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Supplementary Figure 3. Representative gating strategy for the analysis of blood and spleen samples from HLA-A2 transgenic mice.

Supplementary Figure 5. Antitumor activity of mCUE-101 in TC-1 tumors.

Purpose:To assess the potential for CUE-101, a novel therapeutic fusion protein, to selectively activate and expand HPV16 E711-20-specific CD8+ T cells as an off-the shelf therapy for the treatment of HPV16-driven tumors, including head and neck squamous cell carcinoma (HNSCC), cervical, and anal cancers.Experimental Design:CUE-101 is an Fc fusion protein composed of a human leukocyte antigen (HLA) complex, an HPV16 E7 peptide epitope, reduced affinity human IL2 molecules, and an effector attenuated human IgG1 Fc domain. Human E7-specific T cells and human peripheral blood mononuclear cells (PBMC) were tested to demonstrate cellular activity and specificity of CUE-101, whereas in vivo activity of CUE-101 was assessed in HLA-A2 transgenic mice. Antitumor efficacy with a murine surrogate (mCUE-101) was tested in the TC-1 syngeneic tumor model.Results:CUE-101 demonstrates selective binding, activation, and expansion of HPV16 E711-20-specific CD8+ T cells from PBMCs relative to nontarget cells. Intravenous administration of CUE-101 induced selective expansion of HPV16 E711-20-specific CD8+ T cells in HLA-A2 (AAD) transgenic mice, and anticancer efficacy and immunologic memory was demonstrated in TC-1 tumor-bearing mice treated with mCUE-101. Combination therapy with anti-PD-1 checkpoint blockade further enhanced the observed efficacy.Conclusions:Consistent with its design, CUE-101 demonstrates selective expansion of an HPV16 E711-20-specific population of cytotoxic CD8+ T cells, a favorable safety profile, and in vitro and in vivo evidence supporting its potential for clinical efficacy in an ongoing phase I trial (NCT03978689).

Supplementary Materials and Methods

Supplementary Figure 4. Antitumor activity of mCUE-101 in established TC-1 tumors.

Supplementary Figure 6. Characterization of E7-specific CD8+ T cells in mice bearing TC-1 tumors.

Supplementary Table 1

Supplementary Figure 1. Characterization of CUE-101 binding properties.

Purpose: To assess the potential for CUE-101, a novel therapeutic fusion protein, to selectively activate and expand HPV16 E711-20-specific CD8+ T cells as an off-the shelf therapy for the treatment of HPV16-driven tumors, including head and neck squamous cell carcinoma (HNSCC), cervical, and anal cancers. Experimental Design: CUE-101 is an Fc fusion protein composed of a human leukocyte antigen (HLA) complex, an HPV16 E7 peptide epitope, reduced affinity human IL2 molecules, and an effector attenuated human IgG1 Fc domain. Human E7-specific T cells and human peripheral blood mononuclear cells (PBMC) were tested to demonstrate cellular activity and specificity of CUE-101, whereas in vivo activity of CUE-101 was assessed in HLA-A2 transgenic mice. Antitumor efficacy with a murine surrogate (mCUE-101) was tested in the TC-1 syngeneic tumor model. Results: CUE-101 demonstrates selective binding, activation, and expansion of HPV16 E711-20-specific CD8+ T cells from PBMCs relative to nontarget cells. Intravenous administration of CUE-101 induced selective expansion of HPV16 E711-20-specific CD8+ T cells in HLA-A2 (AAD) transgenic mice, and anticancer efficacy and immunologic memory was demonstrated in TC-1 tumor-bearing mice treated with mCUE-101. Combination therapy with anti-PD-1 checkpoint blockade further enhanced the observed efficacy. Conclusions: Consistent with its design, CUE-101 demonstrates selective expansion of an HPV16 E711-20-specific population of cytotoxic CD8+ T cells, a favorable safety profile, and in vitro and in vivo evidence supporting its potential for clinical efficacy in an ongoing phase I trial (NCT03978689).

Background Activation of T cells requires a specific peptide/HLA (human leukocyte antigen) signal presented by an interacting immune or target cell along with engagement of co-stimulatory molecules or cytokine receptors. Cue Biopharma has developed a proprietary biologics platform, termed Immuno-STAT™ (Selective Targeting and Alteration of T cells), wherein a singular protein framework incorporates peptide/HLA complexes and co-stimulatory or cytokine signals. The CUE-100 series Immuno-STATs selectively deliver rationally engineered IL-2 molecules to antigen-specific T cells. The IL-2 molecules in the CUE-100 series Immuno-STATs contain mutations that attenuate binding to IL-2 receptors alpha and beta, which minimizes activation of regulatory T cells (Tregs) and the irrelevant non-antigen-specific T cell repertoire. We have demonstrated that CUE-100 series Immuno-STATs specific for different antigenic peptides (from HPV16, WT1, MART-1, CMV, Flu, and HIV) induce expansion of functional, oligoclonal, antigen-specific repertoires from human PBMCs. The lead clinical candidate CUE-101, presenting the E711-20 peptide from HPV-16 in the context of HLA-A*02:01, is currently being tested in a Phase 1 clinical trial in recurrent/metastatic head and neck cancer patients with evidence of dose-proportional PK, early pharmacodynamic effects and signals of clinical activity. Methods CUE-100 series Immuno-STATs were tested with human PBMCs to demonstrate specific T cell activation and expansion. Expanded T cell clonality was assessed by single cell TCR sequencing. Responses of T cells to peptide-presenting targets was evaluated by cytokine staining and by assessing their ability to kill target cells. In vivo activity of CUE-100 series Immuno-STATs was assessed in HLA-A2 transgenic mice. Results Data demonstrate that the CUE-100 series Immuno-STATs selectively activate antigen-specific CD8+ T cells. Signaling, cell-based assays and cytokine release studies confirmed functional attenuation of the IL-2 components of the CUE-100 series, which allows for a favorable safety and selectivity profile. Immuno-STATs demonstrated robust expansion of CD8+ T cells after primary stimulation of unprimed hPBMCs, or re-stimulation of hPBMCs after initial cognate peptide stimulation. In addition, CUE-100 series Immuno-STATs expanded CD8+ T cells in naive HLA-A*02 transgenic mice. In both cases the expanded T cells exhibited a polyfunctional response upon challenge with peptide-presenting target cells. Conclusions The presented data suggests that CUE-100 series Immuno-STATs have the potential to enhance anti-tumor immune responses by inducing a robust antigen-specific, oligoclonal, polyfunctional T cell repertoire. Early validation of CUE-100 series Immuno-STATs is obtained through the emerging signs of pharmacodynamic and clinical activity in the ongoing Phase I trial with CUE-101. Reference Quayle SN, Girgis N, Thapa DR, et al. CUE-101, a Novel HPV16 E7-pHLA-IL-2-Fc Fusion protein, enhances tumor antigen specific T cell activation for the treatment of HPV16-driven malignancies. Clin Cancer Res 2020;26:1953–64.

WT1 has been ranked first amongst 75 tumor associated antigens in an effort by the National Cancer Institute to prioritize cancer antigens for therapeutic targeting. Development of novel modalities to target WT1 provide a significant opportunity to address high unmet medical need in WT1-positive hematological and solid tumor malignancies, including AML, ovarian, endometrial, breast, lung, colorectal and pancreatic cancer. We have developed two novel fusion proteins, termed Immuno-STATs (Selective Targeting and Activation of T cells), that are comprised of either HLA-A*02 or HLA-A*24 molecules (human leukocyte antigen) presenting peptide epitopes derived from WT1. Each CUE-102 Immuno-STAT molecule also contains four copies of affinity-attenuated human interleukin-2 (IL-2), and an effector attenuated human immunoglobulin G (IgG1) Fc domain. We present here the biochemical characterization of these molecules and their bioactivity across a variety of in vitro and in vivo studies, demonstrating their ability to selectively expand, from a naïve or pre-primed T cell repertoire, a polyfunctional population of WT1 peptide-specific CD8+ T cells capable of recognizing and responding to WT1 peptide-presenting target cells. Human peripheral blood mononuclear cells (PBMC) and WT1-specific CD8+ T cells were utilized to demonstrate cellular activity and specificity of CUE-102 Immuno-STATs. Repertoire analysis of WT1-specific T cells was done by single cell TCR sequencing. In vivo activity of CUE-102 molecules was assessed in HLA transgenic mice. CUE-102 Immuno-STATs presenting HLA-A*02 or A*24 specificities demonstrated selective binding and TCR triggering of WT1-specific CD8+ T cells. Signaling, cell-based assays and cytokine release studies confirmed significant functional attenuation of the IL-2 components of CUE-102. Primary stimulation of unprimed hPBMCs, or re-stimulation of hPBMCs after initial WT1 peptide stimulation, led to robust expansion of WT1-specific CD8+ T cells and demonstrated the ability of CUE-102 Immuno-STATs to stimulate and expand antigen-specific T cells from both a primed and pre-primed T cell repertoire. The expanded T cells exhibited a polyfunctional response upon challenge with WT1-presenting target cells, confirming that CUE-102 Immuno-STATs selectively expanded WT1-specific CD8+ T cells. The repertoire of the expanded cells, their polyfunctionality and ability to recognize and respond to WT1 peptide-presenting target cells suggest that CUE-102 Immuno-STATs have the potential to enhance anti-tumor immunity in patients with WT1-positive malignancies. Citation Format: Dharma Thapa, Alex Histed, Jonathan Soriano, Luke Witt, Zohra Merazga, Natasha Girgis, Miguel Moreta, Fulvio Diaz, Fan Zhao, Melissa Kemp, Paige Ruthardt, Anish Suri, Steve Quayle, John Ross, Saso Cemerski. CUE-102 Immuno-STATs for selective targeting and expansion of WT1-specific T cells for the treatment of HLA-A02+and/or HLA-A24+patients with WT1+ malignancies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6699.

High-throughput screening has become a mainstay of small-molecule probe and early drug discovery. The question of how to build and evolve efficient screening collections systematically for cell-based and biochemical screening is still unresolved. It is often assumed that chemical structure diversity leads to diverse biological performance of a library. Here, we confirm earlier results showing that this inference is not always valid and suggest instead using biological measurement diversity derived from multiplexed profiling in the construction of libraries with diverse assay performance patterns for cell-based screens. Rather than using results from tens or hundreds of completed assays, which is resource intensive and not easily extensible, we use high-dimensional image-based cell morphology and gene expression profiles. We piloted this approach using over 30,000 compounds. We show that small-molecule profiling can be used to select compound sets with high rates of activity and diverse biological performance.

Unbiased binding assays involving small-molecule microarrays were used to identify compounds that display unique patterns of selectivity among members of the zinc-dependent histone deacetylase family of enzymes. A novel, hydroxyquinoline-containing compound, BRD4354, was shown to preferentially inhibit activity of HDAC5 and HDAC9 in vitro. Inhibition of deacetylase activity appears to be time-dependent and reversible. Mechanistic studies suggest that the compound undergoes zinc-catalyzed decomposition to an ortho-quinone methide, which covalently modifies nucleophilic cysteines within the proteins. The covalent nature of the compound–enzyme interaction has been demonstrated in experiments with biotinylated probe compound and with electrospray ionization–mass spectrometry.

Post-translational modifications of histones alter chromatin structure and play key roles in gene expression and specification of cell states. Small molecules that target chromatin-modifying enzymes selectively are useful as probes and have promise as therapeutics, although very few are currently available. G9a (also named euchromatin histone methyltransferase 2 (EHMT2)) catalyzes methylation of lysine 9 on histone H3 (H3K9), a modification linked to aberrant silencing of tumor-suppressor genes, among others. Here, we report the discovery of a novel histone methyltransferase inhibitor, BRD4770. This compound reduced cellular levels of di- and trimethylated H3K9 without inducing apoptosis, induced senescence, and inhibited both anchorage-dependent and -independent proliferation in the pancreatic cancer cell line PANC-1. ATM-pathway activation, caused by either genetic or small-molecule inhibition of G9a, may mediate BRD4770-induced cell senescence. BRD4770 may be a useful tool to study G9a and its role in senescence and cancer cell biology.

Histone deacetylases (HDACs) are enzymes involved in many important biological functions. They have been linked to a variety of cancers, psychiatric disorders, and other diseases. Since small molecules can serve as probes to study the relevant biological roles of HDACs, novel scaffolds are necessary to develop more efficient, selective drug candidates. Screening libraries of molecules may yield structurally diverse probes that bind these enzymes and modulate their functions in cells. Here we report a small molecule with a novel hydroxy-pyrimidine scaffold that inhibits multiple HDAC enzymes and modulates acetylation levels in cells. Analogs were synthesized in an effort to evaluate structure-activity relationships.

During group B streptococcal infection, the alpha C protein (ACP) on the bacterial surface binds to host cell surface heparan sulfate proteoglycans (HSPGs) and facilitates entry of bacteria into human epithelial cells. Previous studies in a Drosophila melanogaster model showed that binding of ACP to the sulfated polysaccharide chains (glycosaminoglycans) of HSPGs promotes host death and is associated with higher bacterial burdens. We hypothesized that ACP-glycosaminoglycan binding might determine infection outcome by altering host responses to infection, such as expression of antimicrobial peptides. As glycosaminoglycans/HSPGs also interact with a number of endogenous secreted signaling molecules in Drosophila , we examined the effects of host and pathogen glycosaminoglycan/HSPG-binding structures in host survival of infection and antimicrobial peptide expression. Strikingly, host survival after infection with wild-type streptococci was enhanced among flies overexpressing the endogenous glycosaminoglycan/HSPG-binding morphogen Decapentaplegic—a transforming growth factor β-like Drosophila homolog of mammalian bone morphogenetic proteins—but not by flies overexpressing a mutant, non-glycosaminoglycan-binding Decapentaplegic, or the other endogenous glycosaminoglycan/HSPG-binding morphogens, Hedgehog and Wingless. While ACP-glycosaminoglycan binding was associated with enhanced transcription of peptidoglycan recognition proteins and antimicrobial peptides, Decapentaplegic overexpression suppressed transcription of these genes during streptococcal infection. Further, the glycosaminoglycan-binding domain of ACP competed with Decapentaplegic for binding to the soluble glycosaminoglycan heparin in an in vitro assay. These data suggest that, in addition to promoting bacterial entry into host cells, ACP competes with Decapentaplegic for binding to glycosaminoglycans/HSPGs during infection and that these bacterial and endogenous glycosaminoglycan-binding structures determine host survival and regulate antimicrobial peptide transcription.

The combination of nanoparticles and biological molecules is of intense interest because of the synergistic properties offered by such newly synthesized composites. Heparin (HP), conjugated to nanomaterials, has recently been investigated for its chemical and biological properties. HP has a number of biological activities that can be enhanced when composited with nanoparticles. In addition, HP improves the biocompatibility of nanoparticles improving their performance in various biological applications. A variety of recent research combines HP and nanomaterials for a myriad of applications. HP has been conjugated to the surface of the nanoparticles, such as magnetic and metallic nanoparticles, or biodegradable and nondegradable synthetic polymers. HP has also been incorporated into the nanoparticles. There are numerous possibilities for material composites and chemistries that incorporate HP. This opens the door for novel applications ranging from improving anticoagulant activity, for anticancer and antitumor therapy, to tissue engineering and biosensors. This review examines the different possibilities of HP-based nanoparticle composites and their medicinal or biological applications.

Aims: Silver nanoparticles exhibit unique antibacterial properties that make these ideal candidates for biological and medical applications. We utilized a clean method involving a single synthetic step to prepare silver nanoparticles that exhibit antimicrobial activity. Materials & methods: These nanoparticles were prepared by reducing silver nitrate with diaminopyridinylated heparin (DAPHP) and hyaluronan (HA) polysaccharides and tested for their efficacy in inhibiting microbial growth. Results & discussion: The resulting silver nanoparticles exhibit potent antimicrobial activity against Staphylococcus aureus and modest activity against Escherichia coli. Silver–HA showed greater antimicrobial activity than silver–DAPHP, while silver–glucose nanoparticles exhibited very weak antimicrobial activity. Neither HA nor DAPHP showed activity against S. aureus or E. coli. Conclusion: These results suggest that DAPHP and HA silver nanoparticles have potential in antimicrobial therapeutic applications.

Metal nanoparticles have been studied for their anticoagulant and anti-inflammatory efficacy in various models. Specifically, gold and silver nanoparticles exhibit properties that make these ideal candidates for biological applications. The typical synthesis of gold and silver nanoparticles incorporates contaminants that could pose further problems. Here we demonstrate a clean method of synthesizing gold and silver nanoparticles that exhibit biological functions. These nanoparticles were prepared by reducing AuCl(4) and AgNO(3) using heparin and hyaluronan as both reducing and stabilizing agents. The particles show stability under physiological conditions and narrow size distributions for heparin particles and wider distribution for hyaluronan particles. Studies show that the heparin nanoparticles exhibit anticoagulant properties. Additionally, either gold- or silver-heparin nanoparticles exhibit local anti-inflammatory properties without any significant effect on systemic hemostasis upon administration in carrageenan-induced paw edema models. In conclusion, gold and silver nanoparticles complexed with heparin demonstrated effective anticoagulant and anti-inflammatory efficacy, having potential in various local applications.

Liquid chro ma tog ra phy–mass spec trom e try was applied to deter mine the action pattern of dif fer ent chon droi tin lyases. Two com mer cial enzymes, chon dro itin ase ABC (Pro teus vul ga ris) and chon dro itinase ACII (Arth ro bac ter au res cens), hav ing action pat terns pre vi ously deter mined by vis cos i me try and gel elec tro pho re sis were first exam ined. Next, the action pat terns of recombinant lyases, chon dro itin ase ABC from Bac te roi des theta i ota omi cron (expressed in Esch e richia coli) and chon dro itin ase AC from Fla vo bac terium hep ar i num (expressed in its original host), were exam ined. Chon droi tin sul fate A (CS-A, also known as chon droi tin-4-sul fate) was used as the sub strate for these four lyases. Ali quots taken at var i ous time points were ana lyzed. The prod ucts of chon dro itin ase ABC (P. vul ga ris) and chon dro itin ase AC (F. hepar i num) con tained unsat u rated oli go sac cha rides of sizes rang ing from disac cha ride to deca sac cha ride, dem on strat ing that both are end o lyt ic enzymes. The prod ucts affor ded by chon dro itin ase ABC (B. thetai ota omi cron) and chon dro itin ase ACII (A. au res cens) con tained pri mar ily unsat u rated disac cha ride. These two ex o lyt ic enzymes showed dif fer ent minor prod ucts, sug gest ing some sub tle spec i fic ity dif fer ences between the actions of these two ex o lyt ic lyases on chon droi tin sul fate A.

Heparin, a widely used anticoagulant, is being rapidly displaced by low-molecular-weight heparins. Recently, certain lots of heparin have been associated with anaphylactoid-type reactions resulting from contamination with oversulfated chondroitin sulfate. This impurity has also contaminated low-molecular-weight heparins obtained by chemical and enzymatic depolymerization of heparin. The sensitivity of oversulfated chondroitin sulfate to five different depolymerization processes similar to ones used in preparing low-molecular-weight heparins is reported.

Annexin A2 and heparin bind to one another with high affinity and in a calcium-dependent manner, an interaction that may play a role in mediating fibrinolysis. In this study, three heparin-derived oligosaccharides of different lengths were co-crystallized with annexin A2 to elucidate the structural basis of the interaction. Crystal structures were obtained at high resolution for uncomplexed annexin A2 and three complexes of heparin oligosaccharides bound to annexin A2. The common heparin-binding site is situated at the convex face of domain IV of annexin A2. At this site, annexin A2 binds up to five sugar residues from the nonreducing end of the oligosaccharide. Unlike most heparin-binding consensus patterns, heparin binding at this site does not rely on arrays of basic residues; instead, main-chain and side-chain nitrogen atoms and two calcium ions play important roles in the binding. Especially significant is a novel calcium-binding site that forms upon heparin binding. Two sugar residues of the heparin derivatives provide oxygen ligands for this calcium ion. Comparison of all four structures shows that heparin binding does not elicit a significant conformational change in annexin A2. Finally, surface plasmon resonance measurements were made for binding interactions between annexin A2 and heparin polysaccharide in solution at pH 7.4 or 5.0. The combined data provide a clear basis for the calcium dependence of heparin binding to annexin A2.

Computational methods for image-based profiling are under active development, but their success hinges on assays that can capture a wide range of phenotypes. We have developed a multiplex cytological profiling assay that “paints the cell” with as many fluorescent markers as possible without compromising our ability to extract rich, quantitative profiles in high throughput. The assay detects seven major cellular components. In a pilot screen of bioactive compounds, the assay detected a range of cellular phenotypes and it clustered compounds with similar annotated protein targets or chemical structure based on cytological profiles. The results demonstrate that the assay captures subtle patterns in the combination of morphological labels, thereby detecting the effects of chemical compounds even though their targets are not stained directly. This image-based assay provides an unbiased approach to characterize compound- and disease-associated cell states to support future probe discovery.

Gene expression in eukaryotic organisms can be influenced by a host of internal and external stimuli. The magnitude and duration of the response can often determine the effect of a diverse array of biological functions, many of which can be driven by gene expression. The catalog of possibilities encoded within an organism's primary DNA sequence is heritable and, by design, is immutable in response to these temporally defined internal and external factors and events. It has become increasingly evident that the primary sequence alone is not the only determinant of phenotype and function. Rather, epigenetic mechanisms can influence gene expression without directly affecting the primary DNA sequence. These epigenetic mechanisms can affect gene expression directly via the modification of DNA, or indirectly via the modification of chromatin (storage state of DNA). The acetylation and deacetylation of the N-terminal tails of histones (the core protein component of chromatin) represents an important epigenetic mechanism which affects chromatin structure and, ultimately genomic response, via gene expression. The regulation of histone acetylation status is tightly balanced by the opposing function of two families of enzymes: the HATs (histone acetyltransferases) and the HDACs (histone deacetylases). The function and regulation of these enzymes, which is highly conserved in evolution, can have a profound effect on a multitude of biological processes including transcription, DNA repair and replication, and cellular differentiation. Keywords: Chromatin; Euchromatin; Heterochromatin; Nucleosome; Histones; Acetylation; Deacetylation; HDACs (histone deacetylases); HATs (histone acetyltranferases); Sirtuins

2011 marks the 10-year anniversary of milestone manuscripts describing drafts of the human genome sequence. Over the past decade, a number of new proteins have been linked to disease-many of which fall into classes that have been historically considered challenging from the perspective of drug discovery. Several of these newly associated proteins lack structural information or strong annotation with regard to function, making development of conventional in vitro functional assays difficult. A recent resurgence in the popularity of simple small molecule binding assays has led to new approaches that do not require knowledge of protein structure or function in advance. Here we briefly review selected methods for executing binding assays that have been used successfully to discover small-molecule probes or drug candidates.

The combination of nanoparticles and biological molecules is of intense interest because of the synergistic properties offered by such newly synthesized composites. Heparin (HP), conjugated to nanomaterials, has recently been investigated for its chemical and biological properties. HP has a number of biological activities that can be enhanced when composited with nanoparticles. In addition, HP improves the biocompatibility of nanoparticles improving their performance in various biological applications. A variety of recent research combines HP and nanomaterials for a myriad of applications. HP has been conjugated to the surface of the nanoparticles, such as magnetic and metallic nanoparticles, or biodegradable and nondegradable synthetic polymers. HP has also been incorporated into the nanoparticles. There are numerous possibilities for material composites and chemistries that incorporate HP. This opens the door for novel applications ranging from improving anticoagulant activity, for anticancer and antitumor therapy, to tissue engineering and biosensors. This review examines the different possibilities of HP-based nanoparticle composites and their medicinal or biological applications.

Silver and gold nanoparticles display unique physical and biological properties that have been extensively studied for biological and medical applications. Typically, gold and silver nanoparticles are prepared by chemical reductants that utilize excess toxic reactants, which need to be removed for biological purposes. We utilized a clean method involving a single synthetic step to prepare metal nanoparticles for evaluating potential effects on angiogenesis modulation. These nanoparticles were prepared by reducing silver nitrate and gold chloride with diaminopyridinyl (DAP)-derivatized heparin (HP) polysaccharides. Both gold and silver nanoparticles reduced with DAPHP exhibited effective inhibition of basic fibroblast growth factor (FGF-2)-induced angiogenesis, with an enhanced anti-angiogenesis efficacy with the conjugation to DAPHP (P

Heparan sulfate has been isolated for the first time from the mosquito Anopheles stephensi, a known vector for Plasmodium parasites, the causative agents of malaria. Chondroitin sulfate, but not dermatan sulfate or hyaluronan, was also present in the mosquito. The glycosaminoglycans were isolated, from salivary glands and midguts of the mosquito in quantities sufficient for disaccharide microanalysis. Both of these organs are invaded at different stages of the Plasmodium life cycle. Mosquito heparan sulfate was found to contain the critical trisulfated disaccharide sequence, -->4)beta-D-GlcNS6S(1-->4)-alpha-L-IdoA2S(1-->, that is commonly found in human liver heparan sulfate, which serves as the receptor for apolipoprotein E and is also believed to be responsible for binding to the circumsporozoite protein found on the surface of the Plasmodium sporozoite. The heparan sulfate isolated from the whole mosquito binds to circumsporozoite protein, suggesting a role within the mosquito for infection and transmission of the Plasmodium parasite.

Listeria monocytogenes, a food-borne pathogen that infects immunocompromised patients, enters and proliferates within mammalian cells by taking advantage of host cell machinery. While entry into macrophages and other phagocytic cells occurs constitutively, intracellular invasion of nonphagocytic cells, such as epithelial and endothelial cells, occurs through induced phagocytosis. Invasion of these nonphagocytic cell types is under the control of the secreted L. monocytogenes protein internalin B (InlB), which directly associates with and activates the receptor tyrosine kinase Met. Activation of Met by InlB has previously been shown to be potentiated by binding of glycosaminoglycans to the GW domains of this protein. We studied the interaction between heparin and full-length InlB as well as a truncated, functional form of InlB to understand the mode of interaction between these two molecules. InlB preferred long-chain (or=dp14) heparin oligosaccharides, and the interaction with heparin fit a complicated binding model with a dissociation constant in the nanomolar range. While there are various explanations for this complicated binding model, one supported by our data involves binding and rebinding of InlB to multiple binding sites on heparin in a positive and weakly cooperative manner. This mode is consistent with enhancement of interaction of InlB with glycosaminoglycans for activation of Met.

The 3-O-sulfonation of glucosamine residues in heparan sulfate (HS) by 3-O-sulfotransferase (3-OST) is a key substitution that is present in HS sequences of biological importance, in particular HS anticoagulant activity. Six different isoforms of 3-OST have been identified that exhibit different substrate specificity. In this paper the affinity and kinetics of the interaction between 3-O-sulfotransferase isoform 1 (3-OST-1) and HS have been examined using surface plasmon resonance (SPR). 3-OST-1 binds with micomolar affinity to HS (K(D) = 2.79 microM), and this interaction is apparently independent of the presence of the coenzyme, 3'-phosphoadenosine 5'-phosphosulfate (PAPS). A conformational change in the complex has also been detected, supporting data from previous studies. Selected 3-OST-1 mutants have provided valuable information of amino acid residues that participate in 3-OST-1 interaction with HS substrate and its catalytic activity. The results from this study contribute to understanding the substrate specificity among the 3-OST isoforms and in the mechanism of 3-OST-1-catalyzed biosynthesis of anticoagulant HS.

The biological roles of heparin (HP) and heparan sulfate (HS) are mediated mainly through their interaction with proteins. In the present work, we provide a rapid method for screening HP/HS–protein interactions providing structural data on the key sulfo groups that participate in the binding. A library of polysaccharides structurally related to HP was prepared by immobilizing the biotinylated N-sulfated K5 polysaccharide (N-sulfoheparosan) on sensor chips followed by selective modification of this polysaccharide with enzymes that participate in HP/HS biosynthesis. The polysaccharides synthesized on the surface of the sensor chips differ in the number and position of sulfo groups present both on uronic acid and glucosamine residues. Surface plasmon resonance was used to measure the interaction of each member of this polysaccharide library with antithrombin III (ATIII), to afford structural information on sulfo groups required for this HP/HS–protein interaction. This method is viewed as widely applicable for the study of the structure–activity relationship (SAR) of HP/HS–protein interactions.

The cover picture shows the binding of an influenza virus to the surface of a host cell. The hemaglutinin (blue spikes) binds to the sialic acid (green hexagons) residues present on the non-reducing end of the surface glycoprotein to gain entry into the cell. Once the cell is infected, the viral neuraminidases (pink pinwheels) cleave the sialic acids to escape. The 1,2,3-triazole-linked sialic acid derivatives (green spheres) were designed to act as non-hydrolyzable inhibitors to block the virus. Details are discussed in the article by R. J. Linhardt et al. on p. 2611 ff.