Bicyclic isothioureas for conjugation with tubulin targeted anticancer agents

Two bicyclic annulated isothiourea derivatives were synthesized using as a key stage either the reaction of isothiocyanate halide with sodium sulfide or cyclization of unsaturated thiourea in the presence of bromine. X-ray molecular structure of N-[(3aSR,7aRS,Z)-hexahydro-1,3-benzothiazol-2(3H)-ylidene]glycine was determined. The conjugate of colchicine with [(3aR,5S,6aS)-2-(tert-butylamino)-3a,5,6,6a-tetrahydro-4H-cyclopenta[d]thiazol-5-yl]methanol obtained demonstrated pronounced cytotoxic effect on cancer cells.     

Versatile site-specific conjugation of small molecules to siRNA using click chemistry

We have previously demonstrated that conjugation of small molecule ligands to small interfering RNAs (siRNAs) and anti-microRNAs results in functional siRNAs and antagomirs in vivo. Here we report on the development of an efficient chemical strategy to make oligoribonucleotide-ligand conjugates using the copper-catalyzed azide-alkyne cycloaddition (CuAAC) or click reaction. Three click reaction approaches were evaluated for their feasibility and suitability for high-throughput synthesis: the CuAAC reaction at the monomer level prior to oligonucleotide synthesis, the solution-phase postsynthetic "click conjugation", and the "click conjugation" on an immobilized and completely protected alkyne-oligonucleotide scaffold. Nucleosides bearing 5'-alkyne moieties were used for conjugation to the 5'-end of the oligonucleotide. Previously described 2'- and 3'-O-propargylated nucleosides were prepared to introduce the alkyne moiety to the 3' and 5' termini and to the internal positions of the scaffold. Azido-functionalized ligands bearing lipophilic long chain alkyls, cholesterol, oligoamine, and carbohydrate were utilized to study the effect of physicochemical characteristics of the incoming azide on click conjugation to the alkyne-oligonucleotide scaffold in solution and on immobilized solid support. We found that microwave-assisted click conjugation of azido-functionalized ligands to a fully protected solid-support bound alkyne-oligonucleotide prior to deprotection was the most efficient "click conjugation" strategy for site-specific, high-throughput oligonucleotide conjugate synthesis tested. The siRNA conjugates synthesized using this approach effectively silenced expression of a luciferase gene in a stably transformed HeLa cell line.     

Epitope alteration by small molecules and applications in drug discovery

Small molecules and antibodies are normally considered separately in drug discovery, except in the case of covalent conjugates. We unexpectedly discovered several small molecules that could inhibit or enhance antibody–epitope interactions which opens new possibilities in drug discovery and therapeutic modulation of auto-antibodies. We first discovered a small molecule, CRANAD-17, that enhanced the binding of an antibody to amyloid beta (Aβ), one of the major hallmarks of Alzheimer''s disease, by stable triplex formation. Next, we found several small molecules that altered antibody–epitope interactions of tau and PD-L1 proteins, demonstrating the generality of this phenomenon. We report a new screening technology for ligand discovery, screening platform based on epitope alteration for drug discovery (SPEED), which is label-free for both the antibody and small molecule. SPEED, applied to an Aβ antibody, led to the discovery of a small molecule, GNF5837, that inhibits Aβ aggregation and another, obatoclax, that binds Aβ plaques and can serve as a fluorescent reporter in brain slices of AD mice. We also found a small molecule that altered the binding between Aβ and auto-antibodies from AD patient serum. SPEED reveals the sensitivity of antibody–epitope interactions to perturbation by small molecules and will have multiple applications in biotechnology and drug discovery.

A screening platform based on epitope alteration for drug discovery (SPEED).     

Substrate-dependent targeting of eukaryotic translation initiation factor 4A by pateamine A: negation of domain-linker regulation of activity

Central to cap-dependent eukaryotic translation initiation is the eIF4F complex, which is composed of the three eukaryotic initiation factors eIF4E, eIF4G, and eIF4A. eIF4A is an RNA-dependent ATPase and an ATP-dependent helicase that unwinds local secondary structure in mRNA to allow binding of the 43S ribosomal complex. The marine natural product pateamine A (PatA) has been demonstrated to inhibit cap-dependent initiation by targeting eIF4A and disrupting its protein-protein interactions while increasing its enzymatic activities. Here we demonstrate that the increased activity is caused by the induction of global conformational changes within eIF4A. Furthermore, binding of PatA is dependent on substrate (RNA and ATP) binding, and the increased activity upon PatA binding is caused by relief of a negative regulatory function of the eIF4A unique domain linker.     

Fluorine in medicinal chemistry: Recent therapeutic applications of fluorinated small molecules

Advances in the development of newly developed fluorinated drugs, approved or in clinical trials, as organized by biological targets/disease states, are reviewed. In a few cases, compounds in early stages of development will be discussed, particularly where new promising targets are involved. Important topics such as anticancer and antiviral drugs were covered in the two previous reviews in this series. Included herein will be fluorinated drugs for treatment of diseases of the central nervous system, various cardiovascular diseases and obesity, antibacterial agents, and antifungal therapy.     

Chemical conjugation with cyclodextrins as a versatile tool for drug delivery

A pillar[5]arene-based N-heterocyclic carbene ligand was prepared by reaction of bromoethoxy pillar[5]arene with excess 1-methylimidazole at 130 °C in the absence of solvent and used as a catalyst for the Suzuki coupling reaction. Excellent yields were obtained when the Suzuki reactions were carried out under ambient atmosphere in ethanol, employing 0.2 mol% ligand, 1 mol% PdCl₂(CH₃CN)₂ and 1.5 mmol of K₂CO₃. The novel pillar[5]arene-based imidazolium salt is a promising material for the construction of highly active supramolecular catalytic systems.     

PDMS absorption of small molecules and consequences in microfluidic applications

Microfluidic devices made out of polydimethylsiloxane (PDMS) have many physical properties that are useful for cell culture applications, such as transparency and gas permeability. Another distinct characteristic of PDMS is its ability to absorb hydrophobic small molecules. Partitioning of molecules into PDMS can significantly change solution concentrations and could potentially alter experimental outcomes. Herein we discuss PDMS absorption and its potential impact on microfluidic experiments.     

Organometallic ruthenium and iridium phosphorus complexes: Synthesis,cellular imaging,organelle targeting and anticancer applications

The use of metal complexes containing phosphorus ligands as anticancer agents has not been well studied. In this work, eight novel half‐sandwich Ir^III and Ru^II compounds with P^P‐chelating ligands have been synthesized and fully characterized, and alongside two crystal structures were reported. All eight complexes displayed highly potent antiproliferative activity, up to nine times more potent than the clinical anticancer drug cisplatin towards A549 lung cancer cells. Complex Ir1 , which has a simpler structure and highly potent antiproliferative activity, was selected to investigate in further mechanistic studies. No hydrolysis and nucleobase binding occurred for complex Ir1 . In order to elucidate subcellular localization, the self‐luminescence of the complex Ir1 was utilized. Ir1 can specifically target lysosomes and facilitate excessive production of reactive oxygen species, resulting in lysosomal membrane permeabilization in A549 cells. Release of cathepsin B and changes in the mitochondria membrane potential also contributed to the observed cytotoxicity of Ir1 , which demonstrated an anticancer action mechanism that was different from that of cisplatin. The favorable results from biological and chemical research demonstrated that these types of complexes hold significant theranostic potential.     

Magnetic microcapsules for targeted delivery of anticancer drugs

To achieve targeted distribution of anticancer drugs with sustained activity, ferromagnetic ethylcellulose microcapsules containing an anticancer drug, mitomycin C (FM-MMC-mc), were prepared by a method based on phase separation principles. Two prototypes of FM-MMC-mc were made: one with the drug as the core and zinc ferrite on its capsular surface (outer type); the other with both the drug and zinc ferrite as the core (inner type). Both preparations provided a sustained-release property and a sensitive response to conventional magnetic force, although certain differences in the release rate of drug, magnetic responsiveness, and particle size were found between the two dosage forms. Animal studies showed that the magnetic microcapsules could be magnetically controlled in the artery and urinary bladder. VX2 tumors in the rabbit hind limb and urinary bladder were successfully treated with magnetic control of FM-MMC-mc. Pharmacokinetic study revealed that the targeting of the microcapsules markedly enhanced the drug absorption into the surrounding tissues for a prolonged period of time. The results indicate the feasibility and effectiveness of the magnetic microcapsules as a targeted drug delivery system.     

Genetic screens and selections for small molecules based on a synthetic riboswitch that activates protein translation

Genetic selection provides the most powerful method to assay large libraries of biomolecules for function. However, harnessing the power of genetic selection for the detection of specific, nonendogenous small-molecule targets in vivo remains a significant challenge. The ability to genetically select for small molecules would provide a reaction-independent mechanism to clone biosynthesis genes from large DNA libraries and greatly facilitate the exploration of large libraries of mutant enzymes for improved synthetic capabilities including altered substrate specificities and enhanced regio- or stereoselectivities. While remarkable progress has been made in developing genetic methods to detect small molecules in vivo, many of these methods rely on engineering small-molecule-protein interactions which remains a difficult problem, and the potential for some of these systems to assay large libraries is limited by the low transformation efficiency and long doubling time of yeast relative to bacteria. Herein, we demonstrate that synthetic riboswitches that activate protein translation in response to a specific small molecule can be used to perform sensitive genetic screens and selections for the presence of small molecules in Escherichia coli. We further demonstrate that the exquisite molecular discrimination properties of aptamers selected in vitro translate directly into an in vivo genetic selection system. Finally, we demonstrate that a cell harboring a synthetic riboswitch with a particular ligand specificity can be selectively amplified from a million-fold larger pool of cells containing mutant riboswitches that respond to a closely related ligand, suggesting that it is possible to use genetic selection in E. coli to discover synthetic riboswitches with new ligand specificities from libraries of mutant riboswitches.     

Labeling proteins with small molecules by site-specific posttranslational modification

We report here the development of a general strategy for site-specific labeling of proteins with small molecules by posttranslational modification enzyme, phosphopantetheinyl transferase Sfp. The target proteins are expressed as fusions to the peptide carrier protein (PCP) excised from nonribosomal peptide synthetase, and Sfp catalyzes the covalent modification of a specific serine residue on PCP by the small molecule-phosphopantetheinyl conjugate. The labeling reaction proceeds with high specificity and efficiency, targeting PCP fusion proteins in the cell lysate. The PCP tag has been shown to be compatible with various proteins, and Sfp-catalyzed PCP modification, compatible with various small-molecule probes conjugated to coenzyme A, highlighting the potential of the PCP tag for site-specific protein labeling with small molecules.     

Concurrent binding and delivery of proteins and lipophilic small molecules using polymeric nanogels

Supramolecular nanoassemblies, which are capable of binding and delivering either lipophilic small molecules or hydrophilic molecules, are of great interest. Concurrently binding and delivering this combination of molecules is cumbersome, because of the opposing supramolecular host requirements. We describe the development of a versatile nanoassembly system that is capable of binding and delivering both, a protein and a lipophilic small molecule, simultaneously inside the cells.     

Microfluidic chip-based liquid chromatography coupled to mass spectrometry for determination of small molecules in bioanalytical applications

The development and integration of microfabricated liquid chromatography (LC) microchips have increased dramatically in the last decade due to the needs of enhanced sensitivity and rapid analysis as well as the rising concern on reducing environmental impacts of chemicals used in various types of chemical and biochemical analyses. Recent development of microfluidic chip-based LC mass spectrometry (chip-based LC-MS) has played an important role in proteomic research for high throughput analysis. To date, the use of chip-based LC-MS for determination of small molecules, such as biomarkers, active pharmaceutical ingredients (APIs), and drugs of abuse and their metabolites, in clinical and pharmaceutical applications has not been thoroughly investigated. This mini-review summarizes the utilization of commercial chip-based LC-MS systems for determination of small molecules in bioanalytical applications, including drug metabolites and disease/tumor-associated biomarkers in clinical samples as well as adsorption, distribution, metabolism, and excretion studies of APIs in drug discovery and development. The different types of commercial chip-based interfaces for LC-MS analysis are discussed first and followed by applications of chip-based LC-MS on biological samples as well as the comparison with other LC-MS techniques.     

Trifunctional 99mTc based radiopharmaceuticals: metal-mediated conjugation of a peptide with a nucleus targeting intercalator

The development of molecular imaging agents with multiple functions has become a major trend in radiopharmaceutical chemistry. We present herein the syntheses of trifunctional compounds, combining an acridine orange (AO) based intercalator with a GRP receptor specific bombesin like peptide (BBN). Metal-mediated conjugation of these two functions via the [2 + 1] approach to the third function, the [M(CO)(3)](+) (M = (99m)Tc, Re) moiety, yielded the final trifunctional molecules. The strongly fluorescent acridine orange, a nuclear targeting agent, has been derivatised with 4-imidazolecarboxylate as a bidentate ligand and bombesin with an isonitrile group as a monodentate ligand. For cell and nuclear uptake studies, [Re(L(1)-BBN)(L(2)-Ical)(CO)(3)] type complexes were synthesized and characterized. For radiopharmaceutical purposes, the (99m)Tc analogues have been prepared in a stepwise synthesis. Fluorescence microscopy studies on PC-3 cells, bearing the BBN receptor, showed high and rapid uptake into the cytoplasm. For the bifunctional molecule, lacking the BBN peptide, no internalization was observed.     

MolAlign: an algorithm for aligning multiple small molecules

In small molecule drug discovery projects, the receptor structure is not always available. In such cases it is enormously useful to be able to align known ligands in the way they bind in the receptor. Here we shall present an algorithm for the alignment of multiple small molecule ligands. This algorithm takes pre-generated conformers as input, and proposes aligned assemblies of the ligands. The algorithm consists of two stages: the first stage is to perform alignments for each pair of ligands, the second stage makes use of the results from the first stage to build up multiple ligand alignment assemblies using a novel iterative procedure. The scoring functions are improved versions of the one mentioned in our previous work. We have compared our results with some recent publications. While an exact comparison is impossible, it is clear that our algorithm is fast and produces very competitive results.     

Targeted degradation of proteins by small molecules: a novel tool for functional proteomics

A novel strategy that targets protein for degradation has recently been developed by exploiting a protein-targeting chimeric molecule ('Protac'). Typically, the chimeric Protac is composed of a small-molecule ligand ('bait') on one end and a synthetic octapeptide on the other. This octapeptide is recognized by E3 ubiquitin ligase pVHL (von Hippel Lindau tumor suppressor protein), thereby recruiting a small molecule-bound protein ('prey') to pVHL for ubiquitination and degradation. Since selective degradation of a cellular protein generates a "loss of function" mutation, this protein knock-out strategy may be useful to study the function of a given protein or to evaluate whether a cellular protein is a potential target for drug intervention, in a manner reminiscent of gene knock-out or siRNA approaches. Herein, we show that a synthetic pentapeptide is sufficient to interact with pVHL E3 ligase, and that the pentapeptide-based Protac efficiently induces ubiquitination and degradation of target protein. Our results also demonstrate that the pentapeptide-based Protac can enter cells efficiently to exerts its biological activity effectively. These results suggest that the synthetic pentapeptide can be used either directly in the preparation of cell-permeable Protacs or as a template to develop peptidomimetic or non-peptide Protacs.     

Bio-click chemistry: enzymatic functionalization of PEGylated capsules for targeting applications

All sorted: The enzyme Sortase?A was used to catalyze functionalization of PEGylated capsules with an activation-specific anti-platelet single-chain antibody (scFv). This enzymatic method allows fast, covalent, and site-directed functionalization of delivery vehicles under mild conditions. Activation-specific anti-platelet scFv-coated PEGylated capsules exhibited a high level of selective binding to thrombi, thus suggesting their potential for thrombosis therapy.     

Ligand-regulated peptides: a general approach for modulating protein-peptide interactions with small molecules

We engineered a novel ligand-regulated peptide (LiRP) system where the binding activity of intracellular peptides is controlled by a cell-permeable small molecule. In the absence of ligand, peptides expressed as fusions in an FKBP-peptide-FRB-GST LiRP scaffold protein are free to interact with target proteins. In the presence of the ligand rapamycin, or the nonimmunosuppressive rapamycin derivative AP23102, the scaffold protein undergoes a conformational change that prevents the interaction of the peptide with the target protein. The modular design of the scaffold enables the creation of LiRPs through rational design or selection from combinatorial peptide libraries. Using these methods, we identified LiRPs that interact with three independent targets: retinoblastoma protein, c-Src, and the AMP-activated protein kinase. The LiRP system should provide a general method to temporally and spatially regulate protein function in cells and organisms.