Cysteine specific targeting of the functionally distinct peroxiredoxin and glutaredoxin proteins by the investigational disulfide BNP7787.

Glutaredoxin (Grx), peroxiredoxin (Prx), and thioredoxin (Trx) are redoxin family proteins that catalyze different types of chemical reactions that impact cell growth and survival through functionally distinct intracellular pathways. Much research is focused on understanding the roles of these redoxin proteins in the development and/or progression of human diseases. Grx and Prx are overexpressed in human cancers, including human lung cancers. BNP7787 is a novel investigational agent that has been evaluated in previous clinical studies, including non-small cell lung cancer (NSCLC) studies. Herein, data from activity assays, mass spectrometry analyses, and X-ray crystallographic studies indicate that BNP7787 forms mixed disulfides with select cysteine residues on Grx and Prx and modulates their function. Studies of interactions between BNP7787 and Trx have been conducted and reported separately. Despite the fact that Trx, Grx, and Prx are functionally distinct proteins that impact oxidative stress, cell proliferation and disease processes through different intracellular pathways, BNP7787 can modify each protein and appears to modulate function through mechanisms that are unique to each target protein. Tumor cells are often genomically heterogeneous containing subpopulations of cancer cells that often express different tumor-promoting proteins or that have multiple dysregulated signaling pathways modulating cell proliferation and drug resistance. A multi-targeted agent that simultaneously modulates activity of proteins important in mediating cell proliferation by functionally distinct intracellular pathways could have many potentially useful therapeutic applications.

Novel covalent modification of human anaplastic lymphoma kinase (ALK) and potentiation of crizotinib-mediated inhibition of ALK activity by BNP7787.

BNP7787 (Tavocept, disodium 2,2'-dithio-bis-ethanesulfonate) is a novel, investigational, water-soluble disulfide that is well-tolerated and nontoxic. In separate randomized multicenter Phase II and Phase III clinical trials in non-small-cell lung cancer (NSCLC) patients, treatment with BNP7787 in combination with standard chemotherapy resulted in substantial increases in the overall survival of patients with advanced adenocarcinoma of the lung in the first-line treatment setting. We hypothesized that BNP7787 might interact with and modify human anaplastic lymphoma kinase (ALK). At least seven different variants of ALK fusions with the gene encoding the echinoderm microtubule-associated protein-like 4 (EML4) are known to occur in NSCLC. EML4-ALK fusions are thought to account for approximately 3% of NSCLC cases. Herein, we report the covalent modification of the kinase domain of human ALK by a BNP7787-derived mesna moiety and the functional consequences of this modification in ALK assays evaluating kinase activity. The kinase domain of the ALK protein crystallizes as a monomer, and BNP7787-derived mesna-cysteine adducts were observed at Cys 1235 and Cys 1156. The BNP7787-derived mesna adduct at Cys 1156 is located in close proximity to the active site and results in substantial disorder of the P-loop and activation loop (A-loop). Comparison with the P-loop of apo-ALK suggests that the BNP7787-derived mesna adduct at Cys 1156 interferes with the positioning of Phe 1127 into a small pocket now occupied by mesna, resulting in a destabilization of the loop's binding orientation. Additionally, in vitro kinase activity assays indicate that BNP7787 inhibits ALK catalytic activity and potentiates the activity of the ALK-targeted drug crizotinib.

Naphthamidine urokinase plasminogen activator inhibitors with improved pharmacokinetic properties.

A series of non-amide-linked 6-substituted-2-naphthamidine urokinase plasminogen activator (uPA) inhibitors are described. These compounds possess excellent binding activities and selectivities with significantly improved pharmacokinetic profiles versus previously described amide-linked inhibitors.

A novel mode of Gleevec binding is revealed by the structure of spleen tyrosine kinase.

Spleen tyrosine kinase (Syk) is a non-receptor tyrosine kinase required for signaling from immunoreceptors in various hematopoietic cells. Phosphorylation of two tyrosine residues in the activation loop of the Syk kinase catalytic domain is necessary for signaling, a phenomenon typical of tyrosine kinase family members. Syk in vitro enzyme activity, however, does not depend on phosphorylation (activation loop tyrosine --> phenylalanine mutants retain catalytic activity). We have determined the x-ray structure of the unphosphorylated form of the kinase catalytic domain of Syk. The enzyme adopts a conformation of the activation loop typically seen only in activated, phosphorylated tyrosine kinases, explaining why Syk does not require phosphorylation for activation. We also demonstrate that Gleevec (STI-571, Imatinib) inhibits the isolated kinase domains of both unphosphorylated Syk and phosphorylated Abl with comparable potency. Gleevec binds Syk in a novel, compact cis-conformation that differs dramatically from the binding mode observed with unphosphorylated Abl, the more Gleevec-sensitive form of Abl. This finding suggests the existence of two distinct Gleevec binding modes: an extended, trans-conformation characteristic of tight binding to the inactive conformation of a protein kinase and a second compact, cis-conformation characteristic of weaker binding to the active conformation. Finally, the Syk-bound cis-conformation of Gleevec bears a striking resemblance to the rigid structure of the nonspecific, natural product kinase inhibitor staurosporine.

Discovery of potent inhibitors of dihydroneopterin aldolase using CrystaLEAD high-throughput X-ray crystallographic screening and structure-directed lead optimization.

Potent inhibitors of 7,8-dihydroneopterin aldolase (DHNA; EC 4.1.2.25) have been discovered using CrystaLEAD X-ray crystallographic high-throughput screening followed by structure-directed optimization. Screening of a 10 000 compound random library provided several low affinity leads and their corresponding X-ray crystal structures bound to the enzyme. The presence of a common structural feature in each of the leads suggested a strategy for the construction of a directed library of approximately 1000 compounds that were screened for inhibitory activity in a traditional enzyme assay. Several lead compounds with IC(50) values of about 1 microM against DHNA were identified, and crystal structures of their enzyme-bound complexes were obtained by cocrystallization. Structure-directed optimization of one of the leads thus identified afforded potent inhibitors with submicromolar IC(50) values.

Identification of novel binding interactions in the development of potent, selective 2-naphthamidine inhibitors of urokinase. Synthesis, structural analysis, and SAR of N-phenyl amide 6-substitution.

The preparation and assessment of biological activity of 6-substituted 2-naphthamidine inhibitors of the serine protease urokinase plasminogen activator (uPA, or urokinase) is described. 2-Naphthamidine was chosen as a starting point based on synthetic considerations and on modeling of substituent vectors. Phenyl amides at the 6-position were found to improve binding; replacement of the amide with other two-atom linkers proved ineffective. The phenyl group itself is situated near the S1' subsite; substitutions off of the phenyl group accessed S1' and other distant binding regions. Three new points of interaction were defined and explored through ring substitution. A solvent-exposed salt bridge with the Asp60A carboxylate was formed using a 4-alkylamino group, improving affinity to K(i) = 40 nM. Inhibitors also accessed two hydrophobic regions. One interaction is characterized by a tight hydrophobic fit made with a small dimple largely defined by His57 and His99; a weaker, less specific interaction involves alkyl groups reaching into the broad prime-side protein binding region near Val41 and the Cys42-Cys58 disulfide, displacing water molecules and leading to small gains in activity. Many inhibitors accessed two of these three regions. Affinities range as low as K(i) = 6 nM, and many compounds had K(i) < 100 nM, while moderate to excellent selectivity was gained versus four of five members of a panel of relevant serine proteases. Also, some selectivity against trypsin was generated via the interaction with Asp60A. X-ray structures of many of these compounds were used to inform our inhibitor design and to increase our understanding of key interactions. In combination with our exploration of 8-substitution patterns, we have identified a number of novel binding interactions for uPA inhibitors.