Phenotypic Screens in Antimalarial Drug Discovery.

Phenotypic high-throughput screens are a valuable tool for identifying new chemical compounds with antimalarial activity. Traditionally, these screens have focused solely on the symptomatic asexual blood stage of the parasite life cycle; however, to discover new medicines for malaria treatment and prevention, robust screening technologies against other parasite life-cycle stages are required. This review highlights recent advances and progress toward phenotypic screening methodologies over the past several years, with a focus on exoerythrocytic stage screens.

Encapsidated atom-transfer radical polymerization in Qß virus-like nanoparticles.

Virus-like particles (VLPs) are unique macromolecular structures that hold great promise in biomedical and biomaterial applications. The interior of the 30 nm-diameter Qß VLP was functionalized by a three-step process: (1) hydrolytic removal of endogenously packaged RNA, (2) covalent attachment of initiator molecules to unnatural amino acid residues located on the interior capsid surface, and (3) atom-transfer radical polymerization of tertiary amine-bearing methacrylate monomers. The resulting polymer-containing particles were moderately expanded in size; however, biotin-derivatized polymer strands were only very weakly accessible to avidin, suggesting that most of the polymer was confined within the protein shell. The polymer-containing particles were also found to exhibit physical and chemical properties characteristic of positively charged nanostructures, including the ability to easily enter mammalian cells and deliver functional small interfering RNA.

Engineered mutations change the structure and stability of a virus-like particle.

The single-coat protein (CP) of bacteriophage Qß self-assembles into T = 3 icosahedral virus-like particles (VLPs), of interest for a wide range of applications. These VLPs are very stable, but identification of the specific molecular determinants of this stability is lacking. To investigate these determinants along with manipulations that confer more capabilities to our VLP material, we manipulated the CP primary structure to test the importance of various putative stabilizing interactions. Optimization of a procedure to incorporate fused CP subunits allowed for good control over the average number of covalent dimers in each VLP. We confirmed that the disulfide linkages are the most important stabilizing elements for the capsid and that acidic conditions significantly enhance the resistance of VLPs to thermal degradation. Interdimer interactions were found to be less important for VLP assembly than intradimer interactions. Finally, a single point mutation in the CP resulted in a population of smaller VLPs in three distinct structural forms.

Guiding plant virus particles to integrin-displaying cells.

Viral nanoparticles (VNPs) are structurally regular, highly stable, tunable nanomaterials that can be conveniently produced in high yields. Unmodified VNPs from plants and bacteria generally do not show tissue specificity or high selectivity in binding to or entry into mammalian cells. They are, however, malleable by both genetic and chemical means, making them useful scaffolds for the display of large numbers of cell- and tissue-targeting ligands, imaging moieties, and/or therapeutic agents in a well-defined manner. Capitalizing on this attribute, we modified the genetic sequence of the Cowpea mosaic virus (CPMV) coat protein to display an RGD oligopeptide sequence derived from human adenovirus type 2 (HAdV-2). Concurrently, wild-type CPMV was modified via NHS acylation and Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) chemistry to attach an integrin-binding cyclic RGD peptide. Both types of particles showed strong and selective affinity for several different cancer cell lines that express RGD-binding integrin receptors.

Cell targeting with hybrid Qß virus-like particles displaying epidermal growth factor.

Structurally uniform protein nanoparticles derived from the self-assembly of viral capsid proteins are attractive platforms for the multivalent display of cell-targeting motifs for use in nanomedicine. Virus-based nanoparticles are of particular interest because the scaffold can be manipulated both genetically and chemically to simultaneously display targeting groups and carry a functional payload. Here, we displayed the human epidermal growth factor (EGF) on the exterior surface of bacteriophage Qß as a C-terminal genetic fusion to the Qß capsid protein. The co-assembly of wild-type Qß and EGF-modified subunits resulted in structurally homogeneous nanoparticles displaying between 5 and 12 copies of EGF on their exterior surface. The particles were found to be amenable to bioconjugation by standard methods as well as the high-fidelity copper-catalyzed azide-alkyne cycloaddition reaction (CuAAC). Such chemical derivatization did not impair the ability of the particles to specifically interact with the EGF receptor. Additionally, the particle-displayed EGF remained biologically active promoting autophosphorylation of the EGF receptor and apoptosis of A431 cells. These results suggest that hybrid Qß-EGF nanoparticles could be useful vehicles for targeted delivery of imaging and/or therapeutic agents.

Synthesis, properties, and applications of diazotrifluropropanoyl-containing photoactive analogs of farnesyl diphosphate containing modified linkages for enhanced stability.

Photoactive analogs of farnesyl diphosphate (FPP) are useful probes in studies of enzymes that employ this molecule as a substrate. Here, we describe the preparation and properties of two new FPP analogs that contain diazotrifluoropropanoyl photophores linked to geranyl diphosphate via amide or ester linkages. The amide-linked analog (3) was synthesized in 32P-labeled form from geraniol in seven steps. Experiments with Saccharomyces cerevisiae protein farnesyltransferase (ScPFTase) showed that 3 is an alternative substrate for the enzyme. Photolysis experiments with [(32)P]3 demonstrate that this compound labels the beta-subunits of both farnesyltransferase and geranylgeranyltransferase (types 1 and 2). However, the amide-linked probe 3 undergoes a rearrangement to a photochemically unreactive isomeric triazolone upon long term storage making it inconvenient to use. To address this stability issue, the ester-linked analog 4 was prepared in six steps from geraniol. Computational analysis and X-ray crystallographic studies suggest that 4 binds to protein farnesyl transferase (PFTase) in a similar fashion as FPP. Compound 4 is also an alternative substrate for PFTase, and a 32P-labeled form selectively photocrosslinks the beta-subunit of ScPFTase as well as E. coli farnesyldiphosphate synthase and a germacrene-producing sesquiterpene synthase from Nostoc sp. strain PCC7120 (a cyanobacterial source). Finally, nearly exclusive labeling of ScPFTase in crude E. coli extract was observed, suggesting that [32P]4 manifests significant selectivity and should hence be useful for identifying novel FPP-utilizing enzymes in crude protein preparations.

Purification of prenylated proteins by affinity chromatography on cyclodextrin-modified agarose.

Although protein prenylation is widely studied, there are few good methods for isolating prenylated proteins from their nonprenylated relatives. We report that crosslinked agarose (e.g., Sepharose) chromatography medium that has been chemically functionalized with beta-cyclodextrin (beta-CD) is extremely effective in affinity chromatography of prenylated proteins. In this study, a variety of proteins with C-terminal prenylation target ("CAAX box") sequences were enzymatically prenylated in vitro with natural and nonnatural prenyl diphosphate substrates. The prenylated protein products could then be isolated from starting materials by gravity chromatography or fast protein liquid chromatography (FPLC) on a beta-CD-Sepharose column. One particular prenylation reaction, farnesylation of an mCherry-CAAX fusion construct, was studied in detail. In this case, purified farnesylated product was unambiguously identified by electrospray mass spectrometry. In addition, when mCherry-CAAX was prenylated with a nonnatural, functional isoprenoid substrate, the functional group was maintained by chromatography on beta-CD-Sepharose, such that the resulting protein could be selectively bound at its C terminus to complementary functionality on a solid substrate. Finally, beta-CD-Sepharose FPLC was used to isolate prenylated mCherry-CAAX from crude HeLa cell lysate as a model for purifying prenylated proteins from cell extracts. We propose that this method could be generally useful to the community of researchers studying protein prenylation.