Sulfur incorporation generally improves Ricin inhibition in pterin-appended glycine-phenylalanine dipeptide mimics.

Several 7-aminoamido-pterins were synthesized to evaluate the electronic and biochemical subtleties observed in the 'linker space' when N-{N-(pterin-7-yl)carbonylglycyl}-l-phenylalanine 1 was bound to the active site of RTA. The gylcine-phenylalanine dipeptide analogs included both amides and thioamides. Decarboxy gly-phe analog 2 showed a 6.4-fold decrease in potency (IC50 = 128 µM), yet the analogous thioamide 7 recovered the lost activity and performed similarly to the parent inhibitor (IC50 = 29 µM). Thiourea 12 exhibited an IC50 nearly six times lower than the oxo analog 13. All inhibitors showed the pterin head-group firmly bound in their X-ray structures yet the pendants were not fully resolved suggesting that all pendants are not firmly bound in the RTA linker space. Calculated log P values do not correlate to the increase in bioactivity suggesting other factors dominate.

Peptide-conjugated pterins as inhibitors of ricin toxin A.

Several 7-peptide-substituted pterins were synthesized and tested as competitive active-site inhibitors of ricin toxin A (RTA). Focus began on dipeptide conjugates, and these results further guided the construction of several tripeptide conjugates. The binding of these compounds to RTA was studied via a luminescence-based kinetic assay, as well as through X-ray crystallography. Despite the relatively polar, solvent exposed active site, several hydrophobic interactions, most commonly π-interactions not predicted by modeling programs, were identified in all of the best-performing inhibitors. Nearly all of these compounds provide IC50 values in the low micromolar range.

Optimized 5-membered heterocycle-linked pterins for the inhibition of Ricin Toxin A.

The optimization of a series of pterin amides for use as Ricin Toxin A (RTA) inhibitors is reported. Based upon crystallographic data of a previous furan-linked pterin, various expanded furans were synthesized, linked to the pterin and tested for inhibition. Concurrently, hetero-analogs of furan were explored, leading to the discovery of more potent triazol-linked pterins. Additionally, we discuss a dramatic improvement in the synthesis of these pterin amides via a dual role by diazabicycloundecene (DBU). This synthetic enhancement facilitates rapid diversification of the previously challenging pterin heterocycle, potentially aiding future medicinal research involving this structure.

Structure-based design of ricin inhibitors.

Ricin is a potent cytotoxin easily purified in large quantities. It presents a significant public health concern due to its potential use as a bioterrorism agent. For this reason, extensive efforts have been underway to develop antidotes against this deadly poison. The catalytic A subunit of the heterodimeric toxin has been biochemically and structurally well characterized, and is an attractive target for structure-based drug design. Aided by computer docking simulations, several ricin toxin A chain (RTA) inhibitors have been identified; the most promising leads belonging to the pterin family. Development of these lead compounds into potent drug candidates is a challenging prospect for numerous reasons, including poor solubility of pterins, the large and highly polar secondary binding pocket of RTA, as well as the enzyme's near perfect catalytic efficiency and tight binding affinity for its natural substrate, the eukaryotic ribosome. To date, the most potent RTA inhibitors developed using this approach are only modest inhibitors with apparent IC(50) values in the 10(-4) M range, leaving significant room for improvement. This review highlights the variety of techniques routinely employed in structure-based drug design projects, as well as the challenges faced in the design of RTA inhibitors.

7-Substituted pterins provide a new direction for ricin A chain inhibitors.

Ricin is a potent toxin found in castor seeds. The A chain, RTA, enzymaticlly depurinates a specific adenosine in ribosomal RNA, inhibiting protein synthesis. Ricin is a known chemical weapons threat having no effective antidote. This makes the discovery of new inhibitors of great importance. We have previously used 6-substituted pterins, such as pteroic acid, as an inhibitor platform with moderate success. We now report the success of 7-carboxy pterin (7CP) as an RTA inhibitor; its binding has been monitored using both kinetic and temperature shift assays and by X-ray crystallography. We also discuss the synthesis of various derivatives of 7CP, and their binding affinity and inhibitory effects, as part of a program to make effective RTA inhibitors.

Real-time investigation of SV40 large T-antigen helicase activity using surface plasmon resonance.

The simian virus 40 (SV40) genome is a model system frequently employed for investigating eukaryotic replication. Large T-antigen (T-ag) is a viral protein responsible for unwinding the SV40 genome and recruiting necessary host factors prior to replication. In addition to duplex unwinding T-ag possesses G-quadruplex DNA helicase activity, the physiological consequence of which is unclear. However, formation of G-quadruplex DNA structures may be involved in genome maintenance and function, and helicase activity to resolve these structures may be necessary for efficient replication. We report the first real-time investigation of SV40 T-ag helicase activity using surface plasmon resonance (SPR). In the presence of ATP, T-ag was observed to bind to immobilized single-stranded DNA, forked duplex DNA, and the human telomeric foldover quadruplex DNA sequence. Inhibition of T-ag duplex helicase activity was observable in real-time and the intramolecular quadruplex was unwound.