Drug-Like Small Molecules That Inhibit Expression of the Oncogenic MicroRNA-21.

We report the discovery of drug-like small molecules that bind specifically to the precursor of the oncogenic and pro-inflammatory microRNA-21 with mid-nanomolar affinity. The small molecules target a local structure at the Dicer cleavage site and induce distinctive structural changes in the RNA, which correlate with specific inhibition of miRNA processing. Structurally conservative single nucleotide substitutions eliminate the conformational change induced by the small molecules, which is also not observed in other miRNA precursors. The most potent of these compounds reduces cellular proliferation and miR-21 levels in cancer cell lines without inhibiting kinases or classical receptors, while closely related compounds without this specific binding activity are inactive in cells. These molecules are highly ligand-efficient (MW < 330) and display specific biochemical and cellular activity by suppressing the maturation of miR-21, thereby providing an avenue toward therapeutic development in multiple diseases where miR-21 is abnormally expressed.

A Slow Dynamic RNA Switch Regulates Processing of microRNA-21.

The microRNAs are non-coding RNAs which post-transcriptionally regulate the expression of many eukaryotic genes, and whose dysregulation is a driver of human disease. Here we report the discovery of a very slow (0.1 s-1) conformational rearrangement at the Dicer cleavage site of pre-miR-21, which regulates the relative concentration of readily- and inefficiently-processed RNA structural states. We show that this dynamic switch is affected by single nucleotide mutations and can be biased by small molecule and peptide ligands, which can direct the microRNA to occupy the inefficiently processed state and reduce processing efficiency. This result reveals a new mechanism of RNA regulation and suggests a chemical approach to suppressing or activating pathogenic microRNAs by selective stabilization of their unprocessed or processed states.

Efficient NMR Screening Approach to Discover Small Molecule Fragments Binding Structured RNA.

We describe a scalable nuclear magnetic resonance (NMR) screening approach to identify and prioritize small molecule fragments that bind to structured RNAs. This approach is target agnostic and, therefore, amenable to many RNA structures and libraries, and it provides initial hits for further synthetic elaboration and structure-based drug discovery efforts. We demonstrate the approach on the pre-miR-21 stem-loop, which is of significant interest in oncology and metabolic diseases. We screened the pre-miR-21 hairpin using a small (420 compounds) commercially available fragment library and identified 18 hits in the first round of triage screening. This was further refined to four fragments which passed all screening cascade filters. Among these four hits, a thiadiazole fragment was demonstrated to bind the Dicer cleavage site of pre-miR-21 by target-detected NMR experiments and through the observation of clear intermolecular NOEs.

Structure of the RNA Specialized Translation Initiation Element that Recruits eIF3 to the 5'-UTR of c-Jun.

Specialized translation initiation is a novel form of regulation of protein synthesis, whereby RNA structures within the 5'-UTR regulate translation rates of specific mRNAs. Similar to internal ribosome entry sites (IRESs), specialized translation initiation requires the recruitment of eukaryotic initiation factor 3 (eIF3), but also requires cap recognition by eIF3d, a new 5'-m7GTP recognizing protein. How these RNA structures mediate eIF3 recruitment to affect translation of specific mRNAs remains unclear. Here, we report the nuclear magnetic resonance (NMR) structure of a stem-loop within the c-JUN 5' UTR recognized by eIF3 and essential for specialized translation initiation of this well-known oncogene. The structure exhibits similarity to eIF3 recognizing motifs found in hepatitis C virus (HCV)-like IRESs, suggesting mechanistic similarities. This work establishes the RNA structural features involved in c-JUN specialized translation initiation and provides a basis to search for small molecule inhibitors of aberrant expression of the proto-oncogenic c-JUN.

An ultra-high affinity ligand of HIV-1 TAR reveals the RNA structure recognized by P-TEFb.

The HIV-1 trans-activator protein Tat binds the trans-activation response element (TAR) to facilitate recruitment of the super elongation complex (SEC) to enhance transcription of the integrated pro-viral genome. The Tat-TAR interaction is critical for viral replication and the emergence of the virus from the latent state, therefore, inhibiting this interaction has long been pursued to discover new anti-viral or latency reversal agents. However, discovering active compounds that directly target RNA with high affinity and selectivity remains a significant challenge; limiting pre-clinical development. Here, we report the rational design of a macrocyclic peptide mimic of the arginine rich motif of Tat, which binds to TAR with low pM affinity and 100-fold selectivity against closely homologous RNAs. Despite these unprecedented binding properties, the new ligand (JB181) only moderately inhibits Tat-dependent reactivation in cells and recruitment of positive transcription elongation factor (P-TEFb) to TAR. The NMR structure of the JB181-TAR complex revealed that the ligand induces a structure in the TAR loop that closely mimics the P-TEFb/Tat1:57/AFF4/TAR complex. These results strongly suggest that high-affinity ligands which bind the UCU bulge are not likely to inhibit recruitment of the SEC and suggest that targeting of the TAR loop will be an essential feature of effective Tat inhibitors.

A Small Cyclic ß-Hairpin Peptide Mimics the Rbfox2 RNA Recognition Motif and Binds to the Precursor miRNA 20b.

The RNA recognition motif (RRM), which is the most abundant RNA-binding motif in eukaryotes, is a well-structured domain of about 90 amino acids, yet the ß2ß3 hairpin, corresponding to strands 2 and 3 of the ß-sheet, and the intervening loop make essential interactions with RNA in many RRM complexes. A series of small cyclic peptide mimics of the ß2ß3 hairpin of Rbfox2 protein that recognize the terminal loop of precursor miR-20b have been designed to investigate whether the full RNA-binding protein can be mimicked with a minimal structurally preorganized peptide. Within a small library of seven cyclic peptides, a peptide with low-micromolar affinity for the miR-20b precursor was found. NMR spectroscopy titration data suggest that this peptide specifically targets the apical loop of pre-miR-20b. This work shows that it is possible to mimic RNA-binding proteins with designed stable peptides, which provide a starting point for designing or evolving small peptide mimetics of RRM proteins.

Comprehensive computational design of ordered peptide macrocycles.

Mixed-chirality peptide macrocycles such as cyclosporine are among the most potent therapeutics identified to date, but there is currently no way to systematically search the structural space spanned by such compounds. Natural proteins do not provide a useful guide: Peptide macrocycles lack regular secondary structures and hydrophobic cores, and can contain local structures not accessible with l-amino acids. Here, we enumerate the stable structures that can be adopted by macrocyclic peptides composed of l- and d-amino acids by near-exhaustive backbone sampling followed by sequence design and energy landscape calculations. We identify more than 200 designs predicted to fold into single stable structures, many times more than the number of currently available unbound peptide macrocycle structures. Nuclear magnetic resonance structures of 9 of 12 designed 7- to 10-residue macrocycles, and three 11- to 14-residue bicyclic designs, are close to the computational models. Our results provide a nearly complete coverage of the rich space of structures possible for short peptide macrocycles and vastly increase the available starting scaffolds for both rational drug design and library selection methods.

A Macrocyclic Peptide Ligand Binds the Oncogenic MicroRNA-21 Precursor and Suppresses Dicer Processing.

MicroRNAs (miRNAs) help orchestrate cellular growth and survival through post-transcriptional mechanisms. The dysregulation of miRNA biogenesis can lead to cellular growth defects and chemotherapeutic resistance and plays a direct role in the development of many chronic diseases. Among these RNAs, miR-21 is consistently overexpressed in most human cancers, leading to the down-regulation of key tumor-suppressing and pro-apoptotic factors, suggesting that inhibition of miR-21 biogenesis could reverse these negative effects. However, targeted inhibition of miR-21 using small molecules has had limited success. To overcome difficulties in targeting RNA secondary structure with small molecules, we developed a class of cyclic ß-hairpin peptidomimetics which bind to RNA stem-loop structures, such as miRNA precursors, with potent affinity and specificity. We screened an existing cyclic peptide library and discovered a lead structure which binds to pre-miR21 with KD = 200 nM and prefers it over other pre-miRNAs. The NMR structure of the complex shows that the peptide recognizes the Dicer cleavage site and alters processing of the precursor to the mature miRNA in vitro and in cultured cells. The structure provides a rationale for the peptide binding activity and clear guidance for further improvements in affinity and targeting.

Identification of a Ligand-Binding Site on the Staphylococcus aureus DnaG Primase C-Terminal Domain.

The interface between the DnaG primase C-terminal domain (CTD) and the N-terminal domain of DnaB helicase is essential for bacterial DNA replication because it allows coordinated priming of DNA synthesis at the replication fork while the DNA is being unwound. Because these two proteins are conserved in all bacteria and distinct from those in eukaryotes, their interface is an attractive antibiotic target. To learn more about this interface, we determined the solution structure and dynamics of the DnaG primase CTD from Staphylococcus aureus, a medically important bacterial species. Comparison with the known primase CTD structures shows there are two biologically relevant conformations, an open conformation that likely binds to DnaB helicase and a closed conformation that does not. The S. aureus primase CTD is in the closed conformation, but nuclear magnetic resonance (NMR) dynamic studies indicate there is considerable movement in the linker between the two subdomains and that N564 is the most dynamic residue within the linker. A high-throughput NMR ligand affinity screen identified potential binding compounds, among which were acycloguanosine and myricetin. Although the affinity for these compounds and adenosine was in the millimolar range, all three bind to a common pocket that is present only on the closed conformation of the CTD. This binding pocket is at the opposite end of helices 6 and 7 from N564, the key hinge residue. The identification of this binding pocket should allow the development of stronger-binding ligands that can prevent formation of the CTD open conformation that binds to DnaB helicase.

Development of Small Molecules with a Noncanonical Binding Mode to HIV-1 Trans Activation Response (TAR) RNA.

Small molecules that bind to RNA potently and specifically are relatively rare. The study of molecules that bind to the HIV-1 transactivation response (TAR) hairpin, a cis-acting HIV genomic element, has long been an important model system for the chemistry of targeting RNA. Here we report the synthesis, biochemical, and structural evaluation of a series of molecules that bind to HIV-1 TAR RNA. A promising analogue, 15, retained the TAR binding affinity of the initial hit and displaced a Tat-derived peptide with an IC50 of 40 µM. NMR characterization of a soluble analogue, 2, revealed a noncanonical binding mode for this class of compounds. Finally, evaluation of 2 and 15 by selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) indicates specificity in binding to TAR within the context of an in vitro-synthesized 365-nt HIV-1 5'-untranslated region (UTR). Thus, these compounds exhibit a novel and specific mode of interaction with TAR, providing important suggestions for RNA ligand design.

Structure based approaches for targeting non-coding RNAs with small molecules.

The increasing appreciation of the central role of non-coding RNAs (miRNAs and long non-coding RNAs) in chronic and degenerative human disease makes them attractive therapeutic targets. This would not be unprecedented: the bacterial ribosomal RNA is a mainstay for antibacterial treatment, while the conservation and functional importance of viral RNA regulatory elements has long suggested they would constitute attractive targets for new antivirals. Oligonucleotide-based chemistry has obvious appeals but also considerable pharmacological limitations that are yet to be addressed satisfactorily. Recent studies identifying small molecules targeting non-coding RNAs may provide an alternative approach to oligonucleotide methods. Here we review recent work investigating new structural and chemical principles for targeting RNA with small molecules.

¹H, ¹³C, and ¹5N NMR assignments for the helicase interaction domain of Staphylococcus aureus DnaG primase.

The interaction between DnaG primase and DnaB helicase is essential for stimulating primer synthesis during bacterial DNA replication. The interaction occurs between the N-terminal domain of helicase and the C-terminal domain of primase. Here we present the (1)H, (13)C, and (15)N backbone and side-chain resonance assignments for the C-terminal helicase interaction domain of Staphylococcus aureus primase.

Correlation between protein function and ligand binding profiles.

We report that proteins with the same function bind the same set of small molecules from a standardized chemical library. This observation led to a quantifiable and rapidly adaptable method for protein functional analysis using experimentally derived ligand binding profiles. Ligand binding is measured using a high-throughput NMR ligand affinity screen with a structurally diverse chemical library. The method was demonstrated using a set of 19 proteins with a range of functions. A statistically significant similarity in ligand binding profiles was only observed between the two functionally identical albumins and between the five functionally similar amylases. This new approach is independent of sequence, structure, or evolutionary information and, therefore, extends our ability to analyze and functionally annotate novel genes.

Bacterial protein structures reveal phylum dependent divergence.

Protein sequence space is vast compared to protein fold space. This raises important questions about how structures adapt to evolutionary changes in protein sequences. A growing trend is to regard protein fold space as a continuum rather than a series of discrete structures. From this perspective, homologous protein structures within the same functional classification should reveal a constant rate of structural drift relative to sequence changes. The clusters of orthologous groups (COG) classification system was used to annotate homologous bacterial protein structures in the Protein Data Bank (PDB). The structures and sequences of proteins within each COG were compared against each other to establish their relatedness. As expected, the analysis demonstrates a sharp structural divergence between the bacterial phyla Firmicutes and Proteobacteria. Additionally, each COG had a distinct sequence/structure relationship, indicating that different evolutionary pressures affect the degree of structural divergence. However, our analysis also shows the relative drift rate between sequence identity and structure divergence remains constant.

PROFESS: a PROtein function, evolution, structure and sequence database.

The proliferation of biological databases and the easy access enabled by the Internet is having a beneficial impact on biological sciences and transforming the way research is conducted. There are approximately 1100 molecular biology databases dispersed throughout the Internet. To assist in the functional, structural and evolutionary analysis of the abundant number of novel proteins continually identified from whole-genome sequencing, we introduce the PROFESS (PROtein Function, Evolution, Structure and Sequence) database. Our database is designed to be versatile and expandable and will not confine analysis to a pre-existing set of data relationships. A fundamental component of this approach is the development of an intuitive query system that incorporates a variety of similarity functions capable of generating data relationships not conceived during the creation of the database. The utility of PROFESS is demonstrated by the analysis of the structural drift of homologous proteins and the identification of potential pancreatic cancer therapeutic targets based on the observation of protein-protein interaction networks. Database URL: http://cse.unl.edu/~profess/

Analysis of metabolomic PCA data using tree diagrams.

Large amounts of data from high-throughput metabolomic experiments are commonly visualized using a principal component analysis (PCA) two-dimensional scores plot. The question of the similarity or difference between multiple metabolic states then becomes a question of the degree of overlap between their respective data point clusters in principal component (PC) scores space. A qualitative visual inspection of the clustering pattern in PCA scores plots is a common protocol. This article describes the application of tree diagrams and bootstrapping techniques for an improved quantitative analysis of metabolic PCA data clustering. Our PCAtoTree program creates a distance matrix with 100 bootstrap steps that describes the separation of all clusters in a metabolic data set. Using accepted phylogenetic software, the distance matrix resulting from the various metabolic states is organized into a phylogenetic-like tree format, where bootstrap values 50 indicate a statistically relevant branch separation. PCAtoTree analysis of two previously published data sets demonstrates the improved resolution of metabolic state differences using tree diagrams. In addition, for metabolomic studies of large numbers of different metabolic states, the tree format provides a better description of similarities and differences between each metabolic state. The approach is also tolerant of sample size variations between different metabolic states.

Structural and functional similarity between the bacterial type III secretion system needle protein PrgI and the eukaryotic apoptosis Bcl-2 proteins.

BACKGROUND: Functional similarity is challenging to identify when global sequence and structure similarity is low. Active-sites or functionally relevant regions are evolutionarily more stable relative to the remainder of a protein structure and provide an alternative means to identify potential functional similarity between proteins. We recently developed the FAST-NMR methodology to discover biochemical functions or functional hypotheses of proteins of unknown function by experimentally identifying ligand binding sites. FAST-NMR utilizes our CPASS software and database to assign a function based on a similarity in the structure and sequence of ligand binding sites between proteins of known and unknown function. METHODOLOGY/PRINCIPAL FINDINGS: The PrgI protein from Salmonella typhimurium forms the needle complex in the type III secretion system (T3SS). A FAST-NMR screen identified a similarity between the ligand binding sites of PrgI and the Bcl-2 apoptosis protein Bcl-xL. These ligand binding sites correlate with known protein-protein binding interfaces required for oligomerization. Both proteins form membrane pores through this oligomerization to release effector proteins to stimulate cell death. Structural analysis indicates an overlap between the PrgI structure and the pore forming motif of Bcl-xL. A sequence alignment indicates conservation between the PrgI and Bcl-xL ligand binding sites and pore formation regions. This active-site similarity was then used to verify that chelerythrine, a known Bcl-xL inhibitor, also binds PrgI. CONCLUSIONS/SIGNIFICANCE: A structural and functional relationship between the bacterial T3SS and eukaryotic apoptosis was identified using our FAST-NMR ligand affinity screen in combination with a bioinformatic analysis based on our CPASS program. A similarity between PrgI and Bcl-xL is not readily apparent using traditional global sequence and structure analysis, but was only identified because of conservation in ligand binding sites. These results demonstrate the unique opportunity that ligand-binding sites provide for the identification of functional relationships when global sequence and structural information is limited.

A multi-step NMR screen for the identification and evaluation of chemical leads for drug discovery.

A multi-step NMR based screening assay is described for identifying and evaluating chemical leads for their ability to bind a target protein. The multi-step NMR assay provides structure-related information while being an integral part of a structure based drug discovery and design program. The fundamental principle of the multi-step NMR assay is to combine distinct 1D and 2D NMR techniques, in such a manner, that the inherent strengths and weakness associated with each technique is complementary to each other in the screen. By taking advantage of the combined strengths of 1D and 2D NMR experiments, it is possible to minimize protein requirements and experiment time and differentiate between non-specific and stoichiometric binders while being able to verify ligand binding, determine a semi-quantitative dissociation constant, identify the ligand binding site and rapidly determine a protein-ligand co-structure. Furthermore, the quality and physical behavior of the ligand is readily evaluated to determine its appropriateness as a chemical lead. The utility of the multi-step NMR assay is demonstrated with the use of PrgI from Salmonella typhimurium and human serum albumin (HSA) as target proteins.

Structure and function of Pseudomonas aeruginosa protein PA1324 (21-170).

Pseudomonas aeruginosa is the prototypical biofilm-forming gram-negative opportunistic human pathogen. P. aeruginosa is causatively associated with nosocomial infections and with cystic fibrosis. Antibiotic resistance in some strains adds to the inherent difficulties that result from biofilm formation when treating P. aeruginosa infections. Transcriptional profiling studies suggest widespread changes in the proteome during quorum sensing and biofilm development. Many of the proteins found to be upregulated during these processes are poorly characterized from a functional standpoint. Here, we report the solution NMR structure of PA1324, a protein of unknown function identified in these studies, and provide a putative biological functional assignment based on the observed prealbumin-like fold and FAST-NMR ligand screening studies. PA1324 is postulated to be involved in the binding and transport of sugars or polysaccharides associated with the peptidoglycan matrix during biofilm formation.

Estimating protein-ligand binding affinity using high-throughput screening by NMR.

Many of today's drug discovery programs use high-throughput screening methods that rely on quick evaluations of protein activity to rank potential chemical leads. By monitoring biologically relevant protein-ligand interactions, NMR can provide a means to validate these discovery leads and to optimize the drug discovery process. NMR-based screens typically use a change in chemical shift or line width to detect a protein-ligand interaction. However, the relatively low throughput of current NMR screens and their high demand on sample requirements generally makes it impractical to collect complete binding curves to measure the affinity for each compound in a large and diverse chemical library. As a result, NMR ligand screens are typically limited to identifying candidates that bind to a protein and do not give any estimate of the binding affinity. To address this issue, a methodology has been developed to rank binding affinities for ligands based on NMR screens that use 1D (1)H NMR line-broadening experiments. This method was demonstrated by using it to estimate the dissociation equilibrium constants for twelve ligands with the protein human serum albumin (HSA). The results were found to give good agreement with previous affinities that have been reported for these same ligands with HSA.