Hsp90 is a molecular chaperone of pivotal importance for multiple cell pathways. ATP-regulated internal dynamics are critical for its function and current anticancer pharmacological approaches block the chaperone by using ATP-competitive inhibitors. In this paper, we propose a general approach to perturb Hsp90 through design of new allosteric modulators that alter the functional dynamics of the protein. We rationally developed a library of 2-phenylbenzofurans that, rather than inhibiting, activate Hsp90 ATPase by targeting an allosteric site that we recently identified, located 65Å from the active site. Analysis of protein responses to first-generation activators was exploited to guide the design of second-generation derivatives with improved ability to stimulate ATP hydrolysis. The molecules’ effects on Hsp90 enzymatic, conformational, co-chaperone and client-binding properties were characterized through biochemical, biophysical and cellular approaches. The new, rationally designed probes act as allosteric activators of the chaperone and affect the viability of cancer cell lines for which proper functioning of Hsp90 is necessary.

Despite the enormous progress achieved by modern medicine, numerous diseases still have a profound impact on public health. Infectious diseases caused by a variety of microorganisms (viruses, fungi and parasites) and bacteria are a global major concern and, because of the emergence, for instance, of multidrug resistance, not only in developing countries. The development of preventative therapies, such as the rational design of novel and more efficient vaccines, might offer a solution to this state of affairs and other associated drawbacks. Vaccination is considered by the World Health Organization to be the most cost‐effective strategy for controlling infectious disease, because it should confer long‐term protective immunity in the population. A second consideration involves cancer. The outstanding progress achieved in the identification and structural characterization of tumour‐associated antigens has prompted their employment in tumour immunotherapy, on the basis of the observation that tumour cells possess specific antigens that can be recognized by an immune system appropriately conditioned to the task. Carbohydrates play key roles in many molecular recognition phenomena and they can affect any kind of interaction with the immune system. Saccharide‐based antigens (bacterial capsular polysaccharides or tumour‐associated carbohydrate antigens, for instance) have therefore been studied and employed in the formulation of vaccines. In recent years there has been increasing use of synthetic saccharide antigens for the formulation of vaccine candidates. These structures are indeed chemically well defined, devoid of biologic contaminants and, in principle, available in large amounts, relative to materials extracted from natural sources. In addition, synthetic saccharide antigens can also serve as haptens in protein conjugates, eliciting highly specific antibodies in animal models and humans. The great potential of synthetic saccharide antigens is attested to by the spectacular success of the Cuban vaccine against Haemophilus influenzae type b. Here we review the major advances in the development of synthetic carbohydrate‐based vaccines targeted against infectious diseases and cancer.

Gram-positive Streptococcus agalactiae or group B Streptococcus (GBS) is a leading cause of invasive infections in pregnant women, newborns, and elderly people. Vaccination of pregnant women represents the best strategy for prevention of neonatal disease, and GBS polysaccharide-based conjugate vaccines are currently under clinical testing. The potential of GBS pilus proteins selected by genome-based reverse vaccinology as protective antigens for anti-streptococcal vaccines has also been demonstrated. Dressing pilus proteins with surface glycan antigens could be an attractive approach to extend vaccine coverage. We have recently developed an efficient method for tyrosine-directed ligation of large glycans to proteins via copper-free azide-alkyne [3 + 2] cycloaddition. This method enables targeting of predetermined sites of the protein, ensuring that protein epitopes are preserved prior to glycan coupling and a higher consistency in glycoconjugate batches. Herein, we compared conjugates of the GBS type II polysaccharide (PSII) and the GBS80 pilus protein obtained by classic lysine random conjugation and by the recently developed tyrosine-directed ligation. PSII conjugated to CRM197, a carrier protein used for vaccines in the market, was used as a control. We found that the constructs made from PSII and GBS80 were able to elicit murine antibodies recognizing individually the glycan and protein epitopes on the bacterial surface. The generated antibodies were efficacious in mediating opsonophagocytic killing of strains expressing exclusively PSII or GBS80 proteins. The two glycoconjugates were also effective in protecting newborn mice against GBS infection following vaccination of the dams. Altogether, these results demonstrated that polysaccharide-conjugated GBS80 pilus protein functions as a carrier comparably to CRM197, while maintaining its properties of protective protein antigen. Glycoconjugation and reverse vaccinology can, therefore, be combined to design vaccines with broad coverage. This approach opens a path to a new generation of vaccines. Tyrosine-ligation allows creation of more homogeneous vaccines, correlation of the immune response to defined connectivity points, and fine-tuning of the conjugation site in glycan-protein conjugates.

SummaryA vaccine to prevent infections from the emerging Neisseria meningitidis X (MenX) is becoming an urgent issue. Recently MenX capsular polysaccharide (CPS) fragments conjugated to CRM197 as carrier protein have been confirmed at preclinical stage as promising candidates for vaccine development. However, more insights about the minimal epitope required for the immunological activity of MenX CPS are needed. We report herein the chemical conjugation of fully synthetic MenX CPS oligomers (monomer, dimer, and trimer) to CRM197. Moreover, improvements in some crucial steps leading to the synthesis of MenX CPS fragments are described. Following immunization with the obtained neoglycoconjugates, the conjugated trimer was demonstrated as the minimal fragment possessing immunogenic activity, even though significantly lower than a pentadecamer obtained from the native polymer and conjugated to the same protein. This finding suggests that oligomers longer than three repeating units are possibly needed to mimic the activity of the native polysaccharide.

Background:The capsule of Group B Streptococcus is an important virulence factor and vaccine target. Results: Analysis of type IX polysaccharide revealed a structure similar to type V and VII structures. Conclusion: The structural and phylogenetic basis for the differentiation between types V, VII, and IX was elucidated. Significance: Determination of the type IX structure is instrumental for the development of a carbohydrate-based vaccine.

Studies on the polymerization mode of Neisseria meningitidis serogroup X capsular polymerase CsxA recently identified a truncated construct that can be immobilized and used for length controlled on-column production of oligosaccharides. Here, we combined the use of a synthetic acceptor bearing an appendix for carrier protein conjugation and the on-column process to a novel chemo-enzymatic strategy. After protein coupling of the size optimized oligosaccharide produced by the one-pot elongation procedure, we obtained a more homogeneous glycoconjugate compared to the one previously described starting from the natural polysaccharide. Mice immunized with the conjugated fully synthetic oligomer elicited functional antibodies comparable to controls immunized with the current benchmark MenX glycoconjugates prepared from the natural capsule polymer or from fragments of it enzymatically elongated. This pathogen-free technology allows the fast total in vitro construction of predefined bacterial polysaccharide fragments. Compared to conventional synthetic protocols, the procedure is more expeditious and drastically reduces the number of purification steps to achieve the oligomers. Furthermore, the presence of a linker for conjugation in the synthetic acceptor minimizes manipulations on the enzymatically produced glycan prior to protein conjugation. This approach enriches the methods for fast construction of complex bacterial carbohydrates.

A small library of nonhydrolyzable mimics of GM1 ganglioside, featuring galactose and sialic acid as pharmacophoric carbohydrate residues, was synthesized and tested. All compounds were synthesized from readily available precursors using high-performance reactions, including click chemistry protocols, and avoiding O-glycosidic bonds. Some of the most active molecules also feature a point of further derivatization that can be used for conjugation with polyvalent aglycons. Their affinity towards cholera toxin was assessed by weak affinity chromatography, which allowed a systematic evaluation and selection of the best candidates. Affinity could be enhanced up to one or two orders of magnitude over the affinity of the individual pharmacophoric sugar residues.

Dedicated to Professor Richard R. Schmidt on the occasion of his 78th anniversary Abstract Serotype X of Neisseria meningitidis bacterium (Men X) recently emerged as a substantial threat to public health. Since anti-meningococcal vaccines currently available or under investigation do not contain antigenic components of Men X capsular polysaccharide, there is the need to develop more comprehensive conjugate vaccines capable to offer higher protection. As a preliminary step towards this goal, the synthesis of three conjugatable Men X capsular polysaccharide fragments is described. The installation of the crucial α-glycosyl phosphodiester linkages is based on the hydrogenphosphonate methodology using pure α-glycosyl hydrogenphosphonates 10 and 12 obtained from hemiacetals 9 and 11, respectively.