Winifred W. Prosise

Molecular views of viral polyprotein processing revealed by the crystal structure of the hepatitis C virus bifunctional protease-helicase

BACKGROUND Hepatitis C virus (HCV) currently infects approximately 3% of the worlds population. HCV RNA is translated into a polyprotein that during maturation is cleaved into functional components. One component, nonstructural protein 3 (NS3), is a 631-residue bifunctional enzyme with protease and helicase activities. The NS3 serine protease processes the HCV polyprotein by both cis and trans mechanisms. The structural aspects of cis processing, the autoproteolysis step whereby the protease releases itself from the polyprotein, have not been characterized. The structural basis for inclusion of protease and helicase activities in a single polypeptide is also unknown. RESULTS We report here the 2.5 A resolution structure of an engineered molecule containing the complete NS3 sequence and the protease activation domain of nonstructural protein 4A (NS4A) in a single polypeptide chain (single chain or scNS3-NS4A). In the molecule, the helicase and protease domains are segregated and connected by a single strand. The helicase necleoside triphosphate and RNA interaction sites are exposed to solvent. The protease active site of scNS3-NS4A is occupied by the NS3 C terminus, which is part of the helicase domain. Thus, the intramolecular complex shows one product of NS3-mediated cleavage at the NS3-NS4A junction of the HCV polyprotein bound at the protease active site. CONCLUSIONS The scNS3-NS4A structure provides the first atomic view of polyprotein cis processing. Both local and global structural rearrangements follow the cis cleavage reaction, and large segments of the polyprotein can be folded prior to proteolytic processing. That the product complex of the cis cleavage reaction exists in a stable molecular conformation suggests autoinhibition and substrate-induced activation mechanisms for regulation of NS3 protease activity.

Structure of full-length human anti-PD1 therapeutic IgG4 antibody pembrolizumab

Immunoglobulin G4 antibodies exhibit unusual properties with important biological consequences. We report the structure of the human full-length IgG4 S228P anti-PD1 antibody pembrolizumab, solved to 2.3-Å resolution. Pembrolizumab is a compact molecule, consistent with the presence of a short hinge region. The Fc domain is glycosylated at the CH2 domain on both chains, but one CH2 domain is rotated 120° with respect to the conformation observed in all reported structures to date, and its glycan chain faces the solvent. We speculate that this new conformation is driven by the shorter hinge. The structure suggests a role for the S228P mutation in preventing the IgG4 arm exchange. In addition, this unusual Fc conformation suggests possible structural diversity between IgG subclasses and shows that use of isolated antibody fragments could mask potentially important interactions, owing to molecular flexibility.

Construction and characterization of a fully active PXR/SRC-1 tethered protein with increased stability

The nuclear xenobiotic receptor PXR is a ligand-inducible transcription factor regulating drug-metabolizing enzymes and transporters and a master switch mediating potentially adverse drug-drug interactions. In addition to binding a coactivator protein such as SRC-1, the C-terminal ligand-binding domain (LBD) is solely responsible for ligand recognition and thus the ligand-dependent downstream effects. In an effort to facilitate structural studies of PXR to understand and abolish the interactions between PXR and its ligands, several recombinant PXR/SRC-1 constructs were designed and evaluated for expression, stability and activity. Expression strategies employing either dual expression or translationally coupled bicistronic expression were found to be unsuitable for producing stable PXR in a stochiometric complex with a peptide derived from SRC-1 (SRC-1p). A single polypeptide chain encompassing PXR and SRC-1p tethered with a peptidyl linker was designed to promote intramolecular complex formation. This tethered protein was overexpressed as a soluble protein and required no additional SRC-1p for further stabilization. X-ray crystal structures in the presence and absence of the known PXR agonist SR-12813 were determined to high resolution. In addition, a circular dichroism-based binding assay was developed to allow rapid evaluation of PXR ligand affinity, making this tethered protein a convenient and effective reagent for the rational attenuation of drug-induced PXR-mediated metabolism.

Discovery of a Potent, Injectable Inhibitor of Aurora Kinases Based on the Imidazo-[1,2-a]-Pyrazine Core

The imidazo-[1,2-a]-pyrazine (1) is a dual inhibitor of Aurora kinases A and B with modest cell potency (IC50 = 250 nM) and low solubility (5 μM). Lead optimization guided by the binding mode led to the acyclic amino alcohol 12k (SCH 1473759), which is a picomolar inhibitor of Aurora kinases (TdF K d Aur A = 0.02 nM and Aur B = 0.03 nM) with improved cell potency (phos-HH3 inhibition IC50 = 25 nM) and intrinsic aqueous solubility (11.4 mM). It also demonstrated efficacy and target engagement in human tumor xenograft mouse models.

Discovery of Novel 3,3-Disubstituted Piperidines as Orally Bioavailable, Potent, and Efficacious HDM2-p53 Inhibitors.

A new subseries of substituted piperidines as p53-HDM2 inhibitors exemplified by 21 has been developed from the initial lead 1. Research focused on optimization of a crucial HDM2 Trp23-ligand interaction led to the identification of 2-(trifluoromethyl)thiophene as the preferred moiety. Further investigation of the Leu26 pocket resulted in potent, novel substituted piperidine inhibitors of the HDM2-p53 interaction that demonstrated tumor regression in several human cancer xenograft models in mice. The structure of HDM2 in complex with inhibitors 3, 10, and 21 is described.

Structure of the Insulin Receptor in Complex with Insulin using Single Particle CryoEM Analysis

Insulin Receptor (IR) mediated signaling is crucial in controlling glucose homeostasis, regulating lipid, protein and carbohydrate metabolism, and modulating brain neurotransmitter levels [1, 2]. Aberrations in Insulin signaling have been associated with a variety of disease states, including diabetes, cancer and Alzheimer’s [1, 3, 4]. IR is composed of two heterodimers ( and  chains), each containing an extracellular portion (ectodomain), a single transmembrane helix (TM), and a cytoplasmic tyrosine kinase domain (TK) (Figure 1). One single disulfide bond links the  and  chains in the monomer, while the dimer is stabilized by two interchain disulfide bonds (Figure 1). Insulin is thought to bind to two distinct sites (per monomer), in a complex process that exhibits negative cooperativity [5]. Insulin binding site 1 was mapped by alanine scanning to portion of the L1 domain (Asp12-Asn15, Leu37, Phe39, Phe64 and Arg65) and to the CT helix (Gln692-Pro718) located at the C-terminal end of the ID domain. Site 2 was mapped to loop regions near the junction between the FNIII-1 and FNIII-2 domains [5 and references therein].

Key steps in the structure-based optimization of the hepatitis C virus NS3/4A protease inhibitor SCH503034

Crystal structures of protease/inhibitor complexes guided optimization of the buried nonpolar surface area thereby maximizing hydrophobic binding. The resulting potent tripeptide inhibitor is in clinical trials.

Inactivation of the human cytomegalovirus protease by diisopropylfluorophosphate

Publisher Summary Cytomegalovirus (CMV) protease is a serine protease, and labeling with diisopropylfluorophosphate (DFP) has identified Ser132 as the active site serine. The structure of the CMV protease containing the diisopropylphosphorylserine at residue 132 (DIP-CMV protease) is likely to resemble that of the tetrahedral transition-state intermediate. As the structure of the DFP-treated serine proteases resembles that of the tetrahedral transition-state intermediate, and the inactivated enzyme would not be susceptible to autoproteolysis, production of DIP-CMV protease would be useful for structure based drug design. The concentrations of DFP sufficient to yield stoichiometric incorporation of inhibitor at the active site of CMV protease, also resulted in substantial incorporation of DIP at a second site or sites. This heterogeneous incorporation would preclude crystallographic studies. For this reason, this chapter has attempted to optimize the conditions for inactivation of CMV protease with DFP, to produce pure DIP-CMV protease with minimum second site incorporation. Initial studies indicated that there was no loss of protease activity in the control samples, even when incubated for 23 hours at 22°C. A 3.8 hour incubation was sufficient to completely inactivate 180 μM protease in the presence of 4.3 mM DFP, while a 2.4 fold excess of the inhibitor inactivated only 50% of the enzyme. This indicates that CMV protease is less reactive with DFP than trypsin or chymotrypsin, and requires an order of magnitude excess of the organophosphate to achieve complete inactivation.