Kristine Svenson

Crystal structures of the two major aggrecan degrading enzymes, ADAMTS4 and ADAMTS5

Aggrecanases are now believed to be the principal proteinases responsible for aggrecan degradation in osteoarthritis. Given their potential as a drug target, we solved crystal structures of the two most active human aggrecanase isoforms, ADAMTS4 and ADAMTS5, each in complex with bound inhibitor and one wherein the enzyme is in apo form. These structures show that the unliganded and inhibitor‐bound enzymes exhibit two essentially different catalytic‐site configurations: an autoinhibited, nonbinding, closed form and an open, binding form. On this basis, we propose that mature aggrecanases exist as an ensemble of at least two isomers, only one of which is proteolytically active.

ATP-Competitive Inhibitors of the Mammalian Target of Rapamycin: Design and Synthesis of Highly Potent and Selective Pyrazolopyrimidines.

The mammalian target of rapamycin (mTOR), a central regulator of growth, survival, and metabolism, is a validated target for cancer therapy. Rapamycin and its analogues, allosteric inhibitors of mTOR, only partially inhibit one mTOR protein complex. ATP-competitive, global inhibitors of mTOR that have the potential for enhanced anticancer efficacy are described. Structural features leading to potency and selectivity were identified and refined leading to compounds with in vivo efficacy in tumor xenograft models.

Catalytically active MAP KAP kinase 2 structures in complex with staurosporine and ADP reveal differences with the autoinhibited enzyme

MAP KAP kinase 2 (MK2), a Ser/Thr kinase, plays a crucial role in the inflammatory process. We have determined the crystal structures of a catalytically active C-terminal deletion form of human MK2, residues 41-364, in complex with staurosporine at 2.7 A and with ADP at 3.2 A, revealing overall structural similarity with other Ser/Thr kinases. Kinetic analysis reveals that the K(m) for ATP is very similar for MK2 41-364 and p38-activated MK2 41-400. Conversely, the catalytic rate and binding for peptide substrate are dramatically reduced in MK2 41-364. However, phosphorylation of MK2 41-364 by p38 restores the V(max) and K(m) for peptide substrate to values comparable to those seen in p38-activated MK2 41-400, suggesting a mechanism for regulation of enzyme activity.

Efficacious 11β-Hydroxysteroid Dehydrogenase Type I Inhibitors in the Diet-Induced Obesity Mouse Model

Cortisol and the glucocorticoid receptor signaling pathway have been implicated in the development of diabetes and obesity. The reduction of cortisone to cortisol is catalyzed by 11beta-hydroxysteroid dehydrogenase type I (11beta-HSD1). 2,4-Disubsituted benzenesulfonamides were identified as potent inhibitors of both the human and mouse enzymes. The lead compounds displayed good pharmacokinetics and ex vivo inhibition of the target in mice. Cocrystal structures of compounds 1 and 20 bound to human 11beta-HSD1 were obtained. Compound 20 was found to achieve high concentrations in target tissues, resulting in 95% inhibition in the ex vivo assay when dosed with a food mix (0.5 mg of drug per g of food) after 4 days. Compound 20 was efficacious in a mouse diet-induced obesity model and significantly reduced fed glucose and fasted insulin levels. Our findings suggest that 11beta-HSD1 inhibition may be a valid target for the treatment of diabetes.

3,5-Dioxopyrazolidines, Novel Inhibitors of UDP-N- Acetylenolpyruvylglucosamine Reductase (MurB) with Activity against Gram-Positive Bacteria

ABSTRACT A series of 3,5-dioxopyrazolidines was identified as novel inhibitors of UDP-N-acetylenolpyruvylglucosamine reductase (MurB). Compounds 1 to 3, which are 1,2-bis(4-chlorophenyl)-3,5-dioxopyrazolidine-4-carboxamides, inhibited Escherichia coli MurB, Staphyloccocus aureus MurB, and E. coli MurA with 50% inhibitory concentrations (IC50s) in the range of 4.1 to 6.8 μM, 4.3 to 10.3 μM, and 6.8 to 29.4 μM, respectively. Compound 4, a C-4-unsubstituted 1,2-bis(3,4-dichlorophenyl)-3,5-dioxopyrazolidine, showed moderate inhibitory activity against E. coli MurB, S. aureus MurB, and E. coli MurC (IC50s, 24.5 to 35 μM). A fluorescence-binding assay indicated tight binding of compound 3 with E. coli MurB, giving a dissociation constant of 260 nM. Structural characterization of E. coli MurB was undertaken, and the crystal structure of a complex with compound 4 was obtained at 2.4 Å resolution. The crystal structure indicated the binding of a compound at the active site of MurB and specific interactions with active-site residues and the bound flavin adenine dinucleotide cofactor. Peptidoglycan biosynthesis studies using a strain of Staphylococcus epidermidis revealed reduced peptidoglycan biosynthesis upon incubation with 3,5-dioxopyrazolidines, with IC50s of 0.39 to 11.1 μM. Antibacterial activity was observed for compounds 1 to 3 (MICs, 0.25 to 16 μg/ml) and 4 (MICs, 4 to 8 μg/ml) against gram-positive bacteria including methicillin-resistant S. aureus, vancomycin-resistant Enterococcus faecalis, and penicillin-resistant Streptococcus pneumoniae.

Three-Dimensional Structure and Biophysical Characterization of Staphylococcus aureus Cell Surface Antigen–Manganese Transporter MntC

MntC is a metal-binding protein component of the Mn²⁺-specific mntABC transporter from the pathogen Staphylococcus aureus. The protein is expressed during the early stages of infection and was proven to be effective at reducing both S. aureus and Staphylococcus epidermidis infections in a murine animal model when used as a vaccine antigen. MntC is currently being tested in human clinical trials as a component of a multiantigen vaccine for the prevention of S. aureus infections. To better understand the biological function of MntC, we are providing structural and biophysical characterization of the protein in this work. The three-dimensional structure of the protein was solved by X-ray crystallography at 2.2Å resolution and suggests two potential metal binding modes, which may lead to reversible as well as irreversible metal binding. Precise Mn²⁺-binding affinity of the protein was determined from the isothermal titration calorimetry experiments using a competition approach. Differential scanning calorimetry experiments confirmed that divalent metals can indeed bind to MntC reversibly as well as irreversibly. Finally, Mn²⁺-induced structural and dynamics changes have been characterized using spectroscopic methods and deuterium-hydrogen exchange mass spectroscopy. Results of the experiments show that these changes are minimal and are largely restricted to the structural elements involved in metal coordination. Therefore, it is unlikely that antibody binding to this antigen will be affected by the occupancy of the metal-binding site by Mn²⁺.

Atypical Antigen Recognition Mode of a Shark Immunoglobulin New Antigen Receptor (IgNAR) Variable Domain Characterized by Humanization and Structural Analysis

Background: Single domain variable regions of shark antibodies (V-NARs) are promising biotherapeutic candidates. Results: A V-NAR specific for human serum albumin was humanized, and its crystal structure in complex with the antigen was solved, revealing an unusual recognition mode. Conclusion: Humanization preserved antigen binding properties and activity of the parental shark antibody. Significance: A structural framework for humanization of shark antibodies was established. The immunoglobulin new antigen receptors (IgNARs) are a class of Ig-like molecules of the shark immune system that exist as heavy chain-only homodimers and bind antigens by their single domain variable regions (V-NARs). Following shark immunization and/or in vitro selection, V-NARs can be generated as soluble, stable, and specific high affinity monomeric binding proteins of ∼12 kDa. We have previously isolated a V-NAR from an immunized spiny dogfish shark, named E06, that binds specifically and with high affinity to human, mouse, and rat serum albumins. Humanization of E06 was carried out by converting over 60% of non-complementarity-determining region residues to those of a human germ line Vκ1 sequence, DPK9. The resulting huE06 molecules have largely retained the specificity and affinity of antigen binding of the parental V-NAR. Crystal structures of the shark E06 and its humanized variant (huE06 v1.1) in complex with human serum albumin (HSA) were determined at 3- and 2.3-Å resolution, respectively. The huE06 v1.1 molecule retained all but one amino acid residues involved in the binding site for HSA. Structural analysis of these V-NARs has revealed an unusual variable domain-antigen interaction. E06 interacts with HSA in an atypical mode that utilizes extensive framework contacts in addition to complementarity-determining regions that has not been seen previously in V-NARs. On the basis of the structure, the roles of various elements of the molecule are described with respect to antigen binding and V-NAR stability. This information broadens the general understanding of antigen recognition and provides a framework for further design and humanization of shark IgNARs.

Naphthyl tetronic acids as multi-target inhibitors of bacterial peptidoglycan biosynthesis.

Since the discovery of penicillin in 1929, many important antibiotic agents have made significant contributions to the prevention and treatment of infections caused by bacteria. Despite these remarkable achievements, infections are still the second-leading cause of death worldwide and remain a major public health problem. Clearly, there is great need for novel antibacterial agents to address resistance problems associated with current antibiotics. Toward this end, three broad strategies have been recently employed in the search for new leads: high-throughput screening of large compound libraries, genomics, and combinatorial biosynthesis. Although some limitations of the former approach to targets in bacterial peptidoglycan biosynthesis have been reported, the peptidoglycan biosynthetic pathway remains an attractive target, validated in the clinic with fosfomycin and vancomycin. Peptidoglycan biosynthesis is a complex process, which involves three main stages: a) cytoplasmic soluble enzymes that include MurA–F, b) membrane-bound enzymes that include MraY and MurG, and finally c) transglycosylases and transpeptidases, which act external to the cytoplasmic membrane. The Mur enzymes are unique to bacteria and are involved in essential functions of both Gram-positive and Gram-negative organisms. Another attractive aspect of Mur enzyme inhibitors is the potential to be bactericidal, leading to cell lysis and bacterial death. Inhibitors of peptidoglycan biosynthesis initiate a complex process of gene expression resulting in the induction of MurA and Mur I in Gram-positive bacteria to compensate for the slower rate of peptidoglycan biosynthesis. Several classes of natural products or their semisynthetic derivatives represented by liposidomycins, amphomycins, and muraymycins are inhibitors of MraY, whereas nisin, ramoplanin, and mersacidin are lipid II inhibitors. In the last decade a few small-molecule inhibitors of the Mur enzymes have been reported, including sesquiterpene lactones, 5-sulfonoxyanthranilic acids T6361 and T6362, UDP-MurNAc (MurA), imidazolinones, 4-thiazolidinones, thienopyrazoles, phosphinates (MurB), peptidosulfonamides, 3-cyanothiophenes (MurF), and d-glutamic acid analogues (Mur I). Despite the discovery of small-molecule inhibitors of various Mur enzymes, many limitations have been noted, including poor antibacterial activities in cells. In parallel, a number of new assay formats for the identification of Mur enzyme inhibitors have been described based on different platforms such as ultra-efficient affinity HTS, LC– MS, TLC, HPLC, and solid-support TLC. Our efforts in identifying Mur enzyme inhibitors were based on an initial pathway screen searching for inhibitors of multiple enzymes, MurA–F. Hits in this assay were evaluated against the individual Mur enzymes for lead optimization. Using this strategy, we identified two classes of inhibitors : 3,5-dioxopyrazolidines and pulvinones, with activities against several of the Mur enzymes. Inhibitors of multiple Mur enzymes are attractive given the essential role of each Mur enzyme in peptidoglycan biosynthesis. This strategy may prevent the development of drug resistance through the multi-target hypothesis. Herein we report on the SAR of the naphthyl tetronic acids and highlight their binding to the E. coli enzyme MurB. The target naphthylfuran-2-ones 5a–k were prepared by a three-step process starting from 3-bromo-4-methoxy-5H-furan2-one (1) and the appropriately substituted aldehydes 2 (Scheme 1). Bromofuranone 1 was acquired by bromination of the commercially available 4-methoxy-5H-furan-2-one with Nbromosuccinimide in carbon tetrachloride at reflux. Deprotonation of 2 at C5 with lithium isopropylcyclohexylamide (LICA) followed by an aldol reaction with substituted aldehydes 2 mediated by anhydrous ZnCl2 afforded diastereomeric alcohols, which were converted into their mesylate or chloride derivatives in situ followed by elimination to generate the exocyclic double bond of 3 in the thermodynamically more stable Z configuration. The key step involved a Suzuki cross-coupling of 3 with aryl boronic acids catalyzed by either [PdACHTUNGTRENNUNG(PPh3)4] or [PdCl2ACHTUNGTRENNUNG(dppf)2] to afford the methoxyfuranones 4a–k. Demethylation of methoxyfuranones 4a–k with lithium bromide in the final step afforded the desired naphthylfuran-2ones 5a–k. Purification by silica gel column chromatography was followed by an acid wash of the collected fractions to restore the acidic functionality. A panel of nine enzymes was used to assess the abilities of the naphthylfuranones to inhibit the Mur enzymes, and thus to define SAR trends for multiple enzyme inhibition (Table 1). The four isomeric bis-naphthyl compounds (Entries 1–4) were evaluated to determine whether there is a preference for aor blinked naphthyl groups at either C3 or C5. The trend seems to favor C3 b and C5 a substitution. Replacement of naphthyl with p-chlorophenyl (Entries 5–8) gave compounds 5e–h with good broad-spectrum activity against the Mur enzymes, thus confirming the desired SAR trend. Further optimization of the C5 a methylidene naphthyl derivative 5h by changing the p[a] Dr. T. S. Mansour, Dr. B. Rasmussen, Dr. G. Krishnamurthy, C. Smeltzer, Dr. Y. Yang, Dr. A. Severin, P. J. Petersen, Dr. G. Singh Medicinal Chemistry, Wyeth Research 401 North Middletown Road, Pearl River, NY 10965 (USA) Fax: (+1)845-602-5580 E-mail : mansout@wyeth.com [b] Dr. C. E. Caufield, K. M. Morris, S. Naughton, S. Antane, Dr. D. Quagliato Wyeth Research, CN 8000, Princeton, NJ 08543 (USA) [c] Dr. R. Chopra, K. Svenson, Dr. J. Bard Wyeth Research, Cambridge, MA 02140 (USA)

Discovery and initial optimization of 5,5'-disubstituted aminohydantoins as potent β-secretase (BACE1) inhibitors

8,8-Diphenyl-2,3,4,8-tetrahydroimidazo[1,5-a]pyrimidin-6-amine (1) was identified through HTS, as a weak (micromolar) inhibitor of BACE1. X-Ray crystallographic studies indicate the 2-aminoimidazole ring forms key H-bonding interactions with Asp32 and Asp228 in the catalytic site of BACE1. Lead optimization using structure-based focused libraries led to the identification of low nanomolar BACE1 inhibitors such as 20b with substituents which extend from the S(1) to the S(3) pocket.

Engineering a Monomeric Fc Domain Modality by N-Glycosylation for the Half-life Extension of Biotherapeutics

Background: The bivalency of IgG and Fc fusion could cause undesired therapeutic properties. Results: We developed a stable monomeric Fc modality by N-glycosylation engineering, enabling the generation of crystal structure. Conclusion: The monomeric Fc prolonged the half-life of Fab domain through the interaction with neonatal Fc receptor. Significance: The monomeric Fc will be used for pharmacokinetics enhancement of biotherapeutics that require monovalent properties. Human IgG is a bivalent molecule that has two identical Fab domains connected by a dimeric Fc domain. For therapeutic purposes, however, the bivalency of IgG and Fc fusion proteins could cause undesired properties. We therefore engineered the conversion of the natural dimeric Fc domain to a highly soluble monomer by introducing two Asn-linked glycans onto the hydrophobic CH3-CH3 dimer interface. The monomeric Fc (monoFc) maintained the binding affinity for neonatal Fc receptor (FcRn) in a pH-dependent manner. We solved the crystal structure of monoFc, which explains how the carbohydrates can stabilize the protein surface and provides the rationale for molecular recognition between monoFc and FcRn. The monoFc prolonged the in vivo half-life of an antibody Fab domain, and a tandem repeat of the monoFc further prolonged the half-life. This monoFc modality can be used to improve the pharmacokinetics of monomeric therapeutic proteins with an option to modulate the degree of half-life extension.