Type-2 Cu sites are found in all the major branches of life and are often involved in the catalysis of oxygen species. Four type-2 Cu protein families are selected as model systems for review: amine oxidases, Cu monooxygenases, nitrite reductase/multicopper oxidase, and CuZn superoxide dismutase. For each model protein, the availability of multiple crystal structures and detailed enzymological studies provides a detailed molecular view of the type-2 Cu site and delineation of the mechanistic role of the Cu in biological function. Comparison of these model proteins leads to the identification of common properties of the Cu sites and insight into the evolution of the trinuclear active site found in multicopper oxidases.
Although efforts to develop a vaccine against HIV have so far met with little success, recent studies of HIV-positive patients with strongly neutralizing sera have shown that the human immune system is capable of producing potent and broadly-neutralizing antibodies (bnAbs), some of which neutralize up to 90 % of HIV strains. These antibodies bind to conserved vulnerable sites on the viral envelope glycoprotein gp120, and identification of these sites has provided tantalizing clues about the design of potentially effective vaccines. Carbohydrates play a key role in this field, as a large fraction of bnAbs bind to carbohydrates or combinations of carbohydrate and peptide elements on gp120. Additionally, carbohydrates partially mask some peptide surfaces recognized by bnAbs. The use of engineered glycoproteins and other glycostructures as vaccines to elicit antibodies with broad neutralizing activity is therefore a key area of interest in HIV vaccine design.
Cryptosporidium parvum is a potential biowarfare agent, an important AIDS pathogen, and a major cause of diarrhea and malnutrition. No vaccines or effective drug treatment exist to combat Cryptosporidium infection. This parasite relies on inosine 5'-monophosphate dehydrogenase (IMPDH) to obtain guanine nucleotides, and inhibition of this enzyme blocks parasite proliferation. Here, we report the first crystal structures of CpIMPDH. These structures reveal the structural basis of inhibitor selectivity and suggest a strategy for further optimization. Using this information, we have synthesized low-nanomolar inhibitors that display 10(3) selectivity for the parasite enzyme over human IMPDH2.
A vast number of biological processes are mediated by multivalent ligand-receptor interactions, including cell adhesion, host invasion by pathogens, pathogen neutralization by host and numerous cell regulatory signaling pathways. [1] Multivalency is especially important for carbohydrate-receptor interactions: whereas individual glycans [2] may bind with low affinity to a single binding site, the clustering of glycans creates a high-avidity interaction with clustered binding sites. This "carbohydrate cluster effect" [1b] has been demonstrated experimentally with synthetic multivalent carbohydrate ligands which bind well to protein targets. These ligands have included oligo-and polyvalent clusters of glycans on diverse scaffolds, including small molecules, dendrimers, polymers and even viral capsids.To date, most glycocluster ligands have been designed for synthetic convenience rather than control of tertiary structure. However, the biological activity of the natural glycocluster may be influenced by tertiary structure and other elements which are not usually addressed in synthetic glycocluster designs, such as: 1) Glycan spacing and orientation -glycans are normally attached to synthetic scaffolds through long flexible linkers, and the scaffolds themselves are often flexible. [3] 2) Glycan internal flexibility -in a natural glycocluster, As an alternative to rational design, we have been interested in directed evolution-based design of glycocluster ligands. Figure 1 outlines this concept: a library of scaffold molecules is glycosylated, generating a library of glycoclusters. The "best" glycoclusters are selected from the pool by binding to the target protein. These selection winners are then replicated to form a second-generation library and the process is repeated for several rounds until the pool is sufficiently enriched in high-affinity binders. We have chosen DNA as our glycocluster scaffolding material because DNA is easy to synthesize, easy to replicate by PCR, can fold into diverse sequence-dependent structures, and is amenable to sequence-specific "glycosylation" by glycan azides using CuAAC [5] ("click") attachment to alkyne-modified nucleobases. Iterative selection/amplification of DNA structures (SELEX) is often performed to obtain DNAs which bind to a target. [6] Our method, by contrast, would yield DNA scaffolds whose major function would be to position and support glycans optimally for target binding. However, these DNAs might also contain elements which would interact directly with the target, mimicking any non-carbohydrate components necessary in the natural ligand.We decided to test this concept in the design of glycoclusters which mimic the epitope of 2G12, an antibody which protects against HIV infection and binds to a cluster of highmannose glycans on the HIV envelope protein gp120. [7] Rationally-designed clusters of these glycans have been tested as vaccines to elicit 2G12-like antibodies, but without success. [8] Our evolution-based design would be the product of the procedure outlined i...
Summary The protozoan parasite Cryptosporidium parvum is a major cause of gastrointestinal disease; no effective drug therapy exists to treat this infection. Curiously, C. parvum IMPDH (CpIMPDH) is most closely related to prokaryotic IMPDHs, suggesting that the parasite obtained its IMPDH gene via horizontal transfer. We previously identified inhibitors of CpIMPDH that do not inhibit human IMPDHs. Here we show that these compounds also inhibit IMPDHs from Helicobacter pylori, Borrelia burgdorferi, and Streptococcus pyogenes, but not from Escherichia coli. Residues Ala165 and Tyr358 comprise a structural motif that defines susceptible enzymes. Importantly, a second generation CpIMPDH inhibitor has bacteriocidal activity on H. pylori but not Escherichia coli. We propose that CpIMPDH-targeted inhibitors can be developed into a new class of antibiotics that will spare some commensal bacteria.
SELMA (SELection with Modified Aptamers) is a directed evolution method which can be used to develop DNA-supported clusters of carbohydrates in which the geometry of clustering is optimized for strong recognition by a lectin of interest. Herein, we report a modification of SELMA which results in glycoclusters which achieve dramatically stronger target recognition (100-fold) with dramatically fewer glycans (2–3-fold). Our first applications of SELMA yielded clusters of 5–10 oligomannose glycans which were recognized by broadly neutralizing HIV antibody 2G12 with moderate affinities (150–500 nM Kd’s). In the present manuscript, we report glycoclusters containing just 3–4 glycans, which are recognized by 2G12 with Kd’s as low as 1.7 nM. These glycoclusters are recognized by 2G12 as tightly as is the HIV envelope protein gp120, and they are the first constructs to achieve this tight recognition with the minimal number of Man9units (3–4) necessary to occupy the binding sites on 2G12. They are thus of great interest as immunogens which might elicit broadly neutralizing antibodies against HIV.
DNA-based nanostructures have received great attention as molecular vehicles for cellular delivery of biomolecules and cancer drugs. Here, we report on the cellular uptake of tubule-like DNA tile-assembled nanostructures 27 nm in length and 8 nm in diameter that carry siRNA molecules, folic acid and fluorescent dyes. In our observations, the DNA structures are delivered to the endosome and do not reach the cytosol of the GFP-expressing HeLa cells that were used in the experiments. Consistent with this observation, no elevated silencing of the GFP gene could be detected. Furthermore, the presence of up to six molecules of folic acid on the carrier surface did not alter the uptake behavior and gene silencing. We further observed several challenges that have to be considered when performing in vitro and in vivo experiments with DNA structures: (i) DNA tile tubes consisting of 42 nt-long oligonucleotides and carrying single- or double-stranded extensions degrade within one hour in cell medium at 37 °C, while the same tubes without extensions are stable for up to eight hours. The degradation is caused mainly by the low concentration of divalent ions in the media. The lifetime in cell medium can be increased drastically by employing DNA tiles that are 84 nt long. (ii) Dyes may get cleaved from the oligonucleotides and then accumulate inside the cell close to the mitochondria, which can lead to misinterpretation of data generated by flow cytometry and fluorescence microscopy. (iii) Single-stranded DNA carrying fluorescent dyes are internalized at similar levels as the DNA tile-assembled tubes used here.
Cryptosporidium parasites are important waterborne pathogens of both humans and animals. The C. parvum and C. hominis genomes indicate that the only route to guanine nucleotides is via inosine 5'-monophosphate dehydrogenase (IMPDH). Thus the inhibition of the parasite IMPDH presents a potential strategy for treating Cryptosporidium infections. A selective benzimidazole-based inhibitor of C. parvum IMPDH (CpIMPDH) was previously identified in a high throughput screen. Here we report a structure-activity relationship study of benzimidazole-based compounds that resulted in potent and selective inhibitors of CpIMPDH. Several compounds display potent antiparasitic activity in vitro.
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