We have recently proposed a novel drug discovery approach based on biophysical screening of focused positional scanning libraries in which each element of the library contained a common binding moiety for the given target or class of targets. In this Letter, we report on the implementation of this approach to target metal containing proteins. In our implementation, we first derived a focused positional scanning combinatorial library of peptide mimetics (of approximately 100,000 compounds) in which each element of the library contained the metal-chelating moiety hydroxamic acid at the C-terminal. Screening of this library by nuclear magnetic resonance spectroscopy in solution allowed the identification of a novel and selective compound series targeting MMP-12. The data supported that our general approach, perhaps applied using other metal chelating agents or other initial binding fragments, may result very effective in deriving novel and selective agents against metalloenzyme.

Streptococcus gordonii and Streptococcus sanguinis, commensal bacteria present in the oral cavity of healthy individuals, upon entry into the bloodstream can become pathogenic, causing infective endocarditis (IE). Sialic acid-binding serine-rich repeat adhesins on the microbial surface represent an important factor of successful infection to cause IE. They contain Siglec-like binding regions (SLBRs) that variously recognize different repertoires of O-glycans, with some strains displaying high selectivity and others broader specificity. We here dissect at an atomic level the mechanism of interaction of SLBR-B and SLBR-H from S. gordonii with a multivarious approach that combines NMR spectroscopy and computational and biophysical studies. The binding pockets of both SLBRs are broad enough to accommodate extensive interactions with sialoglycans although with key differences related to strain specificity. Furthermore, and significantly, the pattern of interactions established by the SLBRs are mechanistically very different from those reported for mammalian Siglecs despite them having a similar fold. Thus, our detailed description of the binding modes of streptococcal Siglec-like adhesins sparks the development of tailored synthetic inhibitors and therapeutics specific for Streptococcal adhesins to counteract IE, without impairing the interplay between Siglecs and glycans.

The spread of multidrug-resistant strains of Neisseria gonorrhoeae, the etiologic agent of gonorrhea, represents a global health emergency. Therefore, the development of a safe and effective vaccine against gonorrhea is urgently needed. In previous studies, murine monoclonal antibody (mAb) 2C7 was raised against gonococcal lipooligosaccharide (LOS). mAb 2C7 elicits complement-dependent bactericidal activity against gonococci, and its glycan epitope is expressed by almost every clinical isolate. Furthermore, we identified a peptide, cyclic peptide 2 (CP2) that mimicked the 2C7 LOS epitope, elicited bactericidal antibodies in mice, and actively protected in a mouse vaginal colonization model. In this study, we performed structural analyses of mAb 2C7 and its complex with the CP2 peptide by X-ray crystallography, NMR spectroscopy, and molecular dynamics (MD) simulations. The crystal structure of Fab 2C7 bound to CP2 showed that the peptide adopted a beta-hairpin conformation and bound the Fab primarily through hydrophobic interactions. We employed NMR spectroscopy and MD simulations to map the 2C7 epitope and identify the bioactive conformation of CP2. We also used small-angle X-ray scattering (SAXS) and native mass spectrometry to obtain further information about the shape and assembly state of the complex. Collectively, our new structural information suggests strategies for humanizing mAb 2C7 as a therapeutic against gonococcal infection and for optimizing peptide CP2 as a vaccine antigen.

Streptococcus gordonii is a Gram-positive bacterial species that typically colonizes the human oral cavity, but can also cause local or systemic diseases. Serine-rich repeat (SRR) glycoproteins exposed on the S. gordonii bacterial surface bind to sialylated glycans on human salivary, plasma, and platelet glycoproteins, which may contribute to oral colonization as well as endocardial infections. Despite a conserved overall domain organization of SRR adhesins, the Siglec-like binding regions (SLBRs) are highly variable, affecting the recognition of a wide range of sialoglycans. SLBR-N from the SRR glycoprotein of S. gordonii UB10712 possesses the remarkable ability to recognize complex core 2 O-glycans. We here employed a multidisciplinary approach, including flow cytometry, native mass spectrometry, isothermal titration calorimetry, NMR spectroscopy from both protein and ligand perspectives, and computational methods, to investigate the ligand specificity and binding preferences of SLBR-N when interacting with mono- and disialylated core 2 O-glycans. We determined the means by which SLBR-N preferentially binds branched α2,3-disialylated core 2 O-glycans: a selected conformation of the 3'SLn branch is accommodated into the main binding site, driving the sTa branch to further interact with the protein. At the same time, SLBR-N assumes an open conformation of the CD loop of the glycan-binding pocket, allowing one to accommodate the entire complex core 2 O-glycan. These findings establish the basis for the generation of novel tools for the detection of specific complex O-glycan structures and pave the way for the design and development of potential therapeutics against streptococcal infections.