John S. Schneekloth

Small Molecule Microarrays Enable the Identification of a Selective, Quadruplex-Binding Inhibitor of MYC Expression

The transcription factor MYC plays a pivotal role in cancer initiation, progression, and maintenance. However, it has proven difficult to develop small molecule inhibitors of MYC. One attractive route to pharmacological inhibition of MYC has been the prevention of its expression through small molecule-mediated stabilization of the G-quadruplex (G4) present in its promoter. Although molecules that bind globally to quadruplex DNA and influence gene expression are well-known, the identification of new chemical scaffolds that selectively modulate G4-driven genes remains a challenge. Here, we report an approach for the identification of G4-binding small molecules using small molecule microarrays (SMMs). We use the SMM screening platform to identify a novel G4-binding small molecule that inhibits MYC expression in cell models, with minimal impact on the expression of other G4-associated genes. Surface plasmon resonance (SPR) and thermal melt assays demonstrated that this molecule binds reversibly to the MYC G4 with single digit micromolar affinity, and with weaker or no measurable binding to other G4s. Biochemical and cell-based assays demonstrated that the compound effectively silenced MYC transcription and translation via a G4-dependent mechanism of action. The compound induced G1 arrest and was selectively toxic to MYC-driven cancer cell lines containing the G4 in the promoter but had minimal effects in peripheral blood mononucleocytes or a cell line lacking the G4 in its MYC promoter. As a measure of selectivity, gene expression analysis and qPCR experiments demonstrated that MYC and several MYC target genes were downregulated upon treatment with this compound, while the expression of several other G4-driven genes was not affected. In addition to providing a novel chemical scaffold that modulates MYC expression through G4 binding, this work suggests that the SMM screening approach may be broadly useful as an approach for the identification of new G4-binding small molecules.

Identification of Biologically Active, HIV TAR RNA-Binding Small Molecules Using Small Molecule Microarrays

Identifying small molecules that selectively bind to structured RNA motifs remains an important challenge in developing potent and specific therapeutics. Most strategies to find RNA-binding molecules have identified highly charged compounds or aminoglycosides that commonly have modest selectivity. Here we demonstrate a strategy to screen a large unbiased library of druglike small molecules in a microarray format against an RNA target. This approach has enabled the identification of a novel chemotype that selectively targets the HIV transactivation response (TAR) RNA hairpin in a manner not dependent on cationic charge. Thienopyridine 4 binds to and stabilizes the TAR hairpin with a Kd of 2.4 μM. Structure–activity relationships demonstrate that this compound achieves activity through hydrophobic and aromatic substituents on a heterocyclic core, rather than cationic groups typically required. Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) analysis was performed on a 365-nucleotide sequence derived from the 5′ untranslated region (UTR) of the HIV-1 genome to determine global structural changes in the presence of the molecule. Importantly, the interaction of compound 4 can be mapped to the TAR hairpin without broadly disrupting any other structured elements of the 5′ UTR. Cell-based anti-HIV assays indicated that 4 inhibits HIV-induced cytopathicity in T lymphocytes with an EC50 of 28 μM, while cytotoxicity was not observed at concentrations approaching 1 mM.

Chemical approaches to controlling intracellular protein degradation.

A major goal of post-genomic research is to understand and control the function of specific proteins within the proteome. The fields of genetics, genomics, and chemical genetics attempt to study this problem from two fundamentally different approaches. The traditional genetic approach or genomic analysis involves the observation of a particular phenotype, identification of the molecule(s) responsible for that phenotype, and subsequent analysis and manipulation of the corresponding genetic sequence. In contrast, chemical genetics attempts to use small molecules as probes to perturb signaling pathways at the molecular level in the hopes of identifying novel proteins responsible for a particular phenotype. These two distinct approaches (among others) have yielded huge advances in the understanding of protein function. Despite their differences, these fields share the same strategy of inactivation of a protein to study its function. Regulation of protein expression can be described as occurring on three basic levels. First, at the genetic level, the strength of a promoter determines the level of a particular gene product. Second, at the post-transcriptional level, stability of mRNA levels lead to increased production of a protein. Finally, at the post-translational level, modifications such as gly-cosylation, phosphorylation, or degradation significantly affect both intracellular levels and the activity of a protein. The most effective way to study the function of a protein has traditionally been to observe the phenotypic change in its absence. Therefore, methods at each of these three levels have been developed to disrupt protein expression. Many ways to control protein function are known, including inducible transcription[1] and methods that affect post-translational modifications[2–4] or inhibit degradation.[5] Although these methods are effective, they are fundamentally different from strategies that lower protein levels post-translationally. As genetic knockout methodology is well understood, this review focuses on recent advances in protein inactivation at the post-translational level, specifically comparing novel chemical and biochemical methods to the post-transcriptional method of inactivation, RNA interference (RNAi).

Natural product inhibitors of the ubiquitin-proteasome pathway.

Natural products continue to be a source of inspiration for chemists and biologists alike. The search for biologically active natural prodcuts has provided troves of information about biological processes, and natural products continue to be some of the most powerful and useful probes of biological processes available. Complex and unusual molecular architectures provide the impetus for new reaction development and push the limits of known synthetic chemistry. In addition to serving as tools for basic research, natural products represent starting points for drug discovery efforts in many cases. This review details the impressive chemical diversity present in the family of molecules that inhibit the proteasome and other aspects of the ubiquitin-proteasome pathway. To date, over 60 natural products that inhibit the ubiquitin-proteasome pathway have been identified. Although early work recognized inhibitors of the proteasome itself, more modern research has revealed that inhibition of many aspects of this critical pathway is possible. One powerful example of this is the inhibition of the interaction between p53 and MDM2 (an E3 ubiquitin ligase). Natural product proteasome inhibitors have served as powerful tools to unravel the intricacies of the ubiquitin-proteasome and related pathways. Several of these natural products have been developed into anticancer drug candidates, and one proteasome inhibitor has already been approved for the treatment of multiple myeloma. Despite the wealth of information available about naturally occurring proteasome inhibitors and related compounds, it is clear that exciting research about this class of molecules will continue well into the future.

Discovery of Inhibitors of MicroRNA-21 Processing Using Small Molecule Microarrays

The identification of small molecules that bind to and perturb the function of microRNAs is an attractive approach for the treatment for microRNA-associated pathologies. However, there are only a few small molecules known to interact directly with microRNAs. Here, we report the use of a small molecule microarray (SMM) screening approach to identify low molecular weight compounds that directly bind to a pre-miR-21 hairpin. Compounds identified using this approach exhibit good affinity for the RNA (ranging from 0.8-2.0 μM) and are not composed of a polycationic scaffold. Several of the highest affinity compounds inhibit Dicer-mediated processing, while in-line probing experiments indicate that the compounds bind to the apical loop of the hairpin, proximal to the Dicer site. This work provides evidence that small molecules can be developed to bind directly to and inhibit miR-21.

Macrophilone A: Structure Elucidation, Total Synthesis, and Functional Evaluation of a Biologically Active Iminoquinone from the Marine Hydroid Macrorhynchia philippina

A previously uncharacterized pyrroloiminoquinone natural product, macrophilone A, was isolated from the stinging hydroid Macrorhynchia philippina. The structure was assigned utilizing long-range NMR couplings and DFT calculations and proved by a concise, five-step total synthesis. Macrophilone A and a synthetic analogue displayed potent biological activity, including increased intracellular reactive oxygen species levels and submicromolar cytotoxicity toward lung adenocarcinoma cells.

Characterization of clinically used oral antiseptics as quadruplex-binding ligands

Abstract Approaches to characterize the nucleic acid-binding properties of drugs and druglike small molecules are crucial to understanding the behavior of these compounds in cellular systems. Here, we use a Small Molecule Microarray (SMM) profiling approach to identify the preferential interaction between chlorhexidine, a widely used oral antiseptic, and the G-quadruplex (G4) structure in the KRAS oncogene promoter. The interaction of chlorhexidine and related drugs to the KRAS G4 is evaluated using multiple biophysical methods, including thermal melt, fluorescence titration and surface plasmon resonance (SPR) assays. Chlorhexidine has a specific low micromolar binding interaction with the G4, while related drugs have weaker and/or less specific interactions. Through NMR experiments and docking studies, we propose a plausible binding mode driven by both aromatic stacking and groove binding interactions. Additionally, cancer cell lines harbouring oncogenic mutations in the KRAS gene exhibit increased sensitivity to chlorhexidine. Treatment of breast cancer cells with chlorhexidine decreases KRAS protein levels, while a KRAS gene transiently expressed by a promoter lacking a G4 is not affected. This work confirms that known ligands bind broadly to G4 structures, while other drugs and druglike compounds can have more selective interactions that may be biologically relevant.

Measuring RNA–Ligand Interactions with Microscale Thermophoresis

In recent years, there has been dramatic growth in the study of RNA. RNA has gone from being known as an intermediate in the central dogma of molecular biology to a molecule with a large diversity of structure and function that is involved in all aspects of biology. As new functions are rapidly discovered, it has become clear that there is a need for RNA-targeting small molecule probes to investigate RNA biology and clarify the potential for therapeutics based on RNA-small molecule interactions. While a host of techniques exist to measure RNA-small molecule interactions, many of these have drawbacks that make them intractable for routine use and are often not broadly applicable. A newer technology called microscale thermophoresis (MST), which measures the directed migration of a molecule and/or molecule-ligand complex along a temperature gradient, can be used to measure binding affinities using very small amounts of sample. The high sensitivity of this technique enables measurement of affinity constants in the nanomolar and micromolar range. Here, we demonstrate how MST can be used to study a range of biologically relevant RNA interactions, including peptide-RNA interactions, RNA-small molecule interactions, and displacement of an RNA-bound peptide by a small molecule.

Chemical and structural studies provide a mechanistic basis for recognition of the MYC G-quadruplex

G-quadruplexes (G4s) are noncanonical DNA structures that frequently occur in the promoter regions of oncogenes, such as MYC, and regulate gene expression. Although G4s are attractive therapeutic targets, ligands capable of discriminating between different G4 structures are rare. Here, we describe DC-34, a small molecule that potently downregulates MYC transcription in cancer cells by a G4-dependent mechanism. Inhibition by DC-34 is significantly greater for MYC than other G4-driven genes. We use chemical, biophysical, biological, and structural studies to demonstrate a molecular rationale for the recognition of the MYC G4. We solve the structure of the MYC G4 in complex with DC-34 by NMR spectroscopy and illustrate specific contacts responsible for affinity and selectivity. Modification of DC-34 reveals features required for G4 affinity, biological activity, and validates the derived NMR structure. This work advances the design of quadruplex-interacting small molecules to control gene expression in therapeutic areas such as cancer.Targeting noncoding nucleic acids with small molecules represents an important and significant challenge in chemical biology and drug discovery. Here the authors characterize DC-34, a small molecule that exhibits selective binding to specific G4 structures, and provide a structural basis for its selectivity

Macrophilones from the Marine Hydroid Macrorhynchia philippina Can Inhibit ERK Cascade Signaling

Six new macrophilone-type pyrroloiminoquines were isolated and identified from an extract of the marine hydroid Macrorhynchia philippina. The proton-deficient and heteroatom-rich structures of macrophilones B-G (2-7) were elucidated by spectroscopic analysis and comparison of their data with those of the previously reported metabolite macrophilone A (1). Compounds 1-7 are the first pyrroloiminoquines to be reported from a hydroid. The macrophilones were shown to inhibit the enzymatic conjugation of SUMO to peptide substrates, and macrophilones A (1) and C (3) exhibit potent and selective cytotoxic properties in the NCI-60 anticancer screen. Bioinformatic analysis revealed a close association of the cytotoxicity profiles of 1 and 3 with two known B-Raf kinase inhibitory drugs. While compounds 1 and 3 showed no kinase inhibitory activity, they resulted in a dramatic decrease in cellular protein levels of selected components of the ERK signal cascade. As such, the chemical scaffold of the macrophilones could provide small-molecule therapeutic leads that target the ERK signal transduction pathway.