Natural‐Product‐Like Spiroketals and Fused Bicyclic Acetals as Potential Therapeutic Agents for B‐Cell Chronic Lymphocytic Leukaemia

B‐cell chronic lymphocytic leukaemia (CLL) is the most common form of leukaemia in the Western world for which no curative treatments are currently available. Purine nucleotide analogues and alkylating agents feature frequently in combination regimens to treat the malignant state, but their use has not led to any significant improvement in patient survival. Consequently, there still remains a need for alternative small‐molecule chemotherapeutics. Natural products are an unparalleled source of drug leads, and an unending inspiration for the design of small‐molecule libraries for drug discovery. The screening of focused libraries of natural‐product‐like spiroketal and fused bicyclic acetal small molecules against primary CLL cells has led to the identification of a small series of novel and potent cytotoxic agents towards primary CLL cells. The validation of the activity of these molecules is delineated through a series of synthesis and screening iterations, whereas preliminary mode of action studies positively indicate their ability to induce cell death via an apoptotic pathway with no evidence of necrosis to further support their potential as novel chemotherapeutic agents.     

Cisplatin Targeting of Bacterial Ribosomal RNA Hairpins

Cisplatin is a clinically important chemotherapeutic agent known to target purine bases in nucleic acids. In addition to major deoxyribonucleic acid (DNA) intrastrand cross-links, cisplatin also forms stable adducts with many types of ribonucleic acid (RNA) including siRNA, spliceosomal RNAs, tRNA, and rRNA. All of these RNAs play vital roles in the cell, such as catalysis of protein synthesis by rRNA, and therefore serve as potential drug targets. This work focused on platination of two highly conserved RNA hairpins from E. coli ribosomes, namely pseudouridine-modified helix 69 from 23S rRNA and the 790 loop of helix 24 from 16S rRNA. RNase T1 probing, MALDI mass spectrometry, and dimethyl sulfate mapping revealed platination at GpG sites. Chemical probing results also showed platination-induced RNA structural changes. These findings reveal solvent and structural accessibility of sites within bacterial RNA secondary structures that are functionally significant and therefore viable targets for cisplatin as well as other classes of small molecules. Identifying target preferences at the nucleotide level, as well as determining cisplatin-induced RNA conformational changes, is important for the design of more potent drug molecules. Furthermore, the knowledge gained through studies of RNA-targeting by cisplatin is applicable to a broad range of organisms from bacteria to human.     

Non-Coding RNA-Driven Regulation of rRNA Biogenesis

Ribosomal RNA (rRNA) biogenesis takes place in the nucleolus, the most prominent condensate of the eukaryotic nucleus. The proper assembly and integrity of the nucleolus reflects the accurate synthesis and processing of rRNAs which in turn, as major components of ribosomes, ensure the uninterrupted flow of the genetic information during translation. Therefore, the abundant production of rRNAs in a precisely functional nucleolus is of outmost importance for the cell viability and requires the concerted action of essential enzymes, associated factors and epigenetic marks. The coordination and regulation of such an elaborate process depends on not only protein factors, but also on numerous regulatory non-coding RNAs (ncRNAs). Herein, we focus on RNA-mediated mechanisms that control the synthesis, processing and modification of rRNAs in mammals. We highlight the significance of regulatory ncRNAs in rRNA biogenesis and the maintenance of the nucleolar morphology, as well as their role in human diseases and as novel druggable molecular targets.     

Receptor‐Based Virtual Screening and Biological Characterization of Human Apurinic/Apyrimidinic Endonuclease (Ape1) Inhibitors

The endonucleolytic activity of human apurinic/apyrimidinic endonuclease (AP endo, Ape1) is a major factor in maintaining the integrity of the genome. Conversely, as an undesired effect, Ape1 overexpression has been linked to resistance to radio‐ and chemotherapeutic treatments in several human tumors. Inhibition of Ape1 using siRNA or the expression of a dominant negative form of the protein has been shown to sensitize cells to DNA‐damaging agents, including various chemotherapeutic agents. Therefore, inhibition of the enzymatic activity of Ape1 might result in a potent antitumor therapy. A number of small molecules have been described as Ape1 inhibitors; however, those compounds are in the early stages of development. Herein we report the identification of new compounds as potential Ape1 inhibitors through a docking‐based virtual screening technique. Some of the compounds identified have in vitro activities in the low‐to‐medium micromolar range. Interaction of these compounds with the Ape1 protein was observed by mass spectrometry. These molecules also potentiate the cytotoxicity of the chemotherapeutic agent methyl methanesulfonate in fibrosarcoma cells. This study demonstrates the power of docking and virtual screening techniques as initial steps in the design of new drugs, and opens the door to the development of a new generation of Ape1 inhibitors.     

Design,Synthesis and Bioevaluation of an EphA2 Receptor‐Based Targeted Delivery System

Because of its overexpression in a range of solid tumors, the EphA2 receptor is a validated target for cancer therapeutics. We recently described a new targeted delivery system based on specific EphA2‐targeting peptides conjugated with the chemotherapeutic agent paclitaxel. Here, we investigate the chemical determinants responsible for the stability and degradation of these agents in plasma. Introducing modifications in both the peptide and the linker between the peptide and paclitaxel resulted in drug conjugates that are both long‐lived in rat plasma and that markedly decrease tumor size in a prostate cancer xenograft model compared with paclitaxel alone treatment. These studies identify critical rate‐limiting degradation sites on the peptide–drug conjugates, enabling the design of agents with increased stability and efficacy. These results provide support for our central hypothesis that peptide–drug conjugates targeting EphA2 represent an innovative and potentially effective strategy to selectively deliver cytotoxic drugs to cancer cells.     

RNA folding pathways and the self-assembly of ribosomes

Many RNAs do not directly code proteins but are nonetheless indispensable to cellular function. These strands fold into intricate three-dimensional shapes that are essential structures in protein synthesis, splicing, and many other processes of gene regulation and expression. A variety of biophysical and biochemical methods are now showing, in real time, how ribosomal subunits and other ribonucleoprotein complexes assemble from their molecular components. Footprinting methods are particularly useful for studying the folding of long RNAs: they provide quantitative information about the conformational state of each residue and require little material. Data from footprinting complement the global information available from small-angle X-ray scattering or cryo-electron microscopy, as well as the dynamic information derived from single-molecule F?rster resonance energy transfer (FRET) and NMR methods. In this Account, I discuss how we have used hydroxyl radical footprinting and other experimental methods to study pathways of RNA folding and 30S ribosome assembly. Hydroxyl radical footprinting probes the solvent accessibility of the RNA backbone at each residue in as little as 10 ms, providing detailed views of RNA folding pathways in real time. In conjunction with other methods such as solution scattering and single-molecule FRET, time-resolved footprinting of ribozymes showed that stable domains of RNA tertiary structure fold in less than 1 s. However, the free energy landscapes for RNA folding are rugged, and individual molecules kinetically partition into folding pathways that lead through metastable intermediates, stalling the folding or assembly process. Time-resolved footprinting was used to follow the formation of tertiary structure and protein interactions in the 16S ribosomal RNA (rRNA) during the assembly of 30S ribosomes. As previously observed in much simpler ribozymes, assembly occurs in stages, with individual molecules taking different routes to the final complex. Interactions occur concurrently in all domains of the 16S rRNA, and multistage protection of binding sites of individual proteins suggests that initial encounter complexes between the rRNA and ribosomal proteins are remodeled during assembly. Equilibrium footprinting experiments showed that one primary binding protein was sufficient to stabilize the tertiary structure of the entire 16S 5'-domain. The rich detail available from the footprinting data showed that the secondary assembly protein S16 suppresses non-native structures in the 16S 5'-domain. In doing so, S16 enables a conformational switch distant from its own binding site, which may play a role in establishing interactions with other domains of the 30S subunit. Together, the footprinting results show how protein-induced changes in RNA structure are communicated over long distances, ensuring cooperative assembly of even very large RNA-protein complexes such as the ribosome.     

Structure‐Based Design of a Eukaryote‐Selective Antiprotozoal Fluorinated Aminoglycoside

Aminoglycosides (AG) are antibiotics that lower the accuracy of protein synthesis by targeting a highly conserved RNA helix of the ribosomal A‐site. The discovery of AGs that selectively target the eukaryotic ribosome, but lack activity in prokaryotes, are promising as antiprotozoals for the treatment of neglected tropical diseases, and as therapies to read‐through point‐mutation genetic diseases. However, a single nucleobase change A1408G in the eukaryotic A‐site leads to negligible affinity for most AGs. Herein we report the synthesis of 6′‐fluorosisomicin, the first 6′‐fluorinated aminoglycoside, which specifically interacts with the protozoal cytoplasmic rRNA A‐site, but not the bacterial A‐site, as evidenced by X‐ray co‐crystal structures. The respective dispositions of 6′‐fluorosisomicin within the bacterial and protozoal A‐sites reveal that the fluorine atom acts only as a hydrogen‐bond acceptor to favorably interact with G1408 of the protozoal A‐site. Unlike aminoglycosides containing a 6′‐ammonium group, 6′‐fluorosisomicin cannot participate in the hydrogen‐bonding pattern that characterizes stable pseudo‐base‐pairs with A1408 of the bacterial A‐sites. Based on these structural observations it may be possible to shift the biological activity of aminoglycosides to act preferentially as antiprotozoal agents. These findings expand the repertoire of small molecules targeting the eukaryotic ribosome and demonstrate the usefulness of fluorine as a design element.     

RNA-Cleaving DNA Thresholder Controlled by Concentrations of miRNA Cancer Marker

Oligonucleotide gene therapy (OGT) agents suppress specific mRNAs in cells and thus reduce the expression of targeted genes. The ability to unambiguously distinguish cancer from healthy cells can solve the low selectivity problem of OGT agents. Cancer RNA markers are expressed in both healthy and cancer cells with a higher expression level in cancer cells. We have designed a DNA-based construct, named DNA thresholder (DTh) that cleaves targeted RNA only at high concentrations of cancer marker RNA and demonstrates low cleavage activity at low marker concentrations. The RNA-cleaving activity can be adjusted within one order of magnitude of the cancer marker RNA concentration by simply redesigning DTh. Importantly, DTh recognizes cancer marker RNA, while cleaving targeted RNA; this offers a possibility to suppress vital genes exclusively in cancer cells, thus triggering their death. DTh is a prototype of computation-inspired molecular device for controlling gene expression and cancer treatment.     

Drug Delivery to the Malaria Parasite Using an Arterolane‐Like Scaffold

Antimalarial agents artemisinin and arterolane act via initial reduction of a peroxide bond in a process likely mediated by ferrous iron sources in the parasite. Here, we report the synthesis and antiplasmodial activity of arterolane‐like 1,2,4‐trioxolanes specifically designed to release a tethered drug species within the malaria parasite. Compared with our earlier drug delivery scaffolds, these new arterolane‐inspired systems are of significantly decreased molecular weight and possess superior metabolic stability. We describe an efficient, concise and scalable synthesis of the new systems, and demonstrate the use of the aminonucleoside antibiotic puromycin as a chemo/biomarker to validate successful drug release in live Plasmodium falciparum parasites. Together, the improved drug‐like properties, more efficient synthesis, and proof of concept using puromycin, suggests these new molecules as improved vehicles for targeted drug delivery to the malaria parasite.     

An in vitro Assay to Measure Targeted Drug Delivery to Bone Mineral

Targeted delivery of drugs to their site of action is a promising strategy to decrease adverse effects and enhance efficacy, but successful applications of this strategy have been scarce. Human bone is a tissue with unique properties due to its high hydroxyapatite mineral content. However, with the exception of bisphosphonates, bone mineral has not been targeted in a successful clinical application of drugs that act on bone, such as anti‐resorptive or bone anabolic agents. Herein we present an NMR‐based in vitro assay to measure binding affinities of small molecules to hydroxyapatite (HAP) or bone powder. Binding was shown to be specific and competitive, and the assay can be carried out in a direct binding format or in competition mode. A selection of clinically relevant bisphosphonates was ranked by their binding affinity for HAP. The binding affinity decreases in the order: pamidronate > alendronate > zoledronate > risedronate > ibandronate. The differences in binding affinities span a factor of 2.1 between pamidronate and ibandronate, consistent with previous studies. The rank order is very similar with bone powder, although the binding capacity of bone powder is smaller and binding kinetics are slower. A zoledronate derivative that lacks the central hydroxy group binds to HAP with 2.3‐fold weaker affinity than zoledronate itself. Any small molecule can be analyzed for its binding to HAP or bone powder, and the binding of common bone‐staining agents such as alizarin and its derivatives was confirmed in the new assay. This assay supports a strategy for targeted delivery of drugs to bone by attaching a bone‐affinity tag to the active drug substance.     

Optimization of Short RNA Aptamers for TNBC Cell Targeting

Triple-negative breast cancer (TNBC) is an aggressive cancer with limited targeted therapies. RNA aptamers, suitably chemically modified, work for therapeutic purposes in the same way as antibodies. We recently generated 2′Fluoro-pyrimidines RNA-aptamers that act as effective recognition elements for functional surface signatures of TNBC cells. Here, we optimized three of them by shortening and proved the truncated aptamers as optimal candidates to enable active targeting to TNBC. By using prediction of secondary structure to guide truncation, we identified structural regions that account for the binding motifs of the full-length aptamers. Their chemical synthesis led to short aptamers with superb nuclease resistance, which specifically bind to TNBC target cells and rapidly internalize into acidic compartments. They interfere with the growth of TNBC cells as mammospheres, thus confirming their potential as anti-tumor agents. We propose sTN145, sTN58 and sTN29 aptamers as valuable tools for selective TNBC targeting and promising candidates for effective treatments, including therapeutic agents and targeted delivery nanovectors.     

DEAD-Box RNA Helicase DDX47 Maintains Midgut Homeostasis in Locusta migratoria

Tissue homeostasis is critical for maintaining organ shape, size, and function. The condition is regulated by the balance between the generation of new cells and the loss of senescent cells, and it involves many factors and mechanisms. The midgut, an important part of the intestinal tract, is responsible for digestion and nutrient absorption in insects. LmDDX47, the ortholog of DEAD-box helicase 47 from Locusta migratoria, is indispensable for sustaining a normal midgut in the nymphs. However, the underlying cellular and molecular mechanisms remain to be elucidated. In this study, LmDDX47 knockdown resulted in atrophy of the midgut and gastric cecum in both nymph and adult locusts. After LmDDX47 knockdown, the number of regenerative and columnar cells in the midgut was significantly reduced, and cell death was induced in columnar tissue. LmDDX47 was localized to the nucleolus; this was consistent with the reduction in 18S rRNA synthesis in the LmDDX47 knockdown group. In addition, the acetylation and crotonylation levels of midgut proteins were significantly increased. Therefore, LmDDX47 could be a key regulator of midgut homeostasis, regulating 18S rRNA synthesis as well as protein acetylation and crotonylation in the migratory locust.     

The molecular interactions that stabilize RNA tertiary structure: RNA motifs, patterns, and networks

RNA molecules adopt specific three-dimensional structures critical to their function. Many essential metabolic processes, including protein synthesis and RNA splicing, are carried out by RNA molecules with elaborate tertiary structures (e.g. 3QIQ, right). Indeed, the ribosome and self-splicing introns are complex RNA machines. But even the coding regions in messenger RNAs and viral RNAs are flanked by highly structured untranslated regions, which provide regulatory information necessary for gene expression. RNA tertiary structure is defined as the three-dimensional arrangement of RNA building blocks, which include helical duplexes, triple-stranded structures, and other components that are held together through connections collectively termed RNA tertiary interactions. The structural diversity of these interactions is now a subject of intense investigation, involving the techniques of NMR, X-ray crystallography, chemical genetics, and phylogenetic analysis. At the same time, many investigators are using biophysical techniques to elucidate the driving forces for tertiary structure formation and the mechanisms for its stabilization. RNA tertiary folding is promoted by maximization of base stacking, much like the hydrophobic effect that drives protein folding. RNA folding also requires electrostatic stabilization, both through charge screening and site binding of metals, and it is enhanced by desolvation of the phosphate backbone. In this Account, we provide an overview of the features that specify and stabilize RNA tertiary structure. A major determinant for overall tertiary RNA architecture is local conformation in secondary-structure junctions, which are regions from which two or more duplexes project. At junctions and other structures, such as pseudoknots and kissing loops, adjacent helices stack on one another, and these coaxial stacks play a major role in dictating the overall architectural form of an RNA molecule. In addition to RNA junction topology, a second determinant for RNA tertiary structure is the formation of sequence-specific interactions. Networks of triple helices, tetraloop-receptor interactions, and other sequence-specific contacts establish the framework for the overall tertiary fold. The third determinant of tertiary structure is the formation of stabilizing stacking and backbone interactions, and many are not sequence specific. For example, ribose zippers allow 2'-hydroxyl groups on different RNA strands to form networks of interdigitated hydrogen bonds, serving to seal strands together and thereby stabilize adjacent substructures. These motifs often require monovalent and divalent cations, which can interact diffusely or through chelation to specific RNA functional groups. As we learn more about the components of RNA tertiary structure, we will be able to predict the structures of RNA molecules from their sequences, thereby obtaining key information about biological function. Understanding and predicting RNA structure is particularly important given the recent discovery that although most of our genome is transcribed into RNA molecules, few of them have a known function. The prevalence of RNA viruses and pathogens with RNA genomes makes RNA drug discovery an active area of research. Finally, knowledge of RNA structure will facilitate the engineering of supramolecular RNA structures, which can be used as nanomechanical components for new materials. But all of this promise depends on a better understanding of the RNA parts list, and how the pieces fit together.     

Tin Carboxylate Complexes of Natural Bacteriochlorin for Combined Photodynamic and Chemotherapy of Cancer è

Photodynamic therapy (PDT) is currently one of the most promising methods of cancer treatment. However, this method has some limitations, including a small depth of penetration into biological tissues, the low selectivity of accumulation, and hypoxia of the tumor tissues. These disadvantages can be overcome by combining PDT with other methods of treatment, such as radiation therapy, neutron capture therapy, chemotherapy, etc. In this work, potential drugs were obtained for the first time, the molecules of which contain both photodynamic and chemotherapeutic pharmacophores. A derivative of natural bacteriochlorophyll a with a tin IV complex, which has chemotherapeutic activity, acts as an agent for PDT. This work presents an original method for obtaining agents of combined action, the structure of which is confirmed by various physicochemical methods of analysis. The method of molecular modeling was used to investigate the binding of the proposed drugs to DNA. In vitro biological tests were carried out on several lines of tumor cells: Hela, A549, S37, MCF7, and PC-3. It was shown that the proposed conjugates of binary action for some cell lines had a dark cytotoxicity that was significantly higher (8–10 times) than the corresponding metal complexes of amino acids, which was explained by the targeted chemotherapeutic action of the tin (IV) complex due to chlorin. The greatest increase in efficiency relative to the initial dipropoxy-BPI was found for the conjugate with lysine as a chelator of the tin cation relative to cell lines, with the following results: S-37 increased 3-fold, MCF-7 3-fold, and Hela 2.4-fold. The intracellular distribution of the obtained agents was also studied by confocal microscopy and showed a diffuse granular distribution with predominant accumulation in the near nuclear region.     

Synthetic Cancer‐Targeting Innate Immune Stimulators Give Insights into Avidity Effects

Multispecific and multivalent antibodies are seen as promising cancer therapeutics, and numerous antibody fragments and derivatives have been developed to exploit avidity effects that result in increased selectivity. Most of these multispecific and multivalent antibody strategies make use of recombinant expression of antigen‐binding modules. In contrast, chemical synthesis and chemoselective ligations can be used to generate a variety of molecules with different numbers and combinations of binding moieties in a modular and homogeneous fashion. In this study we synthesized a series of targeted immune system engagers (ISErs) by using solid‐phase peptide synthesis and chemoselective ligations. To explore avidity effects, we constructed molecules bearing different numbers and combinations of two “binder” peptides that target ephrin A2 and integrin α₃ receptors and an “effector” peptide that binds to formyl peptide receptors and stimulates an immune response. We investigated various strategies for generating multivalent and multispecific targeted innate immune stimulators and studied their activities in terms of binding to cancer cells and stimulation of immune cells. This study gives insights into the influence that multivalency and receptor density have on avidity effects and is useful for the design of potential anticancer therapeutics.     

Metal Sulfide Semiconductor Nanomaterials and Polymer Microgels for Biomedical Applications

The development of nanomaterials with therapeutic and/or diagnostic properties has been an active area of research in biomedical sciences over the past decade. Nanomaterials have been identified as significant medical tools with potential therapeutic and diagnostic capabilities that are practically impossible to accomplish using larger molecules or bulk materials. Fabrication of nanomaterials is the most effective platform to engineer therapeutic agents and delivery systems for the treatment of cancer. This is mostly due to the high selectivity of nanomaterials for cancerous cells, which is attributable to the porous morphology of tumour cells which allows nanomaterials to accumulate more in tumour cells more than in normal cells. Nanomaterials can be used as potential drug delivery systems since they exist in similar scale as proteins. The unique properties of nanomaterials have drawn a lot of interest from researchers in search of new chemotherapeutic treatment for cancer. Metal sulfide nanomaterials have emerged as the most used frameworks in the past decade, but they tend to aggregate because of their high surface energy which triggers the thermodynamically favoured interaction. Stabilizing agents such as polymer and microgels have been utilized to inhibit the particles from any aggregations. In this review, we explore the development of metal sulfide polymer/microgel nanocomposites as therapeutic agents against cancerous cells.     

Fluorescent Probes for G‐Quadruplex Structures

Mounting evidence supports the presence of biologically relevant G‐quadruplexes in single‐cell organisms, but the existence of endogenous G‐quadruplex structures in mammalian cells remains highly controversial. This is due, in part, to the common misconception that DNA and RNA molecules are passive information carriers with relatively little structural or functional complexity. For those working in the field, however, the lack of available tools for characterizing DNA structures in vivo remains a major limitation to addressing fundamental questions about structure–function relationships of nucleic acids. In this review, we present progress towards the direct detection of G‐quadruplex structures by using small molecules and modified oligonucleotides as fluorescent probes. While most development has focused on cell‐permeable probes that selectively bind to G‐quadruplex structures with high affinity, these same probes can induce G‐quadruplex folding, thereby making the native conformation of the DNA or RNA molecule (i.e., in the absence of probe) uncertain. For this reason, modified oligonucleotides and fluorescent base analogues that serve as “internal” fluorescent probes are presented as an orthogonal means for detecting conformational changes, without necessarily perturbing the equilibria between G‐quadruplex, single‐stranded, and duplex DNA. The major challenges and motivation for the development of fluorescent probes for G‐quadruplex structures are presented, along with a summary of the key photophysical, biophysical, and biological properties of reported examples.     

Proteasome Inhibitors with Photocontrolled Activity

Proteasome inhibitors are widely used in cancer treatment as chemotherapeutic agents. However, their employment often results in severe side effects, due to their non‐specific cytotoxicity towards healthy tissue. This problem might be overcome by using a photopharmacological approach, that is, by attaining external, dynamic, spatiotemporal photocontrol over the activity of a cytotoxic agent, achieved by the introduction of a photoswitchable moiety into its molecular structure. Here we describe the design, synthesis, and activity of photoswitchable proteasome inhibitors. Substantial differences in proteasome inhibitory activity in cell extracts were observed before and after irradiation with light. The presented results show potential for the development of chemotherapeutic agents that can be switched on and off with light, constituting a new strategy for spatiotemporally modulating proteasomal activity.