Andrew Prussia

Functional Interaction between Paramyxovirus Fusion and Attachment Proteins

Paramyxovirinae envelope glycoproteins constitute a premier model to dissect how specific and dynamic interactions in multisubunit membrane protein complexes can control deep-seated conformational rearrangements. However, individual residues that determine reciprocal specificity of the viral attachment and fusion (F) proteins have not been identified. We have developed an assay based on a pair of canine distemper virus (CDV) F proteins (strains Onderstepoort (ODP) and Lederle) that share ∼95% identity but differ in their ability to form functional complexes with the measles virus (MV) attachment protein (H). Characterization of CDV F chimeras and mutagenesis reveals four residues in CDV F-ODP (positions 164, 219, 233, and 317) required for productive interaction with MV H. Mutating these residues to the Lederle type disrupts triggering of F-ODP by MV H without affecting functionality when co-expressed with CDV H. Co-immunoprecipitation shows a stronger physical interaction of F-ODP than F-Lederle with MV H. Mutagenesis of MV F highlights the MV residues homologous to CDV F residues 233 and 317 as determinants for physical glycoprotein interaction and fusion activity under homotypic conditions. In assay reversal, the introduction of sections of the CDV H stalk into MV H shows a five-residue fragment (residues 110–114) to mediate specificity for CDV F-Lederle. All of the MV H stalk chimeras are surface-expressed, show hemadsorption activity, and trigger MV F. Combining the five-residue H chimera with the CDV F-ODP quadruple mutant partially restores activity, indicating that the residues identified in either glycoprotein contribute interdependently to the formation of functional complexes. Their localization in structural models of F and H suggests that placement in particular of F residue 233 in close proximity to the 110–114 region of H is structurally conceivable.

Probing the Spatial Organization of Measles Virus Fusion Complexes

ABSTRACT The spatial organization of metastable paramyxovirus fusion (F) and attachment glycoprotein hetero-oligomers is largely unknown. To further elucidate the organization of functional fusion complexes of measles virus (MeV), an archetype of the paramyxovirus family, we subjected central predictions of alternative docking models to experimental testing using three distinct approaches. Carbohydrate shielding through engineered N-glycans indicates close proximity of a membrane-distal, but not membrane-proximal, section of the MeV attachment (H) protein stalk domain to F. Directed mutagenesis of this section identified residues 111, 114, and 118 as modulators of avidity of glycoprotein interactions and determinants of F triggering. Stalk-length variation through deletion or insertion of HR elements at positions flanking this section demonstrates that the location of the stalk segment containing these residues cannot be altered in functional fusion complexes. In contrast, increasing the distance between the H head domains harboring the receptor binding sites and this section through insertion of structurally rigid α-helical domains with a pitch of up to approximately 75 Å downstream of stalk position 118 partially maintains functionality in transient expression assays and supports efficient growth of recombinant virions. In aggregate, these findings argue against specific protein-protein contacts between the H head and F head domains but instead support a docking model that is characterized by short-range contacts between the prefusion F head and the attachment protein stalk, possibly involving H residues 111, 114, and 118, and extension of the head domain of the attachment protein above prefusion F.

Farnesyl transferase inhibitors impair chromosomal maintenance in cell lines and human tumors by compromising CENP-E and CENP-F function

Farnesyl transferase inhibitors (FTI) exhibit anticancer activity as a single agent in preclinical studies and show promise in combination with other therapeutics in clinical trials. Previous studies show that FTIs arrest cancer cells in mitosis; however, the mechanism by which this occurs is unclear. Here, we observed that treatment of various cancer cell lines with the FTI lonafarnib caused mitotic chromosomal alignment defects, leaving cells in a pseudometaphase state, whereby both aligned chromosomes and chromosomes juxtaposed to the spindle poles (termed “lagging chromosomes”) were observed in the same cell. To determine how this occurs, we investigated the functionality of two farnesylated mitotic proteins, CENP-E and CENP-F, which mediate chromosomal capture and alignment. The data show that lonafarnib in proliferating cancer cells depletes CENP-E and CENP-F from metaphase but not prometaphase kinetochores. Loss of CENP-E and CENP-F metaphase localization triggered aberrant chromosomal maintenance, causing aligned chromosomes to be prematurely released from the spindle equator and become lagging chromosomes, resulting in a mitotic delay. Furthermore, lonafarnib treatment reduces sister kinetochore tension and activates the BubR1 spindle checkpoint, suggesting that farnesylation of CENP-E and CENP-F is critical for their functionality in maintaining kinetochore-microtubule interactions. Importantly, apparently similar chromosomal alignment defects were observed in head and neck tumors samples from a phase I trial with lonafarnib, providing support that lonafarnib disrupts chromosomal maintenance in human cancers. Lastly, to examine how farnesylation could regulate CENP-E in mediating kinetochore-microtubule attachments, we examined possible docking motifs of a farnesyl group on the outer surface of the microtubule. This analysis revealed three hydrophobic patches on the tubulin dimer for insertion of a farnesyl group, alluding to the possibility of an association between a farnesyl group and the microtubule. [Mol Cancer Ther 2007;6(4):1317–28]

Systematic Approaches towards the Development of Host-Directed Antiviral Therapeutics

Since the onset of antiviral therapy, viral resistance has compromised the clinical value of small-molecule drugs targeting pathogen components. As intracellular parasites, viruses complete their life cycle by hijacking a multitude of host-factors. Aiming at the latter rather than the pathogen directly, host-directed antiviral therapy has emerged as a concept to counteract evolution of viral resistance and develop broad-spectrum drug classes. This approach is propelled by bioinformatics analysis of genome-wide screens that greatly enhance insights into the complex network of host-pathogen interactions and generate a shortlist of potential gene targets from a multitude of candidates, thus setting the stage for a new era of rational identification of drug targets for host-directed antiviral therapies. With particular emphasis on human immunodeficiency virus and influenza virus, two major human pathogens, we review screens employed to elucidate host-pathogen interactions and discuss the state of database ontology approaches applicable to defining a therapeutic endpoint. The value of this strategy for drug discovery is evaluated, and perspectives for bioinformatics-driven hit identification are outlined.

Two Domains That Control Prefusion Stability and Transport Competence of the Measles Virus Fusion Protein

ABSTRACT Most viral glycoproteins mediating membrane fusion adopt a metastable native conformation and undergo major conformational changes during fusion. We previously described a panel of compounds that specifically prevent fusion induced by measles virus (MV), most likely by interfering with conformational rearrangements of the MV fusion (F) protein. To further elucidate the basis of inhibition and better understand the mechanism of MV glycoprotein-mediated fusion, we generated and characterized resistant MV variants. Spontaneous mutations conferring drug resistance were confirmed in transient assays and in the context of recombinant virions and were in all cases located in the fusion protein. Several mutations emerged independently at F position 462, which is located in the C-terminal heptad repeat (HR-B) domain. In peptide competition assays, all HR-B mutants at residue 462 revealed reduced affinity for binding to the HR-A core complex compared to unmodified HR-B. Combining mutations at residue 462 with mutations in the distal F head region, which we had previously identified as mediating drug resistance, causes intracellular retention of the mutant proteins. The transport competence and activity of the mutants can be restored, however, by incubation at reduced temperature or in the presence of the inhibitory compounds, indicating that the F escape mutants have a reduced conformational stability and that the inhibitors stabilize a transport-competent conformation of the F trimer. The data support the conclusion that residues located in the head domain of the F trimer and the HR-B region contribute jointly to controlling F conformational stability.

Design of a Small-Molecule Entry Inhibitor with Activity against Primary Measles Virus Strains

ABSTRACT The incidence of measles virus (MV) infection has been significantly reduced in many nations through extensive vaccination; however, the virus still causes significant morbidity and mortality in developing countries. Measles outbreaks also occur in some developed countries that have failed to maintain high vaccine coverage rates. While vaccination is essential in preventing the spread of measles, case management would greatly benefit from the use of therapeutic agents to lower morbidity. Thus, the development of new therapeutic strategies is desirable. We previously reported the generation of a panel of small-molecule MV entry inhibitors. Here we show that our initial lead compound, although providing proof of concept for our approach, has a short half-life (<16 h) under physiological conditions. In order to combine potent antiviral activity with increased compound stability, a targeted library of candidate molecules designed on the structural basis of the first lead has been synthesized and tested against MV. We have identified an improved lead with low toxicity and high stability (half-life ≫ 16 h) that prevents viral entry and hence infection. This compound shows high MV specificity and strong activity (50% inhibitory concentration = 0.6 to 3.0 μM, depending on the MV genotype) against a panel of wild-type MV strains representative of viruses that are currently endemic in the field.

Reversible Inhibition of the Fusion Activity of Measles Virus F Protein by an Engineered Intersubunit Disulfide Bridge

ABSTRACT In search of target sites for the development of paramyxovirus inhibitors, we have engineered disulfide bridges to introduce covalent links into the prefusion F protein trimer of measles virus. F-Edm-452C/460C, predicted to bridge head and stalk domains of different F monomers, shows a high degree of proteolytic maturation and surface expression, predominantly as stable, dithiothreitol-sensitive trimers, but no fusion activity. Reduction of disulfide bridges partially restores activity. These findings underscore the importance of reversible intersubunit interactions between the stalk and head domains for F activity. Noncovalent small molecules mimicking this behavior may constitute a potent strategy for preventing paramyxovirus entry.

Monocarbonyl curcumin analogues: Heterocyclic pleiotropic kinase inhibitors that mediate anticancer properties

Curcumin is a biologically active component of curry powder. A structurally related class of mimetics possesses similar anti-inflammatory and anticancer properties. Mechanism has been examined by exploring kinase inhibition trends. In a screen of 50 kinases relevant to many forms of cancer, one member of the series (4, EF31) showed ≥85% inhibition for 10 of the enzymes at 5 μM, while 22 of the proteins were blocked at ≥40%. IC50 values for an expanded set of curcumin analogues established a rank order of potencies, and analyses of IKKβ and AKT2 enzyme kinetics for 4 revealed a mixed inhibition model, ATP competition dominating. Our curcumin mimetics are generally selective for Ser/Thr kinases. Both selectivity and potency trends are compatible with protein sequence comparisons, while modeled kinase binding site geometries deliver a reasonable correlation with mixed inhibition. Overall, these analogues are shown to be pleiotropic inhibitors that operate at multiple points along cell signaling pathways.

Potent Non-Nucleoside Inhibitors of the Measles Virus RNA-Dependent RNA Polymerase Complex

Measles virus (MV) is one of the most infectious pathogens known. In spite of the existence of a vaccine, approximately 350000 deaths/year result from MV or associated complications. Antimeasles compounds could conceivably diminish these statistics and provide a therapy that complements vaccine treatment. We recently described a high-throughput screening hit compound 1 (16677) against MV-infected cells with the capacity to eliminate viral reproduction at 250 nM by inhibiting the action of the viruss RNA-dependent RNA polymerase complex (RdRp). The compound, 1-methyl-3-(trifluoromethyl)- N-[4-sulfonylphenyl]-1 H-pyrazole-5-carboxamide, 1 carries a critical CF 3 moiety on the 1,2-pyrazole ring. Elaborating on the preliminary structure-activity (SAR) study, the present work presents the synthesis and SAR of a much broader range of low nanomolar nonpeptidic MV inhibitors and speculates on the role of the CF 3 functionality.

Measles Virus Entry Inhibitors: A Structural Proposal for Mechanism of Action and the Development of Resistance†

Previously, we developed a panel of nonpeptidic compounds specifically preventing fusion of the measles virus (MV) with target cells at IC(50) values of 0.6-3 muM. Mutations in the MV fusion protein (MV F) that render resistance to these blockers were described. The structural basis for both inhibition and resistance was unclear in the earlier work because of the availability of a structural model for only the postfusion conformation of MV F. We have now developed structural models for both pre- and postfusion conformers of the latter protein trimer. The models allow investigation of the large-scale conformational changes occurring in the MV fusion machinery and, in conjunction with antisera binding studies, provide a rationale for how inhibitors may arrest a conformational intermediate by interfering with the formation of interactions between the heptad repeat B (HR-B) linker and DIII domains. The models also show that resistance to inhibition can be explained by a predicted destabilizing effect of the mutations on the HR-B domain within the trimeric prefusion structure. This viewpoint is supported by the temperature-dependent differential fusion activities of MV F variants harboring these mutations.