Discovery of a Broad-Spectrum Antiviral Compound That Inhibits Pyrimidine Biosynthesis and Establishes a Type 1 Interferon-Independent Antiviral State.

Viral emergence and reemergence underscore the importance of developing efficacious, broad-spectrum antivirals. Here, we report the discovery of tetrahydrobenzothiazole-based compound 1, a novel, broad-spectrum antiviral lead that was optimized from a hit compound derived from a cytopathic effect (CPE)-based antiviral screen using Venezuelan equine encephalitis virus. Compound 1 showed antiviral activity against a broad range of RNA viruses, including alphaviruses, flaviviruses, influenza virus, and ebolavirus. Mechanism-of-action studies with metabolomics and molecular approaches revealed that the compound inhibits host pyrimidine synthesis and establishes an antiviral state by inducing a variety of interferon-stimulated genes (ISGs). Notably, the induction of the ISGs by compound 1 was independent of the production of type 1 interferons. The antiviral activity of compound 1 was cell type dependent with a robust effect observed in human cell lines and no observed antiviral effect in mouse cell lines. Herein, we disclose tetrahydrobenzothiazole compound 1 as a novel lead for the development of a broad-spectrum, antiviral therapeutic and as a molecular probe to study the mechanism of the induction of ISGs that are independent of type 1 interferons.

Discovery of a novel compound with anti-venezuelan equine encephalitis virus activity that targets the nonstructural protein 2.

Alphaviruses present serious health threats as emerging and re-emerging viruses. Venezuelan equine encephalitis virus (VEEV), a New World alphavirus, can cause encephalitis in humans and horses, but there are no therapeutics for treatment. To date, compounds reported as anti-VEEV or anti-alphavirus inhibitors have shown moderate activity. To discover new classes of anti-VEEV inhibitors with novel viral targets, we used a high-throughput screen based on the measurement of cell protection from live VEEV TC-83-induced cytopathic effect to screen a 340,000 compound library. Of those, we identified five novel anti-VEEV compounds and chose a quinazolinone compound, CID15997213 (IC50 = 0.84 µM), for further characterization. The antiviral effect of CID15997213 was alphavirus-specific, inhibiting VEEV and Western equine encephalitis virus, but not Eastern equine encephalitis virus. In vitro assays confirmed inhibition of viral RNA, protein, and progeny synthesis. No antiviral activity was detected against a select group of RNA viruses. We found mutations conferring the resistance to the compound in the N-terminal domain of nsP2 and confirmed the target residues using a reverse genetic approach. Time of addition studies showed that the compound inhibits the middle stage of replication when viral genome replication is most active. In mice, the compound showed complete protection from lethal VEEV disease at 50 mg/kg/day. Collectively, these results reveal a potent anti-VEEV compound that uniquely targets the viral nsP2 N-terminal domain. While the function of nsP2 has yet to be characterized, our studies suggest that the protein might play a critical role in viral replication, and further, may represent an innovative opportunity to develop therapeutic interventions for alphavirus infection.

Validated Preclinical Mouse Model for Therapeutic Testing against Multidrug-Resistant Pseudomonas aeruginosa Strains.

The rise in infections caused by antibiotic-resistant bacteria is outpacing the development of new antibiotics. The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) are a group of clinically important bacteria that have developed resistance to multiple antibiotics and are commonly referred to as multidrug resistant (MDR). The medical and research communities have recognized that, without new antimicrobials, infections by MDR bacteria will soon become a leading cause of morbidity and death. Therefore, there is an ever-growing need to expedite the development of novel antimicrobials to combat these infections. Toward this end, we set out to refine an existing mouse model of pulmonary Pseudomonas aeruginosa infection to generate a robust preclinical tool that can be used to rapidly and accurately predict novel antimicrobial efficacy. This refinement was achieved by characterizing the virulence of a panel of genetically diverse MDR P. aeruginosa strains in this model, by both 50% lethal dose (LD50) analysis and natural history studies. Further, we defined two antibiotic regimens (aztreonam and amikacin) that can be used as comparators during the future evaluation of novel antimicrobials, and we confirmed that the model can effectively differentiate between successful and unsuccessful treatments, as predicted by in vitro inhibitory data. This validated model represents an important tool in our arsenal to develop new therapies to combat MDR P. aeruginosa strains, with the ability to provide rapid preclinical evaluation of novel antimicrobials and support data from clinical studies during the investigational drug development process. IMPORTANCE The prevalence of antibiotic resistance among bacterial pathogens is a growing problem that necessitates the development of new antibiotics. Preclinical animal models are important tools to facilitate and speed the development of novel antimicrobials. Successful outcomes in animal models not only justify progression of new drugs into human clinical trials but also can support FDA decisions if clinical trial sizes are small due to a small population of infections with specific drug-resistant strains. However, in both cases the preclinical animal model needs to be well characterized and provide robust and reproducible data. Toward this goal, we have refined an existing mouse model to better predict the efficacy of novel antibiotics. This improved model provides an important tool to better predict the clinical success of new antibiotics.

Mobilization of hematopoietic progenitor cells by yeast-derived beta-glucan requires activation of matrix metalloproteinase-9.

Poly-(1,6)-beta-d-glucopyranosyl-(1,3)-beta-d-glucopyranose (PGG) beta-glucan is a soluble yeast-derived polysaccharide that has previously been shown to induce hematopoietic progenitor cell (HPC) mobilization. However, the mobilizing mechanism of action remains unknown. Here, we confirmed that PGG beta-glucan alone or in combination with granulocyte colony-stimulating factor (G-CSF) mobilizes HPC into the periphery. Optimal mobilizing effects were seen 24-48 hours after PGG beta-glucan doses of 4.8-9.6 mg/kg. Animals treated with G-CSF and PGG beta-glucan showed a collaborative effect in HPC mobilization compared with G-CSF treatment alone. Additional studies demonstrated that neither complement 3 nor complement receptor 3 played a role in this effect and that PGG beta-glucan treatment did not induce proinflammatory cytokine secretion. However, bone marrow cells from PGG beta-glucan-treated mice secreted abundant matrix metalloproteinase-9 (MMP-9), and PGG beta-glucan-induced HPC mobilization was abrogated in MMP-9 knockout mice. Moreover, we demonstrated that both hematopoietic and nonhematopoietic cells contributed to MMP-9 secretion upon PGG beta-glucan treatment. In addition, HPCs mobilized by PGG beta-glucan had similar levels of engraftment in host and lineage differentiation capability compared with those mobilized by G-CSF. Thus, PGG beta-glucan is an agent that enhances HPC mobilization and may improve the outcome of clinical stem cell transplantation.

Yeast-derived beta-glucan augments the therapeutic efficacy mediated by anti-vascular endothelial growth factor monoclonal antibody in human carcinoma xenograft models.

PURPOSE: Bevacizumab is a recombinant IgG1 humanized monoclonal antibody against vascular endothelial growth factor (VEGF). Its proposed mechanism of action is independent of immune effector functions. Many human carcinomas not only secrete VEGF but also express membrane-bound VEGF. In addition, VEGF receptors are expressed on tumor cells. It is hypothesized that bevacizumab could bind membrane-bound VEGF or VEGF-VEGF receptor complexes on tumors, thereby initiating potential immunologic consequences. We previously showed that yeast-derived beta-glucan functions with antitumor antibodies that activate complement to recruit complement receptor 3-expressing leukocytes capable of mediating complement receptor 3-dependent cellular cytotoxicity of tumors opsonized with iC3b. In the current study, the therapeutic efficacy mediated by combining bevacizumab with yeast-derived beta-glucan was studied in human carcinoma xenograft models. EXPERIMENTAL DESIGN: Human tumor cell lines were screened for membrane-bound VEGF expression both in vitro and in vivo. Complement activation mediated by bevacizumab was examined. Tumor cell lines positive or negative for membrane-bound VEGF expression were implanted in severe combined immunodeficient mice to establish xenograft models. Tumor-bearing mice were treated with different regimens. Tumor regression and long-term survival were recorded. RESULTS: Human ovarian carcinoma SKOV-3 cells expressed membrane-bound VEGF both in vitro and in vivo. Bevacizumab was bound to membrane-bound VEGF, activated complement, and synergized with beta-glucan to elicit cellular cytotoxicity in vitro. In vivo study showed that beta-glucan could significantly augment the therapeutic efficacy mediated by bevacizumab. CONCLUSIONS: Yeast-derived beta-glucan can synergize with anti-VEGF monoclonal antibody bevacizumab for the treatment of cancer with membrane-bound VEGF expression.

Combined yeast {beta}-glucan and antitumor monoclonal antibody therapy requires C5a-mediated neutrophil chemotaxis via regulation of decay-accelerating factor CD55.

Administration of a combination of yeast-derived beta-glucan with antitumor monoclonal antibodies (mAb) has significant therapeutic efficacy in a variety of syngeneic murine tumor models. We have now tested this strategy using human carcinomas implanted in immunocompromised severe combined immunodeficient mice. Combined immunotherapy was therapeutically effective in vivo against NCI-H23 human non-small-cell lung carcinomas, but this modality was surprisingly ineffective against SKOV-3 human ovarian carcinomas. Whereas NCI-H23 tumors responded to this combination therapy with increased intratumoral neutrophil infiltration and C5a production, these responses were lacking in treated SKOV-3 tumors. Further results suggested that SKOV-3 tumors were protected by up-regulation of the membrane complement regulatory protein CD55 (decay-accelerating factor). Blockade of CD55 in vitro led to enhanced deposition of C activation product C3b and increased cytotoxicity mediated by beta-glucan-primed neutrophils. In vivo, administration of anti-CD55 mAb along with beta-glucan and anti-Her-2/neu mAb caused tumor regression and greatly improved long-term survival in animals bearing the previously resistant SKOV-3 tumors. This was accompanied by increased intratumoral neutrophil accumulation and C5a production. We conclude that CD55 suppresses tumor killing by antitumor mAb plus beta-glucan therapy (and, perhaps, in other circumstances). These results suggest a critical role for CD55 to regulate iC3b and C5a release and in turn to influence the recruitment of beta-glucan-primed neutrophils eliciting killing activity.

Development and evaluation of murine lung-specific disease models for Pseudomonas aeruginosa applicable to therapeutic testing.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen capable of causing a wide range of disease manifestations, including severe bacterial pneumonia. Recently, clinics have reported a rise in nosocomial infections with multidrug resistant (MDR) species, including MDR strains of P. aeruginosa. In order to quickly evaluate the efficacy of new therapeutics for MDR infections, highly reproducible and validated animal models need to be developed for pre-clinical testing. Here, we describe the characterization of two murine models to study MDR P. aeruginosa respiratory disease. We evaluated and compared these models using a non-invasive intratracheal instillation method and established the 50% lethal dose, course of infection, biometric parameters of disease and degree of pneumonia development for each model. Further, we tested meropenem as a proof-of-concept therapeutic and report efficacy data that suggests that the leukopenic model could serve a robust pre-clinical model to test novel therapeutics.

Hematopoietic stem cells from the marrow of mice treated with Flt3 ligand are significantly expanded but exhibit reduced engraftment potential.

BACKGROUND: Hematopoietic stem cells (HSC) can be significantly expanded by hematopoietic growth factors. Flt3 ligand (FL) is a hematopoietic growth factor that induces proliferation and mobilization of HSC into the peripheral blood. We previously reported that FL-mobilized HSC exhibit superior engraftment potential. The engraftment potential of FL-expanded HSC in the bone marrow compartment has not been evaluated. In this study, we investigated the effect of in vivo administration of FL on the engraftment potential of HSC expanded in the marrow. METHODS: B10.BR (H-2k) donor mice were treated for 10 days with 10 microg of FL per day. Partially conditioned allogeneic B10 (H-2b) recipients received whole bone marrow. Purified HSC (c-Kit+/Sca1+/lin-) from the marrow were also transplanted in ablated syngeneic B10.BR recipients. RESULTS: FL treatment significantly expanded HSC in the marrow compartment. The absolute number of T cells and granulocytes were unchanged whereas dendritic cells, facilitating cells, and HSC were significantly increased in the bone marrow of donor mice treated with FL compared with untreated mice. Mice conditioned with 700 cGy and transplanted with FL-treated allogeneic bone marrow showed a significantly lower rate of engraftment (14%) compared with recipients of bone marrow from untreated mice (100%). Syngeneic recipients transplanted with 500, 1000, 2000, or 3000 purified HSC from FL-treated donors also showed reduced long-term survival compared with mice transplanted with HSC from untreated donors. Cell cycle analysis revealed that significantly more bone marrow HSC were in cycle after FL treatment compared with unmanipulated controls. CONCLUSION: These data show that FL treatment for 10 days induces proliferation of HSC but reduces the engraftment potential of HSC harvested from the marrow. The reduced syngeneic engraftment of HSC indicates that FL treatment induces intrinsic changes in HSC, resulting in failure of long-term engraftment or self-renewal despite no change in characteristic phenotype of HSC.

To stay or to leave: Stem cells and progenitor cells navigating the S1P gradient.

Most hematopoietic stem progenitor cells (HSPCs) reside in bone marrow (BM), but a small amount of HSPCs have been found to circulate between BM and tissues through blood and lymph. Several lines of evidence suggest that sphingosine-1-phosphate (S1P) gradient triggers HSPC egression to blood circulation after mobilization from BM stem cell niches. Stem cells also visit certain tissues. After a temporary 36 h short stay in local tissues, HSPCs go to lymph in response to S1P gradient between lymph and tissue and eventually enter the blood circulation. S1P also has a role in the guidance of the primitive HSPCs homing to BM in vivo, as S1P analogue FTY720 treatment can improve HSPC BM homing and engraftment. In stress conditions, various stem cells or progenitor cells can be attracted to local injured tissues and participate in local tissue cell differentiation and tissue rebuilding through modulation the expression level of S1P(1), S1P(2) or S1P(3) receptors. Hence, S1P is important for stem cells circulation in blood system to accomplish its role in body surveillance and injury recovery.

Therapeutic potential of various beta-glucan sources in conjunction with anti-tumor monoclonal antibody in cancer therapy.

Combined beta-glucan with anti-tumor mAb therapy has demonstrated therapeutic efficacy in murine tumor models. The current study was designed to compare the therapeutic efficacy of various sources of beta-glucans. Our studies demonstrated that yeast beta-glucan, in combination with anti-tumor mAb, resulted in significantly smaller tumor burdens and achieved enhanced long-term survival compared to mAb alone or beta-glucan extracts from mushrooms. Further studies indicated that yeast beta-glucan particle was superior to mushroom extracts in inducing cytokine secretion, particularly IL-12 production in dendritic cells (DCs). In addition, results showed that cytokine production was markedly decreased in MyD88-deficient macrophages and DCs but not in complement receptor 3 (CR3)-deficient mice. Our data suggest that yeast beta-glucan demonstrates much stronger adjuvant activity compared to mushroom beta-glucan extracts in tumor therapy. This effect of yeast beta-glucan may be in part ascribed to the cytokine secretion by DCs and macrophages and bioavailability of active beta-glucan moiety.

Yeast beta-glucan amplifies phagocyte killing of iC3b-opsonized tumor cells via complement receptor 3-Syk-phosphatidylinositol 3-kinase pathway.

Anti-tumor mAbs hold promise for cancer therapy, but are relatively inefficient. Therefore, there is a need for agents that might amplify the effectiveness of these mAbs. One such agent is beta-glucan, a polysaccharide produced by fungi, yeast, and grains, but not mammalian cells. Beta-glucans are bound by C receptor 3 (CR3) and, in concert with target-associated complement fragment iC3b, elicit phagocytosis and killing of yeast. Beta-glucans may also promote killing of iC3b-opsonized tumor cells engendered by administration of anti-tumor mAbs. In this study, we report that tumor-bearing mice treated with a combination of beta-glucan and an anti-tumor mAb show almost complete cessation of tumor growth. This activity evidently derives from a 25-kDa fragment of beta-glucan released by macrophage processing of the parent polysaccharide. This fragment, but not parent beta-glucan, binds to neutrophil CR3, induces CBRM 1/5 neoepitope expression, and elicits CR3-dependent cytotoxicity. These events require phosphorylation of the tyrosine kinase, Syk, and consequent PI3K activation because beta-glucan-mediated CR3-dependent cytotoxicity is greatly decreased by inhibition of these signaling molecules. Thus, beta-glucan enhances tumor killing through a cascade of events, including in vivo macrophage cleavage of the polysaccharide, dual CR3 ligation, and CR3-Syk-PI3K signaling. These results are important inasmuch as beta-glucan, an agent without evident toxicity, may be used to amplify tumor cell killing and may open new opportunities in the immunotherapy of cancer.

Beta-glucan enhances complement-mediated hematopoietic recovery after bone marrow injury.

Myelotoxic injury in the bone marrow (BM) as a consequence of total body irradiation (TBI) or granulocyte colony-stimulating factor (G-CSF) mobilization results in the deposition of iC3b on BM stroma (stroma-iC3b). In the present study, we have examined how stroma-iC3b interacts with hematopoietic progenitor cells (HPCs) and the role of complement (C) and complement receptor 3 (CR3) in BM injury/repair. We demonstrate here that stroma-iC3b tethers HPCs via the inserted (I) domain of HPC complement receptor 3 (CR3, CD11b/CD18, Mac-1). Following irradiation, stroma-iC3b was observed in the presence of purified IgM and normal mouse serum (NMS), but not serum from Rag-2(-/-) mice, implicating a role for antibody (Ab) and the classic pathway of C activation. Furthermore, a novel role for soluble yeast beta-glucan, a ligand for the CR3 lectin-like domain (LLD), in the priming of CR3(+) HPC is suggested. Soluble yeast beta-glucan could enhance the proliferation of tethered HPCs, promote leukocyte recovery following sublethal irradiation, and increase the survival of lethally irradiated animals following allogeneic HPC transplantation in a CR3-dependent manner. Taken together, these observations suggest a novel role for C, CR3, and beta-glucan in the restoration of hematopoiesis following injury.

Matching at the MHC class I K locus is essential for long-term engraftment of purified hematopoietic stem cells: a role for host NK cells in regulating HSC engraftment.

The events that regulate engraftment and long-term repopulating ability of hematopoietic stem cells (HSCs) after transplantation are not well defined. We report for the first time that major histocompatibility complex (MHC) class I K plays a critical role in HSC engraftment via interaction with recipient natural killer (NK) cells. Durable engraftment of purified HSCs requires MHC class I K matching between HSC donor and recipient. In the absence of MHC class I K matching, HSCs exhibit impaired long-term engraftment (P =.01). Dependence on MHC class I K matching is eliminated in B6 beige mice that lack NK cell function, as well as in wild-type mice depleted of NK cells, implicating a possible regulatory role of NK cells for HSC engraftment. The coadministration of CD8+/T-cell receptor-negative (TCR-) graft facilitating cells (FCs) matched at MHC class I K to the HSC donor overcomes the requirement for MHC class I K matching between HSCs and recipient. These data demonstrate that FCs inhibit NK cell effects on the HSCs. Notably, FCs do not suppress the cytotoxic activity of activated NK cells. Enhanced green fluorescent protein-positive (EGFP+) FCs persist for one month following allogeneic transplantation, making cold target inhibition an unlikely mechanism. Therefore, MHC class I may play a critical role in the initiating events that dictate HSC engraftment and/or NK-mediated rejection following allogeneic transplantation.

CD8(+), alphabeta-TCR(+), and gammadelta-TCR(+) cells in the recipient hematopoietic environment mediate resistance to engraftment of allogeneic donor bone marrow.

Historically, conditioning for engraftment of hematopoietic stem cells has been nonspecific. In the present study, we characterized which cells in the recipient hematopoietic microenvironment prevent allogeneic marrow engraftment. Mice defective in production of alphabeta-TCR(+), gammadelta-TCR(+), alphabeta- plus gammadelta-TCR(+), CD8(+), or CD4(+) cells were transplanted with MHC-disparate allogeneic bone marrow. Conditioning with 500 cGy total body irradiation (TBI) plus a single dose of cyclophosphamide (CyP) on day +2 establishes chimerism in normal recipients. When mice were conditioned with 300 cGy TBI plus a single dose of CyP on day +2, all engrafted, except wild-type controls and those defective in production of CD4(+) T cells. Mice lacking both alphabeta- and gammadelta-TCR(+) cells engrafted without conditioning, suggesting that both alphabeta- and gammadelta-TCR T cells in the host play critical and nonredundant roles in preventing engraftment of allogeneic bone marrow. CD8 knockout (KO) mice engrafted without TBI, but only if they received CyP on day +2 relative to the marrow infusion, showing that a CD8(-) cell was targeted by the CyP conditioning. The CD8(+) cell effector function is mechanistically different from that for conventional T cells, and independent of CD4(+) T helper cells because CD4 KO mice require substantially higher levels of conditioning than the other KO phenotypes. These results suggest that a number of cell populations with different mechanisms of action mediate resistance to engraftment of allogeneic marrow. Targeting of specific recipient cellular populations may permit conditioning approaches to allow mixed chimerism with minimal morbidity and could potentially avoid the requirement for myelotoxic agents altogether.