Deconstructing Best-in-Class Neoglycoclusters as a Tool for Dissecting Key Multivalent Processes in Glycosidase Inhibition

Multivalency represents an appealing option to modulate selectivity in enzyme inhibition and transform moderate glycosidase inhibitors into highly potent ones. The rational design of multivalent inhibitors is however challenging because global affinity enhancement relies on several interconnected local mechanistic events, whose relative impact is unknown. So far, the largest multivalent effects ever reported for a non-polymeric glycosidase inhibitor have been obtained with cyclopeptoid-based inhibitors of Jack bean alfa-mannosidase (JBalfa-man). Here, we report a structure-activity relationship (SAR) study based on the top-down deconstruction of best-in-class multivalent inhibitors. This approach provides a valuable tool to understand the complex interdependent mechanisms underpinning the inhibitory multivalent effect. Combining SAR experiments, binding stoichiometry assessments, thermodynamic modelling and atomistic simulations allowed us to establish the significant contribution of statistical rebinding mechanisms and the importance of several key parameters, including inhitope accessibility, topological restrictions, and electrostatic interactions. Our findings indicate that strong chelate-binding, resulting from the formation of a cross-linked complex between a multivalent inhibitor and two dimeric JBα-man molecules, is not a sufficient condition to reach high levels of affinity enhancements. The deconstruction approach thus offers unique opportunities to better understand multivalent binding and provides important guidelines for the design of potent and selective multiheaded inhibitors

Immunosuppression (IST) Can Be Safely Ceased during Chemotherapy for PTLD in Renal Transplant Patients

Aim. The optimal management of IST in renal transplant patients with PTLD is uncertain. As chemotherapy regimens used for PTLD are in themselves immunosuppressive, IST may not be required during this phase of treatment. Subsequent long-term reduction in IST is important to prevent relapse. We examined whether a protocol (instituted in 1994) of ceasing IST during chemotherapy for PTLD and recommencing IST at reduced doses after chemotherapy (calcineurin inhibitor at 50%, prednisolone 25% increment in serum creatinine (assumed secondary to chronic allograft nephropathy) compared to 4 controls. 10 cases had normal functional allografts with no signifi cant decrement in renal function. The cumulative rate of renal allograft failure requiring change of treatment to dialysis at 22yrs post-transplant was 34% vs 63% for cases and controls, respectively (

Calcineurin A versus NS5A-TP2/HD Domain Containing 2: A Case Study of Site-directed Low-frequency Random Mutagenesis for Dissecting Target Specificity of Peptide Aptamers

We have previously identified a peptide aptamer (named “R5G42”) via functional selection, for its capacity to slow cell proliferation. A yeast two-hybrid screen of human cDNA libraries, using Apta R5G42 as “bait”, allowed identification of two binding proteins with very different functions: Calcineurin A (CnA) (PP2B/PPP3CA), a protein phosphatase well characterized e.g. for its role in the immune response, and NS5A-TP2/HDDC2, a much less studied protein, induced subsequent to Hepatitis C virus Non-structural protein 5A expression in HepG2 hepatocellular carcinoma cells, with no known activity. Our objective, in the present study, was to dissect their specificity in order to have tools with which to be able to better characterize the actions of the peptide aptamers towards their individual targets. This was achieved by the selection of random mutants of the variable loop, derived from R5G42, evaluating their specificity towards CnA and NS5A-TP2, and analyzing their sequence. An interdisciplinary approach, involving biomolecular computer simulations with integration of the sequence data and yeast two hybrid binding phenotypes of these mutants, yielded two structurally-distinct conformers affording the potential molecular basis of the binding diversity of R5G42. Evaluation of the biological impact of CnA vs NS5A-TP2-specific peptide aptamers indicated that while both contributed to the anti-proliferative effect of R5G42, CnA-binding was essential to stimulate nuclear translocation of NFAT, indicative of activation of endogenous CnA. By dissecting target specificity of R5G42, we have generated novel tools with which to study each target individually. Apta-C8 is the first exogenous molecule reported, capable of directly activating CnA independently of binding to NS5A-TP2, whereas Apta-E1 is the first molecule reported that will allow dissection of the function of NS5A-TP2, serving as an example of the usefulness of peptide aptamer technology for investigating signalling pathways