Development of a GalNAc-Tyrosine-Specific Monoclonal Antibody and Detection of Tyrosine O-GalNAcylation in Numerous Human Tissues and Cell Lines.

Glycosylation is a vital post-translational modification involved in a range of biological processes including protein folding, signaling, and cell-cell interactions. In 2011, a new type of O-linked glycosylation was discovered, wherein the side-chain oxygen of tyrosine is modified with a GalNAc residue (GalNAc-Tyr). At present, very little is known about GalNAc-Tyr prevalence, function, or biosynthesis. Herein, we describe the design and synthesis of a GalNAc-Tyr-derived hapten and its use in generating a GalNAc-Tyr selective monoclonal antibody. The antibody, G10C, has an unusually high affinity (app KD = 100 pM) and excellent selectivity for GalNAc-Tyr. We also obtained a crystal structure of the G10C Fab region in complex with 4-nitrophenyl-N-acetyl-α-d-galactosaminide (a small molecule mimic of GalNAc-Tyr) providing insights into the structural basis for high affinity and selectivity. Using this antibody, we discovered that GalNAc-Tyr is widely expressed in most human tissues, indicating that it is a ubiquitous and underappreciated post-translational modification. Localization to specific cell types and organ substructures within those tissues indicates that GalNAc-Tyr is likely regulated in a cell-specific manner. GalNAc-Tyr was also observed in a variety of cell lines and primary cells but was only present on the external cell surface in certain cancer cell lines, suggesting that GalNAc-Tyr localization may be altered in cancer cells. Collectively, the results shed new light on this under-studied form of glycosylation and provide access to new tools that will enable expanded biochemical and clinical investigations.

Extrachromosomal DNA amplicons in antimalarial-resistant Plasmodium falciparum.

Extrachromosomal (ec) DNAs are genetic elements that exist separately from the genome. Since ecDNA can carry beneficial genes, they are a powerful adaptive mechanism in cancers and many pathogens. For the first time, we report ecDNA contributing to antimalarial resistance in Plasmodium falciparum, the most virulent human malaria parasite. Using pulse field gel electrophoresis combined with PCR-based copy number analysis, we detected two ecDNA elements that differ in migration and structure. Entrapment in the electrophoresis well and low susceptibility to exonucleases revealed that the biologically relevant ecDNA element is large and complex in structure. Using deep sequencing, we show that ecDNA originates from the chromosome and expansion of an ecDNA-specific sequence may improve its segregation or expression. We speculate that ecDNA is maintained using established mechanisms due to shared characteristics with the mitochondrial genome. Implications of ecDNA discovery in this organism are wide-reaching due to the potential for new strategies to target resistance development.

Enhanced Binding and Reduced Immunogenicity of Glycoconjugates Prepared via Solid-State Photoactivation of Aliphatic Diazirine Carbohydrates.

Biological conjugation is an important tool employed for many basic research and clinical applications. While useful, common methods of biological conjugation suffer from a variety of limitations, such as (a) requiring the presence of specific surface-exposed residues, such as lysines or cysteines, (b) reducing protein activity, and/or (c) reducing protein stability and solubility. Use of photoreactive moieties including diazirines, azides, and benzophenones provide an alternative, mild approach to conjugation. Upon irradiation with UV and visible light, these functionalities generate highly reactive carbenes, nitrenes, and radical intermediates. Many of these will couple to proteins in a non-amino-acid-specific manner. The main hurdle for photoactivated biological conjugation is very low yield. In this study, we developed a solid-state method to increase conjugation efficiency of diazirine-containing carbohydrates to proteins. Using this methodology, we produced multivalent carbohydrate-protein conjugates with unaltered protein charge and secondary structure. Compared to carbohydrate conjugates prepared with amide linkages to lysine residues using standard NHS conjugation, the photoreactive prepared conjugates displayed up to 100-fold improved binding to lectins and diminished immunogenicity in mice. These results indicate that photoreactive bioconjugation could be especially useful for in vivo applications, such as lectin targeting, where high binding affinity and low immunogenicity are desired.

Sphingosine Kinase 2 Inhibition and Blood Sphingosine 1-Phosphate Levels.

Sphingosine 1-phosphate (S1P) levels are significantly higher in blood and lymph than in tissues. This S1P concentration difference is necessary for proper lymphocyte egress from secondary lymphoid tissue and to maintain endothelial barrier integrity. Studies with mice lacking either sphingosine kinase (SphK) type 1 and 2 indicate that these enzymes are the sole biosynthetic source of S1P, but they play different roles in setting S1P blood levels. We have developed a set of drug-like SphK inhibitors, with differing selectivity for the two isoforms of this enzyme. Although all SphK inhibitors tested decrease S1P when applied to cultured U937 cells, only those inhibitors with a bias for SphK2 drove a substantial increase in blood S1P in mice and this rise was detectable within minutes of administration of the inhibitor. Blood S1P also increased in response to SphK2 inhibitors in rats. Mass-labeled S1P was cleared more slowly after intravenous injection into SphK2 inhibitor-treated mice or mice lacking a functional SphK2 gene; thus, the increased accumulation of S1P in the blood appears to result from the decreased clearance of S1P from the blood. Therefore, SphK2 appears to have a function independent of generating S1P in cells. Our results suggest that differential SphK inhibition with a drug might afford a method to manipulate blood S1P levels in either direction while lowering tissue S1P levels.

Structure-activity relationship studies of the lipophilic tail region of sphingosine kinase 2 inhibitors.

Sphingosine-1-phosphate (S1P) is a ubiquitous, endogenous small molecule that is synthesized by two isoforms of sphingosine kinase (SphK1 and 2). Intervention of the S1P signaling pathway has attracted significant attention because alteration of S1P levels is linked to several disease states including cancer, fibrosis, and sickle cell disease. While intense investigations have focused on developing SphK1 inhibitors, only a limited number of SphK2-selective agents have been reported. Herein, we report our investigations on the structure-activity relationship studies of the lipophilic tail region of SLR080811, a SphK2-selective inhibitor. Our studies demonstrate that the internal phenyl ring is a key structural feature that is essential in the SLR080811 scaffold. Further, we show the dependence of SphK2 activity and selectivity on alkyl tail length, suggesting a larger lipid binding pocket in SphK2 compared to SphK1.

Direct long read visualization reveals metabolic interplay between two antimalarial drug targets.

Increases in the copy number of large genomic regions, termed genome amplification, are an important adaptive strategy for malaria parasites. Numerous amplifications across the Plasmodium falciparum genome contribute directly to drug resistance or impact the fitness of this protozoan parasite. During the characterization of parasite lines with amplifications of the dihydroorotate dehydrogenase (DHODH) gene, we detected increased copies of an additional genomic region that encompassed 3 genes (~5 kb) including GTP cyclohydrolase I (GCH1 amplicon). While this gene is reported to increase the fitness of antifolate resistant parasites, GCH1 amplicons had not previously been implicated in any other antimalarial resistance context. Here, we further explored the association between GCH1 and DHODH copy number. Using long read sequencing and single read visualization, we directly observed a higher number of tandem GCH1 amplicons in parasites with increased DHODH copies (up to 9 amplicons) compared to parental parasites (3 amplicons). While all GCH1 amplicons shared a consistent structure, expansions arose in 2-unit steps (from 3 to 5 to 7, etc copies). Adaptive evolution of DHODH and GCH1 loci was further bolstered when we evaluated prior selection experiments; DHODH amplification was only successful in parasite lines with pre-existing GCH1 amplicons. These observations, combined with the direct connection between metabolic pathways that contain these enzymes, lead us to propose that the GCH1 locus is beneficial for the fitness of parasites exposed to DHODH inhibitors. This finding highlights the importance of studying variation within individual parasite genomes as well as biochemical connections of drug targets as novel antimalarials move towards clinical approval.

Probing the substitution pattern of indole-based scaffold reveals potent and selective sphingosine kinase 2 inhibitors.

Elevated levels of sphingosine 1-phosphate (S1P) and increased expression of sphingosine kinase isoforms (SphK1 and SphK2) have been implicated in a variety of disease states including cancer, inflammation, and autoimmunity. Consequently, the S1P signaling axis has become an attractive target for drug discovery. Selective inhibition of either SphK1 or SphK2 has been demonstrated to be effective in modulating S1P levels in animal models. While SphK1 inhibitors have received much attention, the development of potent and selective SphK2 inhibitors are emerging. Previously, our group reported a SphK2 naphthalene-based selective inhibitor, SLC5081308, which displays approximately 7-fold selectivity for hSphK2 over hSphK1 and has a SphK2 Ki value of 1.0 µM. To improve SphK2 potency and selectivity, we designed, synthesized, and evaluated a series of indole-based compounds derived from SLC5081308. After investigating substitution patterns around the indole ring, we discovered that 1,5-disubstitution promoted optimal binding in the SphK2 substrate binding site and subsequent inhibition of enzymatic activity. Our studies led to the identification of SLC5101465 (6r, SphK2 Ki = 90 nM, >110 fold selective for SphK2 over SphK1). Molecular modeling studies revealed key nonpolar interactions with Val308, Phe548, His556, and Cys533 and hydrogen bonds with both Asp211 and Asp308 as responsible for the high SphK2 inhibition and selectivity.

Structure-Activity Relationship Studies and Molecular Modeling of Naphthalene-Based Sphingosine Kinase 2 Inhibitors.

The two isoforms of sphingosine kinase (SphK1 and SphK2) are the only enzymes that phosphorylate sphingosine to sphingosine-1-phosphate (S1P), which is a pleiotropic lipid mediator involved in a broad range of cellular processes including migration, proliferation, and inflammation. SphKs are targets for various diseases such as cancer, fibrosis, and Alzheimer's and sickle cell disease. Herein, we disclose the structure-activity profile of naphthalene-containing SphK inhibitors and molecular modeling studies that reveal a key molecular switch that controls SphK selectivity.