Structural characterization of M8C10, a neutralizing antibody targeting a highly conserved prefusion-specific epitope on the metapneumovirus fusion trimerization interface.

IMPORTANCE: Human metapneumovirus (hMPV) is a common pathogen causing lower respiratory tract infections worldwide and can develop severe symptoms in high-risk populations such as infants, the elderly, and immunocompromised patients. There are no approved hMPV vaccines or neutralizing antibodies available for therapeutic or prophylactic use. The trimeric hMPV fusion F protein is the major target of neutralizing antibodies in human sera. Understanding the immune recognition of antibodies to hMPV-F antigen will provide critical insights into developing efficacious hMPV monoclonal antibodies and vaccines.

A streamlined, automated workflow to screen and triage large numbers of baculoviruses for protein expression.

The baculovirus expression system is a powerful and widely used method to generate large quantities of recombinant protein. However, challenges exist in workflows utilizing either liquid baculovirus stocks or the Titerless Infected-Cells Preservation and Scale-Up (TIPS) method, including the time and effort to generate baculoviruses, screen for protein expression and store large numbers of baculovirus stocks. To mitigate these challenges, we have developed a streamlined, hybrid workflow which utilizes high titer liquid virus stocks for rapid plate-based protein expression screening, followed by a TIPS-based scale-up for larger protein production efforts. Additionally, we have automated each step in this screening workflow using a custom robotic system. With these process improvements, we have significantly reduced the time, effort and resources required to manage large baculovirus generation and expression screening campaigns.

Development of a robust crystallization platform for immune receptor TREM2 using a crystallization chaperone strategy.

TREM2 has been identified by genomic analysis as a potential and novel target for the treatment of Alzheimer's disease. To enable structure-based screening of potential small molecule therapeutics, we sought to develop a robust crystallization platform for the TREM2 Ig-like domain. A systematic set of constructs containing the structural chaperone, maltose binding protein (MBP), fused to the Ig domain of TREM2, were evaluated in parallel expression and purification, followed by crystallization studies. Using protein crystallization and high-resolution diffraction as a readout, a MBP-TREM2 Ig fusion construct was identified that generates reproducible protein crystals diffracting at 2.0 Å, which makes it suitable for soaking of potential ligands. Importantly, analysis of crystal packing interfaces indicates that most of the surface of the TREM2 Ig domain is available for small molecule binding. A proof of concept co-crystallization study with a small library of fragments validated potential utility of this system for the discovery of new TREM2 therapeutics.

Structural characterization of nonactive site, TrkA-selective kinase inhibitors.

Current therapies for chronic pain can have insufficient efficacy and lead to side effects, necessitating research of novel targets against pain. Although originally identified as an oncogene, Tropomyosin-related kinase A (TrkA) is linked to pain and elevated levels of NGF (the ligand for TrkA) are associated with chronic pain. Antibodies that block TrkA interaction with its ligand, NGF, are in clinical trials for pain relief. Here, we describe the identification of TrkA-specific inhibitors and the structural basis for their selectivity over other Trk family kinases. The X-ray structures reveal a binding site outside the kinase active site that uses residues from the kinase domain and the juxtamembrane region. Three modes of binding with the juxtamembrane region are characterized through a series of ligand-bound complexes. The structures indicate a critical pharmacophore on the compounds that leads to the distinct binding modes. The mode of interaction can allow TrkA selectivity over TrkB and TrkC or promiscuous, pan-Trk inhibition. This finding highlights the difficulty in characterizing the structure-activity relationship of a chemical series in the absence of structural information because of substantial differences in the interacting residues. These structures illustrate the flexibility of binding to sequences outside of-but adjacent to-the kinase domain of TrkA. This knowledge allows development of compounds with specificity for TrkA or the family of Trk proteins.

Identification of novel bifunctional HIV-1 reverse transcriptase inhibitors.

OBJECTIVES: The increasing prevalence of mutations in HIV-1 reverse transcriptase (RT) that confer resistance to existing NRTIs and NNRTIs underscores the need to develop RT inhibitors with novel mode-of-inhibition and distinct resistance profiles. METHODS: Biochemical assays were employed to identify inhibitors of RT activity and characterize their mode of inhibition. The antiviral activity of the inhibitors was assessed by cell-based assays using laboratory HIV-1 isolates and MT4 cells. RT variants were purified via avidin affinity columns. RESULTS: Compound A displayed equal or greater potency against many common NNRTI-resistant RTs (K103N and Y181C RTs) relative to WT RT. Despite possessing certain NNRTI-like properties, such as being unable to inhibit an engineered variant of RT lacking an NNRTI-binding pocket, we found that compound A was dependent on Mg2+ for binding to RT. Optimization of compound A led to more potent analogues, which retained similar activities against WT and K103N mutant viruses with submicromolar potency in a cell-based assay. One of the analogues, compound G, was crystallized in complex with RT and the structure was determined at 2.6 Å resolution. The structure indicated that compound G simultaneously interacts with the active site (Asp186), the highly conserved primer grip region (Leu234 and Trp229) and the NNRTI-binding pocket (Tyr188). CONCLUSIONS: These findings reveal a novel class of RT bifunctional inhibitors that are not sensitive to the most common RT mutations, which can be further developed to address the deficiency of current RT inhibitors.

Indole acids as a novel PDE2 inhibitor chemotype that demonstrate pro-cognitive activity in multiple species.

An internal HTS effort identified a novel PDE2 inhibitor series that was subsequently optimized for improved PDE2 activity and off-target selectivity. The optimized lead, compound 4, improved cognitive performance in a rodent novel object recognition task as well as a non-human primate object retrieval task. In addition, co-crystallization studies of close analog of 4 in the PDE2 active site revealed unique binding interactions influencing the high PDE isoform selectivity.

The Vaccinia Virus H3 Envelope Protein, a Major Target of Neutralizing Antibodies, Exhibits a Glycosyltransferase Fold and Binds UDP-Glucose.

UNLABELLED: The highly conserved H3 poxvirus protein is a major target of the human antibody response against poxviruses and is likely a key contributor to protection against infection. Here, we present the crystal structure of H3 from vaccinia virus at a 1.9-Å resolution. H3 looks like a glycosyltransferase, a family of enzymes that transfer carbohydrate molecules to a variety of acceptor substrates. Like glycosyltransferases, H3 binds UDP-glucose, as shown by saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopy, and this binding requires Mg(2+) Mutation of the glycosyltransferase-like metal ion binding motif in H3 greatly diminished its binding to UDP-glucose. We found by flow cytometry that H3 binds to the surface of human cells but does not bind well to cells that are deficient in surface glycosaminoglycans. STD NMR experiments using a heparin sulfate decasaccharide confirmed that H3 binds heparin sulfate. We propose that a surface of H3 with an excess positive charge may be the binding site for heparin. Heparin binding and glycosyltransferase activity may be involved in the function of H3 in the poxvirus life cycle. IMPORTANCE: Poxviruses are under intense research because of bioterrorism concerns, zoonotic infections, and the side effects of existing smallpox vaccines. The smallpox vaccine using vaccinia virus has been highly successful, but it is still unclear why the vaccine is so effective. Studying the antigens that the immune system recognizes may allow a better understanding of how the vaccine elicits immunity and how improved vaccines can be developed. Poxvirus protein H3 is a major target of the immune system. The H3 crystal structure shows that it has a glycosyltransferase protein fold. We demonstrate that H3 binds the sugar nucleotide UDP-glucose, as do glycosyltransferases. Our experiments also reveal that H3 binds cell surface molecules that are involved in the attachment of poxviruses to cells. These structural and functional studies of H3 will help in designing better vaccines and therapeutics.

Selective CD4+ T cell help for antibody responses to a large viral pathogen: deterministic linkage of specificities.

Antibody responses are critical components of protective immune responses to many pathogens, but parameters determining which proteins are targeted remain unclear. Vaccination with individual MHC-II-restricted vaccinia virus (VACV, smallpox vaccine) epitopes revealed that CD4(+) T cell help to B cells was surprisingly nontransferable to other virion protein specificities. Many VACV CD4(+) T cell responses identified in an unbiased screen targeted antibody virion protein targets, consistent with deterministic linkage between specificities. We tested the deterministic linkage model by efficiently predicting new vaccinia MHC II epitopes (830% improved efficiency). Finally, we showed CD4(+) T cell help was limiting for neutralizing antibody development and protective immunity in vivo. In contrast to the standard model, these data indicate individual proteins are the unit of B cell-T cell recognition for a large virus. Therefore, MHC restriction is a key selective event for the antiviral antibody response and is probably important for vaccine development to large pathogens.

The identification of a novel lead class for phosphodiesterase 2 inhibition by fragment-based drug design.

We have identified a novel PDE2 inhibitor series using fragment-based screening. Pyrazolopyrimidine fragment 1, while possessing weak potency (Ki = 22.4 µM), exhibited good binding efficiencies (LBE = 0.49, LLE = 4.48) to serve as a start for structure-based drug design. With the assistance of molecular modeling and X-ray crystallography, this fragment was developed into a series of potent PDE2 inhibitors with good physicochemical properties. Compound 16, a PDE2 selective inhibitor, was identified that exhibited favorable rat pharmacokinetic properties.

Structure guided design of a series of selective pyrrolopyrimidinone MARK inhibitors.

The initial structure activity relationships around an isoindoline uHTS hit will be described. Information gleaned from ligand co-crystal structures allowed for rapid refinements in both MARK potency and kinase selectivity. These efforts allowed for the identification of a compound with properties suitable for use as an in vitro tool compound for validation studies on MARK as a viable target for Alzheimer's disease.

Optimization of microtubule affinity regulating kinase (MARK) inhibitors with improved physical properties.

Inhibition of microtubule affinity regulating kinase (MARK) represents a potentially attractive means of arresting neurofibrillary tangle pathology in Alzheimer's disease. This manuscript outlines efforts to optimize a pyrazolopyrimidine series of MARK inhibitors by focusing on improvements in potency, physical properties and attributes amenable to CNS penetration. A unique cylcyclohexyldiamine scaffold was identified that led to remarkable improvements in potency, opening up opportunities to reduce MW, Pgp efflux and improve pharmacokinetic properties while also conferring improved solubility.

Profiling of hMPV F-specific antibodies isolated from human memory B cells.

Human metapneumovirus (hMPV) belongs to the Pneumoviridae family and is closely related to respiratory syncytial virus (RSV). The surface fusion (F) glycoprotein mediates viral fusion and is the primary target of neutralizing antibodies against hMPV. Here we report 113 hMPV-F specific monoclonal antibodies (mAbs) isolated from memory B cells of human donors. We characterize the antibodies' germline usage, epitopes, neutralization potencies, and binding specificities. We find that unlike RSV-F specific mAbs, antibody responses to hMPV F are less dominant against the apex of the antigen, and the majority of the potent neutralizing mAbs recognize epitopes on the side of hMPV F. Furthermore, neutralizing epitopes that differ from previously defined antigenic sites on RSV F are identified, and multiple binding modes of site V and II mAbs are discovered. Interestingly, mAbs that bind preferentially to the unprocessed prefusion F show poor neutralization potency. These results elucidate the immune recognition of hMPV infection and provide novel insights for future hMPV antibody and vaccine development.

The structure of the poxvirus A33 protein reveals a dimer of unique C-type lectin-like domains.

The current vaccine against smallpox is an infectious form of vaccinia virus that has significant side effects. Alternative vaccine approaches using recombinant viral proteins are being developed. A target of subunit vaccine strategies is the poxvirus protein A33, a conserved protein in the Chordopoxvirinae subfamily of Poxviridae that is expressed on the outer viral envelope. Here we have determined the structure of the A33 ectodomain of vaccinia virus. The structure revealed C-type lectin-like domains (CTLDs) that occur as dimers in A33 crystals with five different crystal lattices. Comparison of the A33 dimer models shows that the A33 monomers have a degree of flexibility in position within the dimer. Structural comparisons show that the A33 monomer is a close match to the Link module class of CTLDs but that the A33 dimer is most similar to the natural killer (NK)-cell receptor class of CTLDs. Structural data on Link modules and NK-cell receptor-ligand complexes suggest a surface of A33 that could interact with viral or host ligands. The dimer interface is well conserved in all known A33 sequences, indicating an important role for the A33 dimer. The structure indicates how previously described A33 mutations disrupt protein folding and locates the positions of N-linked glycosylations and the epitope of a protective antibody.

Structural basis for the inhibition of RNase H activity of HIV-1 reverse transcriptase by RNase H active site-directed inhibitors.

HIV/AIDS continues to be a menace to public health. Several drugs currently on the market have successfully improved the ability to manage the viral burden in infected patients. However, new drugs are needed to combat the rapid emergence of mutated forms of the virus that are resistant to existing therapies. Currently, approved drugs target three of the four major enzyme activities encoded by the virus that are critical to the HIV life cycle. Although a number of inhibitors of HIV RNase H activity have been reported, few inhibit by directly engaging the RNase H active site. Here, we describe structures of naphthyridinone-containing inhibitors bound to the RNase H active site. This class of compounds binds to the active site via two metal ions that are coordinated by catalytic site residues, D443, E478, D498, and D549. The directionality of the naphthyridinone pharmacophore is restricted by the ordering of D549 and H539 in the RNase H domain. In addition, one of the naphthyridinone-based compounds was found to bind at a second site close to the polymerase active site and non-nucleoside/nucleotide inhibitor sites in a metal-independent manner. Further characterization, using fluorescence-based thermal denaturation and a crystal structure of the isolated RNase H domain reveals that this compound can also bind the RNase H site and retains the metal-dependent binding mode of this class of molecules. These structures provide a means for structurally guided design of novel RNase H inhibitors.

The essential mosquito-stage P25 and P28 proteins from Plasmodium form tile-like triangular prisms.

P25 and P28 proteins are essential for Plasmodium parasites to infect mosquitoes and are leading candidates for a transmission-blocking malaria vaccine. The Plasmodium vivax P25 is a triangular prism that could tile the parasite surface. The residues forming the triangle are conserved in P25 and P28 from all Plasmodium species. A cocrystal structure shows that a transmission-blocking antibody uses only its heavy chain to bind Pvs25 at a vertex of the triangle.

The PD-1/PD-L1 complex resembles the antigen-binding Fv domains of antibodies and T cell receptors.

Signaling through the programmed death 1 (PD-1) inhibitory receptor upon binding its ligand, PD-L1, suppresses immune responses against autoantigens and tumors and plays an important role in the maintenance of peripheral immune tolerance. Release from PD-1 inhibitory signaling revives "exhausted" virus-specific T cells in chronic viral infections. Here we present the crystal structure of murine PD-1 in complex with human PD-L1. PD-1 and PD-L1 interact through the conserved front and side of their Ig variable (IgV) domains, as do the IgV domains of antibodies and T cell receptors. This places the loops at the ends of the IgV domains on the same side of the PD-1/PD-L1 complex, forming a surface that is similar to the antigen-binding surface of antibodies and T cell receptors. Mapping conserved residues allowed the identification of residues that are important in forming the PD-1/PD-L1 interface. Based on the structure, we show that some reported loss-of-binding mutations involve the PD-1/PD-L1 interaction but that others compromise protein folding. The PD-1/PD-L1 interaction described here may be blocked by antibodies or by designed small-molecule drugs to lower inhibitory signaling that results in a stronger immune response. The immune receptor-like loops offer a new surface for further study and potentially the design of molecules that would affect PD-1/PD-L1 complex formation and thereby modulate the immune response.

The cysteine-rich interdomain region from the highly variable plasmodium falciparum erythrocyte membrane protein-1 exhibits a conserved structure.

Plasmodium falciparum malaria parasites, living in red blood cells, express proteins of the erythrocyte membrane protein-1 (PfEMP1) family on the red blood cell surface. The binding of PfEMP1 molecules to human cell surface receptors mediates the adherence of infected red blood cells to human tissues. The sequences of the 60 PfEMP1 genes in each parasite genome vary greatly from parasite to parasite, yet the variant PfEMP1 proteins maintain receptor binding. Almost all parasites isolated directly from patients bind the human CD36 receptor. Of the several kinds of highly polymorphic cysteine-rich interdomain region (CIDR) domains classified by sequence, only the CIDR1alpha domains bind CD36. Here we describe the CD36-binding portion of a CIDR1alpha domain, MC179, as a bundle of three alpha-helices that are connected by a loop and three additional helices. The MC179 structure, containing seven conserved cysteines and 10 conserved hydrophobic residues, predicts similar structures for the hundreds of CIDR sequences from the many genome sequences now known. Comparison of MC179 with the CIDR domains in the genome of the P. falciparum 3D7 strain provides insights into CIDR domain structure. The CIDR1alpha three-helix bundle exhibits less than 20% sequence identity with the three-helix bundles of Duffy-binding like (DBL) domains, but the two kinds of bundles are almost identical. Despite the enormous diversity of PfEMP1 sequences, the CIDR1alpha and DBL protein structures, taken together, predict that a PfEMP1 molecule is a polymer of three-helix bundles elaborated by a variety of connecting helices and loops. From the structures also comes the insight that DBL1alpha domains are approximately 100 residues larger and that CIDR1alpha domains are approximately 100 residues smaller than sequence alignments predict. This new understanding of PfEMP1 structure will allow the use of better-defined PfEMP1 domains for functional studies, for the design of candidate vaccines, and for understanding the molecular basis of cytoadherence.

Epsilon substituted lysinol derivatives as HIV-1 protease inhibitors.

A series of HIV-1 protease inhibitors containing an epsilon substituted lysinol backbone was synthesized. Two novel synthetic routes using N-boc-L-glutamic acid alpha-benzyl ester and 2,6-diaminopimelic acid were developed. Incorporation of this epsilon substituent enabled access to the S2 pocket of the enzyme, affording high potency inhibitors. Modeling studies and synthetic efforts suggest the potency increase is due to both conformational bias and van der Waals interactions with the S2 pocket.

Redundancy and plasticity of neutralizing antibody responses are cornerstone attributes of the human immune response to the smallpox vaccine.

The smallpox vaccine is widely considered the gold standard for human vaccines, yet the key antibody targets in humans remain unclear. We endeavored to identify a stereotypic, dominant, mature virion (MV) neutralizing antibody target in humans which could be used as a diagnostic serological marker of protective humoral immunity induced by the smallpox vaccine (vaccinia virus [VACV]). We have instead found that diversity is a defining characteristic of the human antibody response to the smallpox vaccine. We show that H3 is the most immunodominant VACV neutralizing antibody target, as determined by correlation analysis of immunoglobulin G (IgG) specificities to MV neutralizing antibody titers. It was determined that purified human anti-H3 IgG is sufficient for neutralization of VACV; however, depletion or blockade of anti-H3 antibodies revealed no significant reduction in neutralization activity, showing anti-H3 IgG is not required in vaccinated humans (or mice) for neutralization of MV. Comparable results were obtained for human (and mouse) anti-L1 IgG and even for anti-H3 and anti-L1 IgG in combination. In addition to H3 and L1, human antibody responses to D8, A27, D13, and A14 exhibited statistically significant correlations with virus neutralization. Altogether, these data indicate the smallpox vaccine succeeds in generating strong neutralizing antibody responses not by eliciting a stereotypic response to a single key antigen but instead by driving development of neutralizing antibodies to multiple viral proteins, resulting in a "safety net" of highly redundant neutralizing antibody responses, the specificities of which can vary from individual to individual. We propose that this is a fundamental attribute of the smallpox vaccine.

A potent broadly neutralizing human RSV antibody targets conserved site IV of the fusion glycoprotein.

Respiratory syncytial virus (RSV) infection is the leading cause of hospitalization and infant mortality under six months of age worldwide; therefore, the prevention of RSV infection in all infants represents a significant unmet medical need. Here we report the isolation of a potent and broadly neutralizing RSV monoclonal antibody derived from a human memory B-cell. This antibody, RB1, is equipotent on RSV A and B subtypes, potently neutralizes a diverse panel of clinical isolates in vitro and demonstrates in vivo protection. It binds to a highly conserved epitope in antigenic site IV of the RSV fusion glycoprotein. RB1 is the parental antibody to MK-1654 which is currently in clinical development for the prevention of RSV infection in infants.