Diana Martik

NUCLEOTIDE-PROMOTED RELEASE OF HMUTSALPHA FROM HETERODUPLEX DNA IS CONSISTENT WITH AN ATP-DEPENDENT TRANSLOCATION MECHANISM

ATP hydrolysis by bacterial and eukaryotic MutS activities is required for their function in mismatch correction, and two different models for the role of ATP in MutS function have been proposed. In the translocation model, based on study of bacterial MutS, ATP binding reduces affinity of the protein for a mismatch and activates secondary DNA binding sites that are subsequently used for movement of the protein along the helix contour in a reaction dependent on nucleotide hydrolysis (Allen, D. J., Makhov, A., Grilley, M., Taylor, J., Thresher, R., Modrich, P., and Griffith, J. D. (1997)EMBO J. 16, 4467–4476). The molecular switch model, based on study of human MutSα, invokes mismatch recognition by the MutSα·ADP complex. After recruitment of downstream repair activities to the MutSα·mismatch complex, ATP binding results in release of MutSα from the heteroduplex (Gradia, S., Acharya, S., and Fishel, R.(1997) Cell 91, 995–1005). To further clarify the function of ATP binding and hydrolysis in human MutSα action, we evaluated the effects of ATP, ADP, and nonhydrolyzable ATP analogs on the lifetime of protein·DNA complexes. All of these nucleotides were found to increase the rate of dissociation of MutSα from oligonucleotide heteroduplexes. These experiments also showed that ADP is not required for mismatch recognition by MutSα, but that the nucleotide alters the dynamics of formation and dissociation of specific complexes. Analysis of the mechanism of ATP-promoted dissociation of MutSα from a 200-base pair heteroduplex demonstrated that dissociation occurs at DNA ends in a reaction dependent on ATP hydrolysis, implying that release from this molecule involves movement of the protein along the helix contour as predicted for a translocation mechanism. In order to reconcile the relatively large rate of movement of MutS homologs along the helix with their modest rate of ATP hydrolysis, we propose a novel mechanism for protein translocation along DNA that supports directional movement over long distances with minimal energy input.

Differential Specificities and Simultaneous Occupancy of Human MutSα Nucleotide Binding Sites

We have examined the permissible nucleotide occupancy states of human MutSα. The MSH2·MSH6 heterodimer binds 1 mol of ADP and 1 mol of adenosine 5′-O-(thiotriphosphate) (ATPγS), with a Kd for each nucleotide of about 1 μm. Anisotropy measurements using BODIPY TR and BODIPY FL fluorescent derivatives of ADP and 5′-adenylyl-β,γ-imidodiphosphate (AMPPNP) also indicate an interaction stoichiometry of 1 mol of ADP and 1 mol of triphosphate analogue per MutSα heterodimer. Di- and triphosphate sites can be simultaneously occupied as judged by sequential filling of the two binding site classes with differentially radiolabeled ADP and ATPγS and by fluorescence resonance energy transfer between BODIPY TR- and BODIPY FL-labeled ADP and AMPPNP. ATP hydrolysis by MutSα is accompanied by a pre-steady-state burst of ADP formation, and analysis of MutSα-bound nucleotide during the first turnover has demonstrated the presence of both ADP and ATP. Simultaneous presence of ADP and a nonhydrolyzable ATP analogue modulates MutSα·heteroduplex interaction in a manner that is distinct from that observed in the presence of ADP or nonhydrolyzable triphosphate alone, and it is unlikely that this effect is due to the presence of a mixed population of binary complexes between MutSα and ADP or a triphosphate analogue. These findings imply that MutSα has two nucleotide binding sites with differential specificities for ADP and ATP and suggest that the ADP·MutSα·ATP ternary complex has an important role in mismatch repair.

Inhibition of plasma kallikrein by a highly specific active site blocking antibody.

Background: Unregulated plasma kallikrein proteolytic activity can result from C1-inhibitor deficiency, causing excessive and potentially fatal edema. Results: The antibody DX-2930 potently and specifically inhibits plasma kallikrein and exhibits a long plasma half-life. Conclusion: An antibody protease inhibitor can lead to potent and specific bioactivity. Significance: DX-2930 could be an effective therapeutic for the prophylactic inhibition of plasma kallikrein-mediated diseases. Plasma kallikrein (pKal) proteolytically cleaves high molecular weight kininogen to generate the potent vasodilator and the pro-inflammatory peptide, bradykinin. pKal activity is tightly regulated in healthy individuals by the serpin C1-inhibitor, but individuals with hereditary angioedema (HAE) are deficient in C1-inhibitor and consequently exhibit excessive bradykinin generation that in turn causes debilitating and potentially fatal swelling attacks. To develop a potential therapeutic agent for HAE and other pKal-mediated disorders, we used phage display to discover a fully human IgG1 monoclonal antibody (DX-2930) against pKal. In vitro experiments demonstrated that DX-2930 potently inhibits active pKal (Ki = 0.120 ± 0.005 nm) but does not target either the zymogen (prekallikrein) or any other serine protease tested. These findings are supported by a 2.1-Å resolution crystal structure of pKal complexed to a DX-2930 Fab construct, which establishes that the pKal active site is fully occluded by the antibody. DX-2930 injected subcutaneously into cynomolgus monkeys exhibited a long half-life (t½ ∼12.5 days) and blocked high molecular weight kininogen proteolysis in activated plasma in a dose- and time-dependent manner. Furthermore, subcutaneous DX-2930 reduced carrageenan-induced paw edema in rats. A potent and long acting inhibitor of pKal activity could be an effective treatment option for pKal-mediated diseases, such as HAE.

Fully human monoclonal antibody inhibitors of the neonatal Fc receptor reduce circulating IgG in non-human primates

The therapeutic management of antibody-mediated autoimmune disease typically involves immunosuppressant and immunomodulatory strategies. However, perturbing the fundamental role of the neonatal Fc receptor (FcRn) in salvaging IgG from lysosomal degradation provides a novel approach – depleting the body of pathogenic immunoglobulin by preventing IgG binding to FcRn and thereby increasing the rate of IgG catabolism. Herein, we describe the discovery and preclinical evaluation of fully human monoclonal IgG antibody inhibitors of FcRn. Using phage display, we identified several potent inhibitors of human-FcRn in which binding to FcRn is pH-independent, with over 1000-fold higher affinity for human-FcRn than human IgG-Fc at pH 7.4. FcRn antagonism in vivo using a human-FcRn knock-in transgenic mouse model caused enhanced catabolism of exogenously administered human IgG. In non-human primates, we observed reductions in endogenous circulating IgG of >60% with no changes in albumin, IgM, or IgA. FcRn antagonism did not disrupt the ability of non-human primates to mount IgM/IgG primary and secondary immune responses. Interestingly, the therapeutic anti-FcRn antibodies had a short serum half-life but caused a prolonged reduction in IgG levels. This may be explained by the high affinity of the antibodies to FcRn at both acidic and neutral pH. These results provide important preclinical proof of concept data in support of FcRn antagonism as a novel approach to the treatment of antibody-mediated autoimmune diseases.