Robert A. Lazarus

Crystal structure of the HGF β-chain in complex with the sema domain of the met receptor

The Met tyrosine kinase receptor and its ligand, hepatocyte growth factor (HGF), play important roles in normal development and in tumor growth and metastasis. HGF‐dependent signaling requires proteolysis from an inactive single‐chain precursor into an active α/β‐heterodimer. We show that the serine protease‐like HGF β‐chain alone binds Met, and report its crystal structure in complex with the Sema and PSI domain of the Met receptor. The Met Sema domain folds into a seven‐bladed β‐propeller, where the bottom face of blades 2 and 3 binds to the HGF β‐chain ‘active site region’. Mutation of HGF residues in the area that constitutes the active site region in related serine proteases significantly impairs HGF β binding to Met. Key binding loops in this interface undergo conformational rearrangements upon maturation and explain the necessity of proteolytic cleavage for proper HGF signaling. A crystallographic dimer interface between two HGF β‐chains brings two HGF β:Met complexes together, suggesting a possible mechanism of Met receptor dimerization and activation by HGF.

A Therapeutic Antibody Targeting BACE1 Inhibits Amyloid-β Production in Vivo

A human antibody inhibits BACE1 activity and Aβ peptide production in cultured neurons and in the central nervous system of mouse and monkey. A Trojan Horse Antibody Scales a Mighty Fortress As impenetrable as the walls of ancient Troy, the tight endothelial cell layer of the blood-brain barrier (BBB) allows only a few select molecules to enter the brain. Unfortunately, this highly effective fortress blocks passage of therapeutic antibodies, limiting their usefulness for treating diseases of the brain and central nervous system. Enter Ryan Watts and his team at Genentech with their ambitious dual goal of making a therapeutic antibody against a popular Alzheimer’s disease drug target, the enzyme β-secretase (BACE1), and developing a strategy to boost the amount of this antibody that enters the brain (Atwal et al. and Yu et al.). BACE1 processes the amyloid precursor protein into amyloid-β (Aβ) peptides including those molecular species that aggregate to form the amyloid plaques found in the brains of Alzheimer’s disease patients. By blocking the activity of BACE1, BACE1 inhibitors should reduce production of the aggregation-prone Aβ peptides, thus decreasing amyloid plaque formation and slowing Alzheimer’s disease progression. Although small-molecule inhibitors of BACE1 have been developed and can readily cross the BBB because of their small size, they do not show sufficient specificity and hence may have toxic side effects. Watts envisaged that a better approach to blocking BACE1 activity might be passive immunization with a highly specific anti-BACE1 antibody. So his team engineered an anti-BACE1 antibody that bound to BACE1 with exquisite specificity and blocked its activity (Atwal et al.). The investigators then showed that this antibody could reduce production of aggregation-prone Aβ peptides in cultured primary neurons. Next, Watts and his colleagues injected the antibody into mice and monkeys and demonstrated a sustained decrease in the concentrations of Aβ peptide in the circulation of these animals and to a lesser extent in the brain. The researchers knew that they must find a way to increase the amount of antibody getting into the brain to reduce Aβ peptide concentrations in the brain sufficiently to obtain a therapeutic effect. So Watts teamed up with fellow Genentechie, Mark Dennis, and they devised an ingenious solution (Yu et al.). The Genentech researchers knew that high-affinity antibodies against the transferrin receptor might be able to cross the BBB using a natural process called receptor-mediated transcytosis. However, when they tested their antibody, they found that although it readily bound to the BBB, it could not detach from the transferrin receptor and hence was not released into the brain. So, they made a series of lower-affinity mouse anti-transferrin receptor antibodies and found variants that could cross the BBB by receptor-mediated transcytosis and were released into the mouse brain once they got across the endothelial cell layer. Next, they designed a bispecific mouse antibody with one arm comprising a low-affinity anti-transferrin receptor antibody and the other arm comprising the high-affinity anti-BACE1 antibody that had shown therapeutic promise in their earlier studies. They demonstrated that their bispecific antibody was able to cross the BBB and reach therapeutic concentrations in the mouse brain. They then showed that this bispecific antibody was substantially more effective at reducing Aβ peptide concentrations in the mouse brain compared to the monospecific anti-BACE1 antibody. This elegant pair of papers not only demonstrates the therapeutic potential of an anti-BACE1 antibody for treating Alzheimer’s disease but also provides a strategy worthy of the ancient Greeks that could be applied to other therapeutic antibodies that require safe passage into the human brain. Reducing production of amyloid-β (Aβ) peptide by direct inhibition of the enzymes that process amyloid precursor protein (APP) is a central therapeutic strategy for treating Alzheimer’s disease. However, small-molecule inhibitors of the β-secretase (BACE1) and γ-secretase APP processing enzymes have shown a lack of target selectivity and poor penetrance of the blood-brain barrier (BBB). Here, we have developed a high-affinity, phage-derived human antibody that targets BACE1 (anti-BACE1) and is anti-amyloidogenic. Anti-BACE1 reduces endogenous BACE1 activity and Aβ production in human cell lines expressing APP and in cultured primary neurons. Anti-BACE1 is highly selective and does not inhibit the related enzymes BACE2 or cathepsin D. Competitive binding assays and x-ray crystallography indicate that anti-BACE1 binds noncompetitively to an exosite on BACE1 and not to the catalytic site. Systemic dosing of mice and nonhuman primates with anti-BACE1 resulted in sustained reductions in peripheral Aβ peptide concentrations. Anti-BACE1 also reduces central nervous system Aβ concentrations in mouse and monkey, consistent with a measurable uptake of antibody across the BBB. Thus, BACE1 can be targeted in a highly selective manner through passive immunization with anti-BACE1, providing a potential approach for treating Alzheimer’s disease. Nevertheless, therapeutic success with anti-BACE1 will depend on improving antibody uptake into the brain.

Production of 2-Keto-L-Gulonate, an Intermediate in L-Ascorbate Synthesis, by a Genetically Modffied Erwinia herbicola

A new metabolic pathway has been created in the microorganism Erwinia herbicola that gives it the ability to produce 2-keto-L-gulonic acid, an important intermediate in the synthesis of L-ascorbic acid. Initially, a Corynebacterium enzyme that could stereoselectively reduce 2,5-diketo-D-gluconic acid to 2-keto-L-gulonic acid was identified and purified. DNA probes based on amino acid sequence information from 2,5-diketo-D-gluconic acid reductase were then used to isolate the gene for this enzyme from a Corynebacterium genomic library. The 2,5-diketo-D-gluconic acid reductase coding region was fused to the Escherichia coli trp promoter and a synthetic ribosome binding site and was then introduced into E. herbicola on a multicopy plasmid. Erwinia herbicola naturally produces 2,5-diketo-D-gluconic acid via glucose oxidation, and when recombinant cells expressing the plasmid-encoded reductase were grown in the presence of glucose, 2-keto-L-gulonic acid was made and released into the culture medium. The data demonstrate the feasibility of creating novel in vivo routes for the synthesis of important specialty chemicals by combining useful metabolic traits from diverse sources in a single organism.

Monovalent antibody design and mechanism of action of onartuzumab, a MET antagonist with anti-tumor activity as a therapeutic agent

Significance Therapeutic antibodies have revolutionized the treatment of human disease. Despite these advances, antibody bivalency limits their utility against some targets. Here, we describe the development of a one-armed (monovalent) antibody, onartuzumab, targeting the receptor tyrosine kinase MET. While initial screening of bivalent antibodies produced agonists of MET, engineering them into monovalent antibodies produced antagonists instead. We explain the structural basis of the mechanism of action with the crystal structure of onartuzumab antigen-binding fragment in complex with MET and HGF-β. These discoveries have led to an additional antibody-based therapeutic option and shed light on the underpinnings of HGF/MET signaling. Binding of hepatocyte growth factor (HGF) to the receptor tyrosine kinase MET is implicated in the malignant process of multiple cancers, making disruption of this interaction a promising therapeutic strategy. However, targeting MET with bivalent antibodies can mimic HGF agonism via receptor dimerization. To address this limitation, we have developed onartuzumab, an Escherichia coli-derived, humanized, and affinity-matured monovalent monoclonal antibody against MET, generated using the knob-into-hole technology that enables the antibody to engage the receptor in a one-to-one fashion. Onartuzumab potently inhibits HGF binding and receptor phosphorylation and signaling and has antibody-like pharmacokinetics and antitumor activity. Biochemical data and a crystal structure of a ternary complex of onartuzumab antigen-binding fragment bound to a MET extracellular domain fragment, consisting of the MET Sema domain fused to the adjacent Plexins, Semaphorins, Integrins domain (MET Sema-PSI), and the HGF β-chain demonstrate that onartuzumab acts specifically by blocking HGF α-chain (but not β-chain) binding to MET. These data suggest a likely binding site of the HGF α-chain on MET, which when dimerized leads to MET signaling. Onartuzumab, therefore, represents the founding member of a class of therapeutic monovalent antibodies that overcomes limitations of antibody bivalency for targets impacted by antibody crosslinking.

Interactions between Hedgehog proteins and their binding partners come into view

Hedgehog (Hh) proteins are secreted signaling molecules that mediate essential tissue-patterning events during embryonic development and function in tissue homeostasis and regeneration throughout life. Hh signaling is regulated by multiple mechanisms, including covalent lipid modification of the Hh protein and interactions with multiple protein and glycan partners. Unraveling the nature and effects of these interactions has proven challenging, but recent structural and biophysical studies of Hh proteins and active fragments of heparin, Ihog, Cdo, Boc, Hedgehog-interacting protein (Hhip), Patched (Ptc), and the monoclonal antibody 5E1 have added a new level of molecular detail to our understanding of how Hh signal response and distribution are regulated within tissues. We review these results and discuss their implications for understanding Hh signaling in normal and disease states.

Suppressor of Fused Regulates Gli Activity through a Dual Binding Mechanism

ABSTRACT The Hedgehog pathway drives proliferation and differentiation by activating the Gli/Ci family of zinc finger transcription factors. Gli/Ci proteins form Hedgehog signaling complexes with other signaling components, including the kinesin-like protein Costal-2, the serine-threonine kinase Fused, and Suppressor of Fused [Su(fu)]. In these complexes Gli/Ci proteins are regulated by cytoplasmic sequestration, phosphorylation, and proteolysis. Here we characterize structural and functional determinants of Su(fu) required for Gli regulation and show that Su(fu) contains at least two distinct domains: a highly conserved carboxy-terminal region required for binding to the amino-terminal ends of the Gli proteins and a unique amino-terminal domain that binds the carboxy-terminal tail of Gli1. While each domain is capable of binding to different Gli1 regions independently, interactions between Su(fu) and Gli1 at both sites are required for cytoplasmic tethering and repression of Gli1. Furthermore, we have solved the crystal structure of the amino-terminal domain of human Su(fu)27-268 at 2.65 Å resolution. This domain forms a concave pocket with a prominent acidic patch. Mutation at Asp159 in the acidic patch disrupts Gli1 tethering and repression while not strongly disrupting binding, indicating that the amino-terminal domain of Su(fu) likely impacts Gli binding through a mechanism distinct from that for tethering and repression. These studies provide a structural basis for understanding the function of Su(fu).

Hedgehog pathway antagonist 5E1 binds hedgehog at the pseudo-active site.

Proper hedgehog (Hh) signaling is crucial for embryogenesis and tissue regeneration. Dysregulation of this pathway is associated with several types of cancer. The monoclonal antibody 5E1 is a Hh pathway inhibitor that has been extensively used to elucidate vertebrate Hh biology due to its ability to block binding of the three mammalian Hh homologs to the receptor, Patched1 (Ptc1). Here, we engineered a murine:human chimeric 5E1 (ch5E1) with similar Hh-binding properties to the original murine antibody. Using biochemical, biophysical, and x-ray crystallographic studies, we show that, like the regulatory receptors Cdon and Hedgehog-interacting protein (Hhip), ch5E1 binding to Sonic hedgehog (Shh) is enhanced by calcium ions. In the presence of calcium and zinc ions, the ch5E1 binding affinity increases 10–20-fold to tighter than 1 nm primarily because of a decrease in the dissociation rate. The co-crystal structure of Shh bound to the Fab fragment of ch5E1 reveals that 5E1 binds at the pseudo-active site groove of Shh with an epitope that largely overlaps with the binding site of its natural receptor antagonist Hhip. Unlike Hhip, the side chains of 5E1 do not directly coordinate the Zn2+ cation in the pseudo-active site, despite the modest zinc-dependent increase in 5E1 affinity for Shh. Furthermore, to our knowledge, the ch5E1 Fab-Shh complex represents the first structure of an inhibitor antibody bound to a metalloprotease fold.

Potent and Selective Kunitz Domain Inhibitors of Plasma Kallikrein Designed by Phage Display

Phage displaying APPI Kunitz domain libraries have been used to design potent and selective active site inhibitors of human plasma kallikrein, a serine protease that plays an important role in both inflammation and coagulation. Selected clones from two Kunitz domain libraries randomized at or near the binding loop (positions 11-13, 15-19, and 34) were sequenced following five rounds of selection on immobilized plasma kallikrein. Invariant preferences for Arg at position 15 and His at position 18 were found, whereas His, Ala, Ala, and Pro were highly preferred residues at positions 13, 16, 17, and 19, respectively. At position 11 Pro, Asp, and Glu were favored, while hydrophobic residues were preferred at position 34. Selected variants, purified by trypsin affinity chromatography and reverse phase high performance liquid chromatography, potently inhibited plasma kallikrein, with apparent equilibrium dissociation constants (K*) ranging from 75 to 300 pM. From sequence and activity data, consensus mutants were constructed by site directed mutagenesis. One such mutant, KALI-DY, which differed from APPI at 6 key residues (T11D, P13H, M17A, I18H, S19P, and F34Y), inhibited plasma kallikrein with a K* = 15 ± 14 pM, representing an increase in binding affinity of more than 10,000-fold compared to APPI. Similar to APPI, the variants also inhibited Factor XIa with high affinity, with K* values ranging from 0.3 to 15 nM; KALI-DY inhibited Factor XIa with a K* = 8.2 ± 3.5 nM. KALI-DY did not inhibit plasmin, thrombin, Factor Xa, Factor XIIa, activated protein C, or tissue factor•Factor VIIa. Consistent with the protease specificity profile, KALI-DY did not prolong the clotting time in a prothrombin time assay, but did prolong the clotting time in an activated partial thromboplastin time assay >3.5-fold at 1 μM.

The factor VII zymogen structure reveals reregistration of beta-strands during activation

BACKGROUND Coagulation factor VIIa (FVIIa) contains a Trypsin-like serine protease domain and initiates the cascade of proteolytic events leading to Thrombin activation and blood clot formation. Vascular injury allows formation of the complex between circulating FVIIa and its cell surface bound obligate cofactor, Tissue Factor (TF). Circulating FVIIa is nominally activated but retains zymogen-like character and requires TF in order to complete the zymogen-to-enzyme transition. The manner in which TF exerts this effect is unclear. The structure of TF/FVIIa is known. Knowledge of the zymogen structure is helpful for understanding the activation transition in this system. RESULTS The 2 A resolution crystal structure of a zymogen form of FVII comprising the EGF2 and protease domains is revealed in a complex with the exosite binding inhibitory peptide A-183 and a vacant active site. The activation domain, which includes the N terminus, differs in ways beyond those that are expected for zymogens in the Trypsin family. There are large differences in the TF binding region. An unprecedented 3 residue shift in registration between beta strands B2 and A2 in the C-terminal beta barrel and hydrogen bonds involving Glu154 provide new insight into conformational changes accompanying zymogen activation, TF binding, and enzymatic competence. CONCLUSIONS TF-mediated allosteric control of the activity of FVIIa can be rationalized. The reregistering beta strand connects the TF binding region and the N-terminal region. The zymogen registration allows H bonds that prevent the N terminus from attaining a key salt bridge with the active site. TF binding may influence an equilibrium by selecting the enzymatically competent registration.

Structural and functional aspects of RGD-containing protein antagonists of glycoprotein IIb-IIIa

In the past few years, knowledge of the structure and function of RGD-containing protein antagonists of the platelet fibrinogen receptor glycoprotein IIb-IIIa has advanced rapidly. The RGD sequence is found at the apex of extended, solvent accessible, and conformationally flexible loops in proteins of vastly different structural frameworks. Conformation of the RGD epitope and the immediate surrounding sequence are critical factors affecting potency and selectivity as integrin antagonists. The RGD sequence has recently been introduced into a number of protein and peptide scaffolds.