Jeremy A. Horst

(+)-SJ733, a clinical candidate for malaria that acts through ATP4 to induce rapid host-mediated clearance of Plasmodium

Significance Useful antimalarial drugs must be rapidly acting, highly efficacious, and have low potential for developing resistance. (+)-SJ733 targets a Plasmodium cation-transporting ATPase, ATP4. (+)-SJ733 cleared parasites in vivo as quickly as artesunate by specifically inducing eryptosis/senescence in infected, treated erythrocytes. Although in vitro selection of pfatp4 mutants with (+)-SJ733 proceeded with moderate frequency, during in vivo selection of pbatp4 mutants, resistance emerged slowly and produced marginally resistant mutants with poor fitness. In addition, (+)-SJ733 met all other criteria for a clinical candidate, including high oral bioavailability, a high safety margin, and transmission blocking activity. These results demonstrate that targeting ATP4 has great potential to deliver useful drugs for malaria eradication. Drug discovery for malaria has been transformed in the last 5 years by the discovery of many new lead compounds identified by phenotypic screening. The process of developing these compounds as drug leads and studying the cellular responses they induce is revealing new targets that regulate key processes in the Plasmodium parasites that cause malaria. We disclose herein that the clinical candidate (+)-SJ733 acts upon one of these targets, ATP4. ATP4 is thought to be a cation-transporting ATPase responsible for maintaining low intracellular Na+ levels in the parasite. Treatment of parasitized erythrocytes with (+)-SJ733 in vitro caused a rapid perturbation of Na+ homeostasis in the parasite. This perturbation was followed by profound physical changes in the infected cells, including increased membrane rigidity and externalization of phosphatidylserine, consistent with eryptosis (erythrocyte suicide) or senescence. These changes are proposed to underpin the rapid (+)-SJ733-induced clearance of parasites seen in vivo. Plasmodium falciparum ATPase 4 (pfatp4) mutations that confer resistance to (+)-SJ733 carry a high fitness cost. The speed with which (+)-SJ733 kills parasites and the high fitness cost associated with resistance-conferring mutations appear to slow and suppress the selection of highly drug-resistant mutants in vivo. Together, our data suggest that inhibitors of PfATP4 have highly attractive features for fast-acting antimalarials to be used in the global eradication campaign.

Caries induced cytokine network in the odontoblast layer of human teeth

BackgroundImmunologic responses of the tooth to caries begin with odontoblasts recognizing carious bacteria. Inflammatory propagation eventually leads to tooth pulp necrosis and danger to health. The present study aims to determine cytokine gene expression profiles generated within human teeth in response to dental caries in vivo and to build a mechanistic model of these responses and the downstream signaling network.ResultsWe demonstrate profound differential up-regulation of inflammatory genes in the odontoblast layer (ODL) in human teeth with caries in vivo, while the pulp remains largely unchanged. Interleukins, chemokines, and all tested receptors thereof were differentially up-regulated in ODL of carious teeth, well over one hundred-fold for 35 of 84 genes. By interrogating reconstructed protein interaction networks corresponding to the differentially up-regulated genes, we develop the hypothesis that pro-inflammatory cytokines highly expressed in ODL of carious teeth, IL-1β, IL-1α, and TNF-α, carry the converged inflammatory signal. We show that IL1β amplifies antimicrobial peptide production in odontoblasts in vitro 100-fold more than lipopolysaccharide, in a manner matching subsequent in vivo measurements.ConclusionsOur data suggest that ODL amplifies bacterial signals dramatically by self-feedback cytokine-chemokine signal-receptor cycling, and signal convergence through IL1R1 and possibly others, to increase defensive capacity including antimicrobial peptide production to protect the tooth and contain the battle against carious bacteria within the dentin.

Community-wide assessment of GPCR structure modelling and ligand docking

Recent breakthroughs in the determination of the crystal structures of G protein-coupled receptors (GPCRs) have provided new opportunities for structure-based drug design strategies targeting this protein family. With the aim of evaluating the current status of GPCR structure prediction and ligand docking, a community-wide, blind prediction assessment — GPCR Dock 2008 — was conducted in coordination with the publication of the crystal structure of the human adenosine A2A receptor bound to the ligand ZM241385. Twenty-nine groups submitted 206 structural models before the release of the experimental structure, which were evaluated for the accuracy of the ligand binding mode and the overall receptor model compared with the crystal structure. This analysis highlights important aspects for success and future development, such as accurate modelling of structurally divergent regions and use of additional biochemical insight such as disulphide bridges in the extracellular loops.

Methicillin-resistant Staphylococcus aureus from dental school clinic surfaces and students

BACKGROUND Methicillin-resistant Staphylococcus aureus (MRSA) isolated from frequently touched dental school clinic surfaces were compared with MRSA isolated nasal cultures of dental students. METHOD Sixty-one dental students and 95 environmental surfaces from 7 clinics were sampled using SANICULT (Starplex Scientific Inc, Etobicoke, Ontario, Canada) swabs. Antimicrobial susceptibility testing was performed, and pulsed-field gel electrophoresis analysis, the mecA gene, multilocus sequence type, and SCCmec type were determined by polymerase chain reaction and sequencing. RESULTS Thirteen (21%) dental students and 8 (8.4%) surfaces were MRSA positive. Three MRSA strains were SCCmec type IV, whereas 3 were nontypeable isolates and Panton-Valentine leukocidin positive (PVL+), and none were USA300. One surface and 1 student isolate shared the same multilocus sequence type ST 8 and were 75% related. Two groups of students carried the same MRSA strains. CONCLUSION The MRSA-positive samples were from 4 of 7 dental clinics. In addition, 21% of the dental students carried MRSA, which is > 10 times higher than the general public and twice as frequent as in other university students. This is the first study to characterize MRSA from dental clinic surfaces and dental students and suggests that both may be reservoirs for MRSA. Further studies are needed to verify this premise.

Protein Meta-Functional Signatures from Combining Sequence, Structure, Evolution, and Amino Acid Property Information

Protein function is mediated by different amino acid residues, both their positions and types, in a protein sequence. Some amino acids are responsible for the stability or overall shape of the protein, playing an indirect role in protein function. Others play a functionally important role as part of active or binding sites of the protein. For a given protein sequence, the residues and their degree of functional importance can be thought of as a signature representing the function of the protein. We have developed a combination of knowledge- and biophysics-based function prediction approaches to elucidate the relationships between the structural and the functional roles of individual residues and positions. Such a meta-functional signature (MFS), which is a collection of continuous values representing the functional significance of each residue in a protein, may be used to study proteins of known function in greater detail and to aid in experimental characterization of proteins of unknown function. We demonstrate the superior performance of MFS in predicting protein functional sites and also present four real-world examples to apply MFS in a wide range of settings to elucidate protein sequence–structure–function relationships. Our results indicate that the MFS approach, which can combine multiple sources of information and also give biological interpretation to each component, greatly facilitates the understanding and characterization of protein function.

Self-assembly of filamentous amelogenin requires calcium and phosphate: from dimers via nanoribbons to fibrils.

Enamel matrix self-assembly has long been suggested as the driving force behind aligned nanofibrous hydroxyapatite formation. We tested if amelogenin, the main enamel matrix protein, can self-assemble into ribbon-like structures in physiologic solutions. Ribbons 17 nm wide were observed to grow several micrometers in length, requiring calcium, phosphate, and pH 4.0-6.0. The pH range suggests that the formation of ion bridges through protonated histidine residues is essential to self-assembly, supported by a statistical analysis of 212 phosphate-binding proteins predicting 12 phosphate-binding histidines. Thermophoretic analysis verified the importance of calcium and phosphate in self-assembly. X-ray scattering characterized amelogenin dimers with dimensions fitting the cross-section of the amelogenin ribbon, leading to the hypothesis that antiparallel dimers are the building blocks of the ribbons. Over 5-7 days, ribbons self-organized into bundles composed of aligned ribbons mimicking the structure of enamel crystallites in enamel rods. These observations confirm reports of filamentous organic components in developing enamel and provide a new model for matrix-templated enamel mineralization.

IGF1R variants associated with isolated single suture craniosynostosis

The genetic contribution to the pathogenesis of isolated single suture craniosynostosis is poorly understood. The role of mutations in genes known to be associated with syndromic synostosis appears to be limited. We present our findings of a candidate gene resequencing approach to identify rare variants associated with the most common forms of isolated craniosynostosis. Resequencing of the coding regions, splice junction sites, and 5′ and 3′ untranslated regions of 27 candidate genes in 186 cases of isolated non‐syndromic single suture synostosis revealed three novel and two rare sequence variants (R406H, R595H, N857S, P190S, M446V) in insulin‐like growth factor I receptor (IGF1R) that are enriched relative to control samples. Mapping the resultant amino acid changes to the modeled homodimer protein structure suggests a structural basis for segregation between these and other disease‐associated mutations found in IGF1R. These data suggest that IGF1R mutations may contribute to the risk and in some cases cause single suture craniosynostosis.

Cementomimetics—constructing a cementum-like biomineralized microlayer via amelogenin-derived peptides

Cementum is the outer-, mineralized-tissue covering the tooth root and an essential part of the system of periodontal tissue that anchors the tooth to the bone. Periodontal disease results from the destructive behavior of the host elicited by an infectious biofilm adhering to the tooth root and left untreated, may lead to tooth loss. We describe a novel protocol for identifying peptide sequences from native proteins with the potential to repair damaged dental tissues by controlling hydroxyapatite biomineralization. Using amelogenin as a case study and a bioinformatics scoring matrix, we identified regions within amelogenin that are shared with a set of hydroxyapatite-binding peptides (HABPs) previously selected by phage display. One 22-amino acid long peptide regions referred to as amelogenin-derived peptide 5 (ADP5) was shown to facilitate cell-free formation of a cementum-like hydroxyapatite mineral layer on demineralized human root dentin that, in turn, supported attachment of periodontal ligament cells in vitro. Our findings have several implications in peptide-assisted mineral formation that mimic biomineralization. By further elaborating the mechanism for protein control over the biomineral formed, we afford new insights into the evolution of protein–mineral interactions. By exploiting small peptide domains of native proteins, our understanding of structure–function relationships of biomineralizing proteins can be extended and these peptides can be utilized to engineer mineral formation. Finally, the cementomimetic layer formed by ADP5 has the potential clinical application to repair diseased root surfaces so as to promote the regeneration of periodontal tissues and thereby reduce the morbidity associated with tooth loss.

Amyloid-like ribbons of amelogenins in enamel mineralization

Enamel, the outermost layer of teeth, is an acellular mineralized tissue that cannot regenerate; the mature tissue is composed of high aspect ratio apatite nanocrystals organized into rods and inter-rod regions. Amelogenin constitutes 90% of the protein matrix in developing enamel and plays a central role in guiding the hierarchical organization of apatite crystals observed in mature enamel. To date, a convincing link between amelogenin supramolecular structures and mature enamel has yet to be described, in part because the protein matrix is degraded during tissue maturation. Here we show compelling evidence that amelogenin self-assembles into an amyloid-like structure in vitro and in vivo. We show that enamel matrices stain positive for amyloids and we identify a specific region within amelogenin that self-assembles into β-sheets. We propose that amelogenin nanoribbons template the growth of apatite mineral in human enamel. This is a paradigm shift from the current model of enamel development.

The NH2-terminal propeptide of type I procollagen acts intracellularly to modulate cell function

The function of the NH2-terminal propeptide of type I procollagen (N-propeptide) is poorly understood. We now show that a recombinant trimeric N-propeptide interacts with transforming growth factor-β1 and BMP2 and exhibits functional effects in stably transfected cells. The synthesis of N-propeptide by COS-7 cells results in an increase in phosphorylation of Akt and Smad3 and is associated with a marked reduction in type I procollagen synthesis and impairment in adhesion. In C2C12 cells, N-propeptide inhibits the osteoblastic differentiation induced by BMP2. Our data suggest that these effects are mediated by the interaction of N-propeptide with an intracellular receptor in the secretory pathway, because they are not observed when recombinant N-propeptide is added to the culture medium of either COS-7 or C2C12 cells. Both the binding of N-propeptide to cytokines and its functional properties are entirely dependent on the exon 2-encoded globular domain, and a mutation that substitutes a serine for a highly conserved cysteine in exon 2 abolishes its function. Our findings suggest that N-propeptide performs an important feedback regulatory function and provides a rationale for the prominence of a homotrimeric form of type I procollagen (α1 trimer) during vertebrate development.