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Human cytomegalovirus (HCMV) encodes multiple surface glycoprotein complexes to infect a variety of cell types. The HCMV Pentamer, composed of gH, gL, UL128, UL130, and UL131A, enhances entry into epithelial, endothelial, and myeloid cells by interacting with the cell surface receptor neuropilin 2 (NRP2). Despite the critical nature of this interaction, the molecular determinants that govern NRP2 recognition remain unclear. Here, we describe the cryo-EM structure of NRP2 bound to Pentamer. The high-affinity interaction between these proteins is calcium dependent and differs from the canonical carboxyl-terminal arginine (CendR) binding that NRP2 typically uses. We also determine the structures of four neutralizing human antibodies bound to the HCMV Pentamer to define susceptible epitopes. Two of these antibodies compete with NRP2 binding, but the two most potent antibodies recognize a previously unidentified epitope that does not overlap the NRP2-binding site. Collectively, these findings provide a structural basis for HCMV tropism and antibody-mediated neutralization.

International audience; Crimean-Congo hemorrhagic fever virus (CCHFV) is the most widespread tick-borne zoonotic virus, with a 30% case fatality rate in humans. Structural information is lacking in regard to the CCHFV membrane fusion glycoprotein Gc-the main target of the host neutralizing antibody response-as well as antibody-mediated neutralization mechanisms. We describe the structure of prefusion Gc bound to the antigen-binding fragments (Fabs) of two neutralizing antibodies that display synergy when combined, as well as the structure of trimeric, postfusion Gc. The structures show the two Fabs acting in concert to block membrane fusion, with one targeting the fusion loops and the other blocking Gc trimer formation. The structures also revealed the neutralization mechanism of previously reported antibodies against CCHFV, providing the molecular underpinnings essential for developing CCHFVspecific medical countermeasures for epidemic preparedness.

Crimean-Congo hemorrhagic fever virus (CCHFV), the only endemic biosafety level 4 pathogen in Europe, is the world’s most widely distributed tick-borne zoonotic virus with a 30% fatality rate in humans, underlining the need for specific therapeutics and vaccines. The CCHFV membrane fusion glycoprotein Gc is the main target of the host neutralizing antibody response. Here we describe the structure of pre-fusion Gc in complex with the antigen-binding fragments (Fabs) corresponding to a therapeutically potent bispecific antibody as well as the structure of unbound Gc in its post-fusion conformation. Our findings suggest that one Fab blocks insertion of the fusion loops into the target membrane, whereas the other blocks formation of the post-fusion trimer. Combined with yeast display of mutagenized Gc, the structures allowed epitope mapping of a large panel of neutralizing antibodies. These data provide the essential molecular underpinnings for developing vaccines and therapeutics against CCHFV.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a priority pathogen that poses a high risk to public health. Due to the high morbidity and mortality rates associated with CCHFV infection, there is an urgent need to develop medical countermeasures for disease prevention and treatment. CCHFV GP38, a secreted glycoprotein of unknown function unique to the Nairoviridae family, was recently shown to be the target of a protective antibody against CCHFV. Here, we present the crystal structure of GP38, which revealed a novel fold with distant homology to another CCHFV glycoprotein that is suggestive of a gene duplication event. We also demonstrate that antibody 13G8 protects STAT1-knockout mice against heterologous CCHFV challenge using a clinical isolate from regions where CCHFV is endemic. Collectively, these data advance our understanding of GP38 structure and antigenicity and should facilitate future studies investigating its function., Crimean-Congo hemorrhagic fever virus (CCHFV) is the causative agent of the most widespread tick-borne viral infection in humans. CCHFV encodes a secreted glycoprotein (GP38) of unknown function that is the target of a protective antibody. Here, we present the crystal structure of GP38 at a resolution of 2.5 Å, which revealed a novel fold primarily consisting of a 3-helix bundle and a β-sandwich. Sequence alignment and homology modeling showed distant homology between GP38 and the ectodomain of Gn (a structural glycoprotein in CCHFV), suggestive of a gene duplication event. Analysis of convalescent-phase sera showed high titers of GP38 antibodies indicating immunogenicity in humans during natural CCHFV infection. The only protective antibody for CCHFV in an adult mouse model reported to date, 13G8, bound GP38 with subnanomolar affinity and protected against heterologous CCHFV challenge in a STAT1-knockout mouse model. Our data strongly suggest that GP38 should be evaluated as a vaccine antigen and that its structure provides a foundation to investigate functions of this protein in the viral life cycle. IMPORTANCE Crimean-Congo hemorrhagic fever virus (CCHFV) is a priority pathogen that poses a high risk to public health. Due to the high morbidity and mortality rates associated with CCHFV infection, there is an urgent need to develop medical countermeasures for disease prevention and treatment. CCHFV GP38, a secreted glycoprotein of unknown function unique to the Nairoviridae family, was recently shown to be the target of a protective antibody against CCHFV. Here, we present the crystal structure of GP38, which revealed a novel fold with distant homology to another CCHFV glycoprotein that is suggestive of a gene duplication event. We also demonstrate that antibody 13G8 protects STAT1-knockout mice against heterologous CCHFV challenge using a clinical isolate from regions where CCHFV is endemic. Collectively, these data advance our understanding of GP38 structure and antigenicity and should facilitate future studies investigating its function.

Platinum-based chemotherapeutics exert their therapeutic efficacy via the formation of DNA adducts which interfere with DNA replication, transcription and cell division and ultimately induce cell death. Repair and tolerance of these Pt-DNA lesions by nucleotide excision repair (NER) and homologous recombination (HR) can substantially reduce the effectiveness of therapy. Inhibition of these repair pathways, therefore, holds the potential to sensitize cancer cells to Pt treatment and increase clinical efficacy. Replication Protein A (RPA) plays essential roles in both NER and HR, along with its role in DNA replication and DNA damage checkpoint activation. Each of these functions is, in part, mediated by RPA binding to single-stranded DNA (ssDNA). Here we report the synthesis and characterization of novel derivatives of RPA small molecule inhibitors and their activity in models of epithelial ovarian cancer (EOC) and non-small cell lung cancer (NSCLC). We have synthesized analogs of our previously reported RPA inhibitor TDRL-505 and determined the structure-activity relationships. These data led us to the identification of TDRL-551, which exhibited a greater than 2-fold increase in in vitro activity. TDRL-551 showed synergy with Pt in tissue culture models of EOC and in vivo efficacy, as a single agent and in combination with platinum, in a NSCLC xenograft model. These data demonstrate the utility of RPA inhibition in EOC and NSCLC and the potential in developing novel anticancer therapeutics that target RPA-DNA interactions.

Targeting DNA repair and the DNA damage response for cancer therapy has recently gained increasing attention with inhibitors of the PI3-K-like kinases in early stage clinical trials. The utility of DNA repair inhibitors can be expanded by their use in combination treatment with DNA damaging chemotherapeutics including cisplatin. We have focused on directly targeting the DNA repair pathway responsible for repairing platinum-induced DNA damage, nucleotide excision repair (NER). We have selected the molecular target XPA, whose role is in the identification and verification of the sites of DNA damage. Clinical validation of XPA has been obtained where high XPA expression in lung, ovarian and lung cancer results in decreased efficacy of platinum therapy. We report the continued development of the X80 class of XPA small molecule inhibitors. In a two-step, iterative process we have identified critical structure activity relationships that have resulted in a 100-fold increase in potency with in vitro IC50 values below 1μM. Analyses of the SARs define the chemical and structural features that impact the interaction with XPA, cellular permeability and contribute to selectivity. Data demonstrate that the X80 class of inhibitors do not interact with DNA but directly bind the XPA protein. Recent production of a sub-fragment of the XPA protein will allow the identification of the direct binding domain to enable continued structure-based design of more potent XPA inhibitors. Supported by NIH grants R01CA180710 and R41CA162648 to JJT. Citation Format: Pamela S. VanderVere-Carozza, Navnath Gavande, Akaash Mishra, John J. Turchi. Small molecule inhibitors targeting the interaction of xeroderma pigmentosum group A protein with cisplatin-damaged DNA. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C58.

Platinum (Pt)-based chemotherapeutics exert their efficacy via the formation of DNA adducts which interfere with DNA replication, transcription and cell division and ultimately induce cell death. DNA repair of Pt-DNA adducts via nucleotide excision repair (NER) or homologous recombination repair (HRR) can substantially reduce the effectiveness of the Pt therapy, which is a major contributor for cellular resistance. Inhibition of these repair pathways, therefore, holds the potential to sensitize cancer cells to Pt-treatment and increase the clinical efficacy. Replication protein A (RPA) is a single-stranded DNA binding protein that plays a crucial role in the NER and HRR pathways apart from its role in replication and DNA damage checkpoint activation. We have pursued the development of small molecule inhibitors of the RPA-DNA interaction as a mechanism to target RPA activity and increase the efficacy of Pt-based cancer chemotherapy. We have developed an inhibitor, TDRL-551, that targets the central oligonucleotide/oligosaccharide binding folds in DNA binding domains A and B (DBD-A/B) of the 70 kDa RPA subunit. This lead compound shows synergy with cisplatin in tissue culture models of epithelial ovarian cancer (EOC) and in vivo efficacy, as a single agent as well as in a combination with platinum, in a lung cancer xenograft model. We have expanded our synthetic scheme and defined structure-activity relationships (SAR) towards optimization of TRDL-551 as an anticancer therapeutic for the treatment of lung and ovarian cancer. Supported by NIH grants R01 CA180710 and R41CA162648 to JJT. Citation Format: Navnath Gavande, Pamela S. VanderVere-Carozza, Akaash Mishra, John J. Turchi. Discovery and development of replication protein A (RPA)-DNA interaction inhibitors for cancer chemotherapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C57.