Exploring the chemical space around 8-mercaptoguanine as a route to new inhibitors of the folate biosynthesis enzyme HPPK

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Kinetic Characterization of a Panel of High-Affinity Monoclonal Antibodies Targeting Ricin and Recombinant Re-Formatting for Biosensor Applications

Ricin is a potent glycoprotein toxin that is structurally composed of two subunits joined via a disulfide bond: a ~30 kDa subunit A (RTA) and a ~32 kDa subunit B (RTB). There are fears of ricin being used as a weapon for warfare and terrorism and, as such, there is an increasing need for the development of immunodiagnostic reagents targeted towards this toxin. This article describes the production and characterization of a panel of six ricin-specific monoclonal IgG antibodies (mAbs), previously selected based upon their ability to inhibit ricin-mediated killing of cultured cells. Subsequent epitope binding analysis using the surface plasmon resonance (SPR) array biosensor (ProteOn XPR36) indicated three distinct, non-competitive binding epitopes (“bins”). The association (ka) and dissociation (kd) rate constants and binding affinities (KD) of each of the mAbs to ricin were also determined by SPR using Biacore T100 instrument. Affinities (KD) ranged from 0.1 nM to 9 nM. We present the coding sequences of the variable domains of the six mAbs, the expression, kinetic and cytotoxicity assays for two recombinant Fab (rFab) fragments and demonstrate a rFab affinity improvement by chain-shuffling. Together, these antibodies and constituent rFabs represent a panel of reagents for high-affinity recognition of ricin with potential national security biosensor applications

Kinetic characterization of a panel of high-affinity monoclonal antibodies targeting ricin and recombinant re-formatting for biosensor applications

Ricin is a potent glycoprotein toxin that is structurally composed of two subunits joined via a disulfide bond: a ~30 kDa subunit A (RTA) and a ~32 kDa subunit B (RTB). There are fears of ricin being used as a weapon for warfare and terrorism and, as such, there is an increasing need for the development of immunodiagnostic reagents targeted towards this toxin. This article describes the production and characterization of a panel of six ricin-specific monoclonal IgG antibodies (mAbs), previously selected based upon their ability to inhibit ricin-mediated killing of cultured cells. Subsequent epitope binding analysis using the surface plasmon resonance (SPR) array biosensor (ProteOn XPR36) indicated three distinct, non-competitive binding epitopes (“bins”). The association (ka) and dissociation (kd) rate constants and binding affinities (KD) of each of the mAbs to ricin were also determined by SPR using Biacore T100 instrument. Affinities (KD) ranged from 0.1 nM to 9 nM. We present the coding sequences of the variable domains of the six mAbs, the expression, kinetic and cytotoxicity assays for two recombinant Fab (rFab) fragments and demonstrate a rFab affinity improvement by chain-shuffling. Together, these antibodies and constituent rFabs represent a panel of reagents for high-affinity recognition of ricin with potential national security biosensor applications

Synthetic scheme for compound 21e.

<p>Synthetic scheme for compound 21e.</p

Thermodynamic parameters for the binding of selected compounds to <i>Sa</i>HPPK as determined by ITC^a.

a<p>Values are the means ± the standard deviation for at least three experiments. All ITC and SPR experiments were performed at 298 K and 293 K, respectively. ^b data from Chhabra <i>et al</i> PlosONE 2012.</p

Structures of <i>C</i>⁸, <i>N</i>⁹ and <i>N</i>⁷-substituted guanine analogues and their binding affinities to <i>Sa</i>HPPK, as determined by SPR.

a<p>in the presence of 10 mM Mg^2+.</p>b<p>in the presence of 10 mM Mg²⁺/1 mM ATP.</p>c<p>nd: not determined.</p

Structure of SaHPPK in complex with 8-MG.

<p>A–C) Structure of <i>Sa</i>HPPK (PDB:1QBC) in complex with 8-MG. A–B Intermolecular interactions between 8-MG and <i>Sa</i>HPPK. C) Surface representation of <i>Sa</i>HPPK showing the bound 8-MG (blue) overlayed with the closed loop L3 (green) and the bound AMPCPP as observed in the <i>Ec</i>HPPK/HMDP/AMPCPP (PDB:1Q0N) complex. D) Ribbon representation of the loop structure of several <i>Ec</i>HPPK structures overlayed with <i>Sa</i>HPPK (yellow) in complex with 8-MG (red) to illustrate the range of conformations in loops L2 and L3. The interaction of the Trp89 (brown) and the phenethyl inhibitor (cyan) is highlighted (PDB:1DY3) and the position of the HMDP (pink) and AMPCPP (pink) from <i>Ec</i>HPPK/HMDP/AMPCPP (PDB:1Q0N). Images were produced using the UCSF Chimera package (<a href="http://www.cgl.ucsf.edu/chimera" target="_blank">www.cgl.ucsf.edu/chimera</a>).</p

HPPK function and known inhibitors.

<p>A) HPPK catalysis. B) Known inhibitors of HPPK.</p