, m(7)GpppG-, and GpppG-RNA, whereas FluA PB2 utilizes methylated G-capped RNA specifically. Biophysical studies with isolated PB2 cap-binding domain (PB2(cap)) confirm that FluB PB2 has expanded mRNA cap recognition capability, although the affinities toward m(7)GTP are significantly reduced when compared with FluA PB2.

Deconstruction of a nutlin: dissecting the binding determinants of a potent protein-protein interaction inhibitor.

Protein-protein interaction (PPI) systems represent a rich potential source of targets for drug discovery, but historically have proven to be difficult, particularly in the lead identification stage. Application of the fragment-based approach may help toward success with this target class. To provide an example toward understanding the potential issues associated with such an application, we have deconstructed one of the best established protein-protein inhibitors, the Nutlin series that inhibits the interaction between MDM2 and p53, into fragments, and surveyed the resulting binding properties using heteronuclear single quantum coherence nuclear magnetic resonance (HSQC NMR), surface plasmon resonance (SPR), and X-ray crystallography.

Metal impurities cause false positives in high-throughput screening campaigns.

Organic impurities in compound libraries are known to often cause false-positive signals in screening campaigns for new leads, but organic impurities do not fully account for all false-positive results. We discovered inorganic impurities in our screening library that can also cause positive signals for a variety of targets and/or readout systems, including biochemical and biosensor assays. We investigated in depth the example of zinc for a specific project and in retrospect in various HTS screens at Roche and propose a straightforward counter screen using the chelator TPEN to rule out inhibition caused by zinc.

Biosensor-based small molecule fragment screening with biolayer interferometry.

Biosensor-based fragment screening is a valuable tool in the drug discovery process. This method is advantageous over many biochemical methods because primary hits can be distinguished from non-specific or non-ideal interactions by examining binding profiles and responses, resulting in reduced false-positive rates. Biolayer interferometry (BLI), a technique that measures changes in an interference pattern generated from visible light reflected from an optical layer and a biolayer containing proteins of interest, is a relatively new method for monitoring small molecule interactions.

Label-free detection of biomolecular interactions using BioLayer interferometry for kinetic characterization.

The analysis of biomolecular interactions is key in the drug development process. Label-free biosensor methods provide information on binding, kinetics, concentration, and the affinity of an interaction. These techniques provide real-time monitoring of interactions between an immobilized ligand (such as a receptor) to an analyte in solution without the use of labels.

Enhancement of the efficacy of an antagonist of an extracellular receptor by attachment to the surface of a biocompatible carrier.

Purpose: In order to improve the in vitro and in vivo efficacy of an integrin antagonist (IA) of the extracellular domain of the alphavbeta3 integrin, a receptor upregulated on tumor neovasculature, the IA was attached to the surface of a dextran-coated liposome (DCL). IA-DCLs were characterized in vitro, and the pharmacokinetic and antitumor properties were assessed in vivo.

Methods: The in vitro binding properties were measured with purified integrin, endothelial cells, and melanoma cells.

A novel antiangiogenesis therapy using an integrin antagonist or anti-Flk-1 antibody coated 90Y-labeled nanoparticles.

Purpose: Integrin alpha(v)beta(3) and vascular endothelial growth factor receptor 2 (Flk-1) have been shown to be involved in tumor-induced angiogenesis. Selective targeting of upregulated alpha(v)beta(3) and Flk-1 on the neovasculature of tumors is a novel antiangiogenesis strategy for treating a wide variety of solid tumors. In the studies described here, we investigated the potential therapeutic efficacy of two three-component treatment regimens using two murine tumor models.