Hepatic FcRn regulates albumin homeostasis and susceptibility to liver injury.

The neonatal crystallizable fragment receptor (FcRn) is responsible for maintaining the long half-life and high levels of the two most abundant circulating proteins, albumin and IgG. In the latter case, the protective mechanism derives from FcRn binding to IgG in the weakly acidic environment contained within endosomes of hematopoietic and parenchymal cells, whereupon IgG is diverted from degradation in lysosomes and is recycled. The cellular location and mechanism by which FcRn protects albumin are partially understood. Here we demonstrate that mice with global or liver-specific FcRn deletion exhibit hypoalbuminemia, albumin loss into the bile, and increased albumin levels in the hepatocyte. In vitro models with polarized cells illustrate that FcRn mediates basal recycling and bidirectional transcytosis of albumin and uniquely determines the physiologic release of newly synthesized albumin into the basal milieu. These properties allow hepatic FcRn to mediate albumin delivery and maintenance in the circulation, but they also enhance sensitivity to the albumin-bound hepatotoxin, acetaminophen (APAP). As such, global or liver-specific deletion of FcRn results in resistance to APAP-induced liver injury through increased albumin loss into the bile and increased intracellular albumin scavenging of reactive oxygen species. Further, protection from injury is achieved by pharmacologic blockade of FcRn-albumin interactions with monoclonal antibodies or peptide mimetics, which cause hypoalbuminemia, biliary loss of albumin, and increased intracellular accumulation of albumin in the hepatocyte. Together, these studies demonstrate that the main function of hepatic FcRn is to direct albumin into the circulation, thereby also increasing hepatocyte sensitivity to toxicity.

Fragment-Based Screening for Enzyme Inhibitors Using Calorimetry.

Isothermal titration calorimetry (ITC) provides a sensitive and accurate means by which to study the thermodynamics of binding reactions. In addition, it enables label-free measurement of enzymatic reactions. The advent of extremely sensitive microcalorimeters have made it increasingly valuable as a tool for hit validation and characterization, but its use in primary screening is hampered by requiring large quantities of reagents and long measurement times. Nanocalorimeters can overcome these limitations of conventional ITC, particularly for screening libraries of 500-1000 compounds such as those encountered in fragment-based lead discovery. This chapter describes how nanocalorimetry and conventional microcalorimetry can be used to screen compound libraries for enzyme inhibitors.

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.

Identification and optimization of PDE10A inhibitors using fragment-based screening by nanocalorimetry and X-ray crystallography.

Fragment-based lead discovery (FBLD) is a technique in which small, low-complexity chemical fragments of 6 to 15 heavy atoms are screened for binding to or inhibiting activity of the target. Hits are then linked and/or elaborated into tightly binding ligands, ideally yielding early lead compounds for drug discovery. Calorimetry provides a label-free method to assay binding and enzymatic activity that is unaffected by the spectroscopic properties of the sample. Conventional microcalorimetry is hampered by requiring large quantities of reagents and long measurement times. Nanocalorimeters can overcome these limitations of conventional isothermal titration calorimetry. Here we use enthalpy arrays, which are arrays of nanocalorimeters, to perform an enzyme activity-based fragment screen for competitive inhibitors of phosphodiesterase 10A (PDE10A). Two dozen fragments with KI <2 mM were identified and moved to crystal soaking trials. All soak experiments yielded high-resolution diffraction, with two-thirds of the fragments yielding high-resolution co-crystal structures with PDE10A. The structural information was used to elaborate fragment hits, yielding leads with KI <1 µM. This study shows how array calorimetry can be used as a prescreening method for fragment-based lead discovery with enzyme targets and paired successfully with an X-ray crystallography secondary screen.

Structure determination of LpxD from the lipopolysaccharide-synthesis pathway of Acinetobacter baumannii.

Acinetobacter baumannii is a Gram-negative bacterium that is resistant to many currently available antibiotics. The protein LpxD is a component of the biosynthetic pathway for lipopolysaccharides in the outer membrane of this bacterium and is a potential target for new antibacterial agents. This paper describes the structure determination of apo forms of LpxD in space groups P2(1) and P4(3)22. These crystals contained six and three copies of the protein molecule in the asymmetric unit and diffracted to 2.8 and 2.7 Šresolution, respectively. A comparison of the multiple protein copies in the asymmetric units of these crystals reveals a common protein conformation and a conformation in which the relative orientation between the two major domains in the protein is altered.

Structure determination of LpxA from the lipopolysaccharide-synthesis pathway of Acinetobacter baumannii.

Acinetobacter baumannii is a Gram-negative pathogenic bacterium which is resistant to most currently available antibiotics and that poses a significant health threat to hospital patients. LpxA is a key enzyme in the biosynthetic pathway of the lipopolysaccharides that are components of the bacterial outer membrane. It is a potential target for antibacterial agents that might be used to fight A. baumannii infections. This paper describes the structure determination of the apo form of LpxA in space groups P2(1)2(1)2(1) and P6(3). These crystal forms contained three and one protein molecules in the asymmetric unit and diffracted to 1.8 and 1.4 Šresolution, respectively. A comparison of the conformations of the independent protein monomers within and between the two crystal asymmetric units revealed very little structural variation across this set of structures. In the P6(3) crystal form the enzymatic site is exposed and is available for the introduction of small molecules of the type used in fragment-based drug discovery and structure-based lead optimization.

Fragment-based screening for inhibitors of PDE4A using enthalpy arrays and X-ray crystallography.

Fragment-based screening has typically relied on X-ray or nuclear magnetic resonance methods to identify low-affinity ligands that bind to therapeutic targets. These techniques are expensive in terms of material and time, so it useful to have a higher throughput method to reliably prescreen a fragment library to identify a subset of compounds for structural analysis. Calorimetry provides a label-free method to assay binding and enzymatic activity that is unaffected by the spectroscopic properties of the sample. Conventional microcalorimetry is hampered by requiring large quantities of reagents and long measurement times. Nanocalorimeters can overcome these limitations of conventional isothermal titration calorimetry. Here we have used enthalpy arrays, which are arrays of nanocalorimeters, to perform an enzyme activity-based fragment screen for competitive inhibitors of phosphodiesterase 4A (PDE4A). Several inhibitors with K ( I ) <2 mM were identified and moved to X-ray crystallization trials. Although the co-crystals did not yield high-resolution data, evidence of binding was observed, and the chemical structures of the hits were consistent with motifs of known PDE4 inhibitors. This study shows how array calorimetry can be used as a prescreening method for fragment-based lead discovery with enzyme targets and provides a list of candidate fragments for inhibition of PDE4A.

The structure of LpxD from Pseudomonas aeruginosa at 1.3†Šresolution.

LpxD is a bacterial protein that is part of the biosynthesis pathway of lipid A and is responsible for transferring 3-hydroxymyristic acid from the R-3-hydroxymyristoyl-acyl carrier protein to the 2-OH group of UDP-3-O-(3-hydroxymyristoyl) glucosamine. The crystal structure of LpxD from Pseudomonas aeruginosa has been determined at high resolution (1.3 Å). The crystal belonged to space group H3, with unit-cell parameters a=b=106.19, c=93.38 Å, and contained one molecule in the asymmetric unit. The structure was solved by molecular replacement using the known structure of LpxD from Escherichia coli (PDB entry 3eh0) as a search model and was refined to Rwork=16.4% (Rfree=18.5%) using 91,655 reflections. The final protein model includes 355 amino-acid residues (including 16 amino acids from a 20 amino-acid N-terminal His tag), one chloride ion and two ethylene glycol molecules.

X-ray crystal structures of monomeric and dimeric peptide inhibitors in complex with the human neonatal Fc receptor, FcRn.

The neonatal Fc receptor, FcRn, is responsible for the long half-life of IgG molecules in vivo and is a potential therapeutic target for the treatment of autoimmune diseases. A family of peptides comprising the consensus motif GHFGGXY, where X is preferably a hydrophobic amino acid, was shown previously to inhibit the human IgG:human FcRn protein-protein interaction (Mezo, A. R., McDonnell, K. A., Tan Hehir, C. A., Low, S. C., Palombella, V. J., Stattel, J. M., Kamphaus, G. D., Fraley, C., Zhang, Y., Dumont, J. A., and Bitonti, A. J. (2008) Proc. Natl. Acad. Sci. U.S.A., 105, 2337-2342). Herein, the x-ray crystal structure of a representative monomeric peptide in complex with human FcRn was solved to 2.6 A resolution. The structure shows that the peptide binds to human FcRn at the same general binding site as does the Fc domain of IgG. The data correlate well with structure-activity relationship data relating to how the peptide family binds to human FcRn. In addition, the x-ray crystal structure of a representative dimeric peptide in complex with human FcRn shows how the bivalent ligand can bridge two FcRn molecules, which may be relevant to the mechanism by which the dimeric peptides inhibit FcRn and increase IgG catabolism in vivo. Modeling of the peptide:FcRn structure as compared with available structural data on Fc and FcRn suggest that the His-6 and Phe-7 (peptide) partially mimic the interaction of His-310 and Ile-253 (Fc) in binding to FcRn, but using a different backbone topology.

Start small and stay small. Minimizing attrition in the clinic with a focus on CNS therapeutics.

Diseases of the central nervous system are among the most devastating to patients and their families. Despite this, treatments for these diseases have lagged behind other therapeutic areas. Although social and economic factors may be partly responsible for the paucity of therapeutic agents, a particularly daunting challenge for CNS drug discovery is the need for compounds to cross the blood brain barrier. Recent analyses of successful drugs have shown that their chemical properties have not changed substantially over the past 40 years while the properties of compounds entering the clinic have become inflated. This property inflation has only exacerbated the challenges of CNS drug discovery as the requirements for delivery to the brain are even more stringent than those for other tissues. New approaches are needed to meet these challenges. In this review, we discuss the merits of fragment based lead discovery and how it may be used to address the challenges of CNS drug discovery. We also summarize compounds discovered by high-throughput screening, substrate evolution and fragment-based lead discovery for well known CNS targets. The results indicate that FBLD may be a key method for discovery of brain penetrable CNS therapeutics.

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.

Interaction with the S1 beta-pocket of urokinase: 8-heterocycle substituted and 6,8-disubstituted 2-naphthamidine urokinase inhibitors.

Several 8-substituted 2-naphthamidine-based inhibitors of the serine protease urokinase plasminogen activator (uPA) are described. Direct attachment of five-membered saturated or unsaturated rings improved inhibitor performance; substitution with sulfones further improved binding profiles. Combination of these substituents or of previously described NH-linked heteroaromatic rings with 6-phenyl amide substituents provided further enhancements to potency and selectivity.

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.