The slow-releasing hydrogen sulfide (H2S) donor, GYY4137, caused concentration-dependent killing of seven different human cancer cell lines (HeLa, HCT-116, Hep G2, HL-60, MCF-7, MV4-11 and U2OS) but did not affect survival of normal human lung fibroblasts (IMR90, WI-38) as determined by trypan blue exclusion. Sodium hydrosulfide (NaHS) was less potent and not active in all cell lines. A structural analogue of GYY4137 (ZYJ1122) lacking sulfur and thence not able to release H2S was inactive. Similar results were obtained using a clonogenic assay. Incubation of GYY4137 (400 µM) in culture medium led to the generation of low (<20 µM) concentrations of H2S sustained over 7 days. In contrast, incubation of NaHS (400 µM) in the same way led to much higher (up to 400 µM) concentrations of H2S which persisted for only 1 hour. Mechanistic studies revealed that GYY4137 (400 µM) incubated for 5 days with MCF-7 but not IMR90 cells caused the generation of cleaved PARP and cleaved caspase 9, indicative of a pro-apoptotic effect. GYY4137 (but not ZYJ1122) also caused partial G2/M arrest of these cells. Mice xenograft studies using HL-60 and MV4-11 cells showed that GYY4137 (100–300 mg/kg/day for 14 days) significantly reduced tumor growth. We conclude that GYY4137 exhibits anti-cancer activity by releasing H2S over a period of days. We also propose that a combination of apoptosis and cell cycle arrest contributes to this effect and that H2S donors should be investigated further as potential anti-cancer agents.

Certain pathogenic bacteria are known to modulate the innate immune response by decorating themselves with sialic acids, which can engage the myelomonocytic lineage inhibitory receptor Siglec-9, thereby evading immunosurveillance. We hypothesized that the well-known up-regulation of sialoglycoconjugates by tumors might similarly modulate interactions with innate immune cells. Supporting this hypothesis, Siglec-9-expressing myelomonocytic cells found in human tumor samples were accompanied by a strong up-regulation of Siglec-9 ligands. Blockade of Siglec-9 enhanced neutrophil activity against tumor cells in vitro. To investigate the function of inhibitory myelomonocytic Siglecs in vivo we studied mouse Siglec-E, the murine functional equivalent of Siglec-9. Siglec-E-deficient mice showed increased in vivo killing of tumor cells, and this effect was reversed by transgenic Siglec-9 expression in myelomonocytic cells. Siglec-Edeficient mice also showed enhanced immunosurveillance of autologous tumors. However, once tumors were established, they grew faster in Siglec-E-deficient mice. In keeping with this, Siglec-E-deficient macrophages showed a propensity toward a tumor-promoting M2 polarization, indicating a secondary role of CD33-related Siglecs in limiting cancer-promoting inflammation and tumor growth. Thus, we define a previously unidentified impact of inhibitory myelomonocytic Siglecs in cancer biology, with distinct roles that reflect the dual function of myelomonocytic cells in cancer progression. In keeping with this, a human polymorphism that reduced Siglec-9 binding to carcinomas was associated with improved early survival in nonsmall-cell lung cancer patients, which suggests that Siglec-9 might be therapeutically targeted within the right time frame and stage of disease.hypersialylation | tumor-associated macrophages | tumor-associated neutrophils | immune evasion | tumor-associated inflammation

Impairment of the formation or action of hydrogen sulfide (H 2 S), an endogenous gasotransmitter, is associated with various diseases, such as hypertension, diabetes mellitus, septic and hemorrhagic shock, and pancreatitis. Cystathionine ␤-synthase and cystathionine ␥-lyase (CSE) are two pyridoxal-5-phosphate (PLP)-dependent enzymes largely responsible for the production of H 2 S in mammals. Inhibition of CSE by DL-propargylglycine (PAG) has been shown to alleviate disease symptoms. Here we report crystal structures of human CSE (hCSE), in apo form, and in complex with PLP and PLP⅐PAG. Structural characterization, combined with biophysical and biochemical studies, provides new insights into the inhibition mechanism of hCSEmediated production of H 2 S. Transition from the open form of apo-hCSE to the closed PLP-bound form reveals large conformational changes hitherto not reported. In addition, PAG binds hCSE via a unique binding mode, not observed in PAG-enzyme complexes previously. The interaction of PAG-hCSE was not predicted based on existing information from known PAG complexes. The structure of hCSE⅐PLP⅐PAG complex highlights the particular importance of Tyr 114 in hCSE and the mechanism of PAG-dependent inhibition of hCSE. These results provide significant insights, which will facilitate the structure-based design of novel inhibitors of hCSE to aid in the development of therapies for diseases involving disorders of sulfur metabolism.

Salmonella Typhi is an exclusive human pathogen that causes typhoid fever. Typhoid toxin is a S. Typhi virulence factor that can reproduce most of the typhoid fever symptoms in experimental animals. Toxicity depends on toxin binding to terminally sialylated glycans on surface glycoproteins. Human glycans are unusual because of the lack of CMAH, which in other mammals converts N-acetylneuraminic acid (Neu5Ac) to N-glycolylneuraminic acid (Neu5Gc). Here we report that typhoid toxin binds to and is toxic towards cells expressing glycans terminated in Neu5Ac (expressed by humans) over glycans terminated in Neu5Gc (expressed by other mammals). Mice constitutively expressing CMAH thus displaying Neu5Gc in all tissues are resistant to typhoid toxin. The atomic structure of typhoid toxin bound to Neu5Ac reveals the structural bases for its binding specificity. These findings provide insight into the molecular bases for Salmonella Typhi’s host specificity and may help the development of therapies for typhoid fever.

Damaged cardiac valves attract blood-borne bacteria, and infective endocarditis is often caused by viridans group streptococci. While such bacteria use multiple adhesins to maintain their normal oral commensal state, recognition of platelet sialoglycans provides an intermediary for binding to damaged valvular endocardium. We use a customized sialoglycan microarray to explore the varied binding properties of phylogenetically related serine-rich repeat adhesins, the GspB, Hsa, and SrpA homologs from Streptococcus gordonii and Streptococcus sanguinis species, which belong to a highly conserved family of glycoproteins that contribute to virulence for a broad range of Gram-positive pathogens. Binding profiles of recombinant soluble homologs containing novel sialic acid-recognizing Siglec-like domains correlate well with binding of corresponding whole bacteria to arrays. These bacteria show multiple modes of glycan, protein, or divalent cation-dependent binding to synthetic glycoconjugates and isolated glycoproteins in vitro. However, endogenous asialoglycan-recognizing clearance receptors are known to ensure that only fully sialylated glycans dominate in the endovascular system, wherein we find these particular streptococci become primarily dependent on their Siglec-like adhesins for glycan-mediated recognition events. Remarkably, despite an excess of alternate sialoglycan ligands in cellular and soluble blood components, these adhesins selectively target intact bacteria to sialylated ligands on platelets, within human whole blood. These preferred interactions are inhibited by corresponding recombinant soluble adhesins, which also preferentially recognize platelets. Our data indicate that circulating platelets may act as inadvertent Trojan horse carriers of oral streptococci to the site of damaged endocardium, and provide an explanation why it is that among innumerable microbes that gain occasional access to the bloodstream, certain viridans group streptococci have a selective advantage in colonizing damaged cardiac valves and cause infective endocarditis.