Novel Mechanism Of Inhibition Research Articles

Platelets regulate ischemia-induced revascularization and angiogenesis by secretion of growth factor–modulating factors

AbstractIn ischemic tissue, platelets can modulate angiogenesis. The specific factors influencing this function, however, are poorly understood. Here, we characterized the complement anaphylatoxin C5a-mediated activation of C5a receptor 1 (C5aR1) expressed on platelets as a potent regulator of ischemia-driven revascularization. We assessed the relevance of the anaphylatoxin receptor C5aR1 on platelets in patients with coronary artery disease as well as those with peripheral artery disease and used genetic mouse models to characterize its significance for ischemia and growth factor–driven revascularization. The presence of C5aR1-expressing platelets was increased in the hindlimb ischemia model. Ischemia-driven angiogenesis was significantly improved in C5aR1−/− mice but not in C5−/− mice, suggesting a specific role of C5aR1. Experiments using the supernatant of C5a-stimulated platelets suggested a paracrine mechanism of angiogenesis inhibition by platelets by means of antiangiogenic CXC chemokine ligand 4 (CXCL4, PF4). Lineage-specific C5aR1 deletion verified that the secretion of CXCL4 depends on C5aR1 ligation on platelets. Using C5aR1−/−CXCL4−/− mice, we observed no additional effect in the revascularization response, underscoring a strong dependence of CXCL4 secretion on the C5a-C5aR1-axis. We identified a novel mechanism for inhibition of neovascularization via platelet C5aR1, which was mediated by the release of antiangiogenic CXCL4.

Identification and structure–activity relationships for a series of N, N-disubstituted 2-aminobenzothiazoles as potent inhibitors of S. aureus

An internal collection of commercial and synthetically derived small molecule compounds was screened against several drug-resistant bacterial pathogens. Compound 1, a known N, N-disubstituted 2-aminobenzothiazole, was found to be a potent inhibitor of Staphylococcus aureus and several associated clinically relevant strains of methicillin-resistant S. aureus suggesting a possible novel mechanism of inhibition. It failed to show activity in any of the Gram-negative pathogens it was tested in. Evaluation in Escherichia coli BW25113 and Pseudomonas aeruginosa PAO1, as well as in their respective hyperporinated and efflux pump-deletion mutants revealed that activity in Gram-negative bacteria is diminished because this benzothiazole scaffold is a substrate for bacterial efflux pumps. Several analogs of 1 were synthesized to generate basic structure–activity relationships for the scaffold which highlighted that the N-propyl imidazole moiety was critical for the observed antibacterial activity.

Targeting platelet GPVI with glenzocimab: a novel mechanism for inhibition.

Platelet glycoprotein VI (GPVI) is attracting interest as a potential target for the development of new antiplatelet molecules with a low bleeding risk. GPVI binding to vascular collagen initiates thrombus formation and GPVI interactions with fibrin promote the growth and stability of the thrombus. In this study, we show that glenzocimab, a clinical stage humanized antibody fragment (Fab) with a high affinity for GPVI, blocks the binding of both ligands through a combination of steric hindrance and structural change. A cocrystal of glenzocimab with an extracellular domain of monomeric GPVI was obtained and its structure determined to a resolution of 1.9 Å. The data revealed that (1) glenzocimab binds to the D2 domain of GPVI, GPVI dimerization was not observed in the crystal structure because glenzocimab prevented D2 homotypic interactions and the formation of dimers that have a high affinity for collagen and fibrin; and (2) the light variable domain of the GPVI-bound Fab causes steric hindrance that is predicted to prevent the collagen-related peptide (CRP)/collagen fibers from extending out of their binding site and preclude GPVI clustering and downstream signaling. Glenzocimab did not bind to a truncated GPVI missing loop residues 129 to 136, thus validating the epitope identified in the crystal structure. Overall, these findings demonstrate that the binding of glenzocimab to the D2 domain of GPVI induces steric hindrance and structural modifications that drive the inhibition of GPVI interactions with its major ligands.

Membrane cholesterol regulates inhibition and substrate transport by the glycine transporter, GlyT2.

Membrane cholesterol binds to and modulates the function of various SLC6 neurotransmitter transporters, including stabilizing the outward-facing conformation of the dopamine and serotonin transporters. Here, we investigate how cholesterol binds to GlyT2 (SLC6A5), modulates glycine transport rate, and influences bioactive lipid inhibition of GlyT2. Bioactive lipid inhibitors are analgesics that bind to an allosteric site accessible from the extracellular solution when GlyT2 adopts an outward-facing conformation. Using molecular dynamics simulations, mutagenesis, and cholesterol depletion experiments, we show that bioactive lipid inhibition of glycine transport is modulated by the recruitment of membrane cholesterol to a binding site formed by transmembrane helices 1, 5, and 7. Recruitment involves cholesterol flipping from its membrane orientation, and insertion of the 3' hydroxyl group into the cholesterol binding cavity, close to the allosteric site. The synergy between cholesterol and allosteric inhibitors provides a novel mechanism of inhibition and a potential avenue for the development of potent GlyT2 inhibitors as alternative therapeutics for the treatment of neuropathic pain and therapeutics that target other SLC6 transporters.

Mechanistic Insights of Sulfonamide‐Based NLRP3 Inhibitors for the Treatment of Neurodegenerative Diseases

Neuroinflammation has proven to play an important role in the pathogenesis of neurodegenerative diseases by encouraging the expression and maturation of pro‐inflammatory regulators. Studies have shown the upregulation of pro‐inflammatory cytokines interleukin (IL)‐1β and IL‐18 in neurodegenerative diseases including Alzheimer’s disease (AD), Parkinson’s disease (PD), multiple sclerosis (MS), traumatic brain injury (TBI), and many more. The NLRP3 (NOD‐, LRR‐ and pyrin domain‐containing protein 3) inflammasome is the key regulator in the maturation and release of these inflammatory cytokines and is therefore an attractive target for suppressing inflammation. Upon assembly and activation of the multimeric NLRP3 inflammasome complex comprised of NLRP3, ASC protein, and pro‐caspase‐1, autoproteolytic cleavage of pro‐caspase‐1 occurs. Activated caspase‐1 is then able to cleave pro‐IL‐1β and pro‐IL‐18 and release their biologically active forms, IL‐1β and IL‐18, consequently leading to an inflammatory cell death known as pyroptosis (Figure 1). Our recent medicinal chemistry campaign has led to a series of novel small molecule inhibitors that are suggested to selectively target NLRP3 and show both in vitro and in vivo activity to inhibit the release of IL‐1β and IL‐18 with sub‐micromolar potencies. Through various assays and photoaffinity labeling experiments using a chemical probe, we have revealed and validated the mechanisms and binding of our lead compound, YQ128, to NLRP3. YQ128 is able to inhibit caspase‐1 and GSDMD cleavage as well as ASC speck formation in mouse macrophages; however, it has no effect on the NLRP3 expression, suggesting its target is upstream of ASC speck and caspase‐1 but downstream of priming. Through target identification studies using a photoaffinity labeling (PAL) technique, we found that YQ128 specifically interacts with NLRP3, but not other components of the NLRP3 inflammasome complex. Competition studies with the parent compound and other known NLRP3 inhibitors suggested the specific and unique binding of YQ128 to NLRP3. The results from other biological assays proposed its binding to the NACHT domain of NLRP3. Using our chemical probe in a pull‐down assay, we also discovered other potential targets within the inflammasome pathway, including a protein that was recently termed “undruggable”. These results suggest a novel mechanism of inhibition by our compounds and further studies are currently underway to improve these inhibitors as potential therapeutics for neuroinflammation in neurodegenerative diseases.

Discovery of Inhibitors of the Lipopolysaccharide Transporter MsbA: From a Screening Hit to Potent Wild-Type Gram-Negative Activity.

The dramatic increase in the prevalence of multi-drug resistant Gram-negative bacterial infections and the simultaneous lack of new classes of antibiotics is projected to result in approximately 10 million deaths per year by 2050. We report on efforts to target the Gram-negative ATP-binding cassette (ABC) transporter MsbA, an essential inner membrane protein that transports lipopolysaccharide from the inner leaflet to the periplasmic face of the inner membrane. We demonstrate the improvement of a high throughput screening hit into compounds with on-target single digit micromolar (μM) minimum inhibitory concentrations against wild-type uropathogenic Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae. A 2.98 Å resolution X-ray crystal structure of MsbA complexed with an inhibitor revealed a novel mechanism for inhibition of an ABC transporter. The identification of a fully encapsulated membrane binding site in Gram-negative bacteria led to unique physicochemical property requirements for wild-type activity.

The antimicrobial drug pyrimethamine inhibits STAT3 transcriptional activity by targeting the enzyme dihydrofolate reductase

Cancer is often characterized by aberrant gene expression patterns caused by the inappropriate activation of transcription factors. Signal transducer and activator of transcription 3 (STAT3) is a key transcriptional regulator of many protumorigenic processes and is persistently activated in many types of human cancer. However, like many transcription factors, STAT3 has proven difficult to target clinically. To address this unmet clinical need, we previously developed a cell-based assay of STAT3 transcriptional activity and performed an unbiased and high-throughput screen of small molecules known to be biologically active in humans. We identified the antimicrobial drug pyrimethamine as a novel and specific inhibitor of STAT3 transcriptional activity. Here, we show that pyrimethamine does not significantly affect STAT3 phosphorylation, nuclear translocation, or DNA binding at concentrations sufficient to inhibit STAT3 transcriptional activity, suggesting a potentially novel mechanism of inhibition. To identify the direct molecular target of pyrimethamine and further elucidate the mechanism of action, we used a new quantitative proteome profiling approach called proteome integral solubility alteration coupled with a metabolomic analysis. We identified human dihydrofolate reductase as a target of pyrimethamine and demonstrated that the STAT3-inhibitory effects of pyrimethamine are the result of a deficiency in reduced folate downstream of dihydrofolate reductase inhibition, implicating folate metabolism in the regulation of STAT3 transcriptional activity. This study reveals a previously unknown regulatory node of the STAT3 pathway that may be important for the development of novel strategies to treat STAT3-driven cancers.

Molecular dynamics simulation of Toxin-Antitoxin (TA) system in Acinetobacter baumannii to explore thenovel mechanism for inhibition of cell wall biosynthesis: Zeta Toxin as an effective therapeutic target.

The majority of bacteria and archaea contains Toxin-Antitoxin system (TA) that codes for the stable Toxin and unstable Antitoxin components forming a complex. The Antitoxin inhibits the catalytic activities of the Toxin. In general, the Antitoxin will be degraded by the proteases leading to the Toxin activation that subsequently targets essential cellular processes, including transcription, translation, replication, cell division, and cell wall biosynthesis. The Zeta Toxin-Epsilon Antitoxin system in ESKAPE pathogen stabilizes the resistance plasmid and promotes pathogenicity. The known TA system in Acinetobacter baumannii are known to be involved in the replication and translation, however, the mechanism of Zeta Toxin-Epsilon Antitoxin in cell wall biosynthesis remains unknown. In the present study, molecular docking and molecular dynamic (MD) simulations were employed to demonstrate whether Zeta Toxin can impair cell wall synthesis in A. baumannii. Further, the degradation mechanism of Antitoxin in the presence and absence of adenosine triphosphate (ATP) molecules are explained through MD simulation. The result reveals that the cleavage of Antitoxin could be possible with the presence of ATP by displaying its response from 20 ns, whereas the Zeta Toxin/Epsilon was unstable after 90 ns. The obtained results demonstrate that Zeta Toxin is "temporarily favorable" for ATP to undergo phosphorylation at UNAG kinase through the substrate tunneling process. The study further evidenced that phosphorylated UNAG prevents the binding of MurA, the enzyme that catalyzes the initial step of bacterial peptidoglycan biosynthesis. Therefore, the present study explores the binding mechanism of Zeta Toxin/Epsilon Antitoxin, which could be beneficial for preventing cell wall biosynthesis as well as for unveiling the alternative treatment options to antibiotics.

The Marine Natural Compound Elatol Is a Novel Protein Translation Inhibitor with Potent Activity Against Aggressive Non-Hodgkin Lymphomas

Prognosis remains poor for patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL) and other aggressive non-Hodgkin lymphomas. Targeted small-molecule signaling inhibitors have been poorly active in this setting due to a heterogeneous pathogenesis in which multiple different but overlapping signals may drive lymphomagenesis. Activation of cap-dependent protein translation is a convergence point of multiple lymphoma drivers, including B-cell receptor (BCR) signaling pathways. Cap-dependent protein translation begins with formation of the eIF4F complex, a heterotrimeric complex comprised of the cap-binding protein eIF4E, the scaffold protein eIF4G, and the ATP-hydrolyzing RNA helicase eIF4A, which is required for expression of many oncogenes, including the common lymphoma driver MYC. Despite evidence that disruption of eIF4F function is toxic to lymphoma cell lines and sensitizes mouse models of lymphoma to chemotherapy, eIF4A inhibitors have not progressed to clinical evaluation. We designed a screen to identify novel eIF4A inhibitors by measuring ATP hydrolysis and screened a collection of ~1,200 natural compounds and purified extracts. The marine natural product elatol was the top hit in our screen, showing selectivity for eIF4A over other DEAD-box family helicases and a panel of 97 common kinases. Elatol inhibits eIF4A's helicase activity in cell-free in vitro assays and binds to the target at a 2:1 ratio through interaction with two key lysine residues that fold together in its RNA-binding groove. It also induced apoptosis in a broad range of cancer cell lines from the NCI-60 (average LD50= 1.9 μM; LD50<1μM in 33/60) cancer cell line and CCLE panels, but our collection of non-Hodgkin lymphoma lines showed the greatest sensitivity as a group (average LD50= 1.3 μM; 9/12 <1μM; range 213-5749nM). In DLBCL cell lines, Elatol treatment reduced global protein translation measured through polysome profiling and O-propargyl puromycin incorporation as well as cap-dependent translation measured by luciferase reporter and western blotting. Elatol also blocked translational activation downstream of BCR stimulation in primary chronic lymphocytic leukemia (CLL) cells. The effects of elatol are similar to the rocaglate silvestrol, which inhibits eIF4A though through a different mechanism, but elatol requires higher concentrations to achieve these effects. Elatol's reduced in vitro potency, however, is off-set by a maximum tolerated in vivo dose to mice of 65mg/kg, compared to silvestrol's typical dosing at 0.2-05 mg/kg. Elatol significantly reduced the growth of DLBCL tumors in a xenograft model. Mechanistically, elatol and silvestrol both clearly function downstream of mTOR for translational inhibition, but unlike silvestrol, elatol causes strong and rapid induction of the stress response transcription factor ATF4. Assessment of drug activity in murine embryonal fibroblasts lacking ATF4 and those with an unphosphorylatable mutant allele of its upstream activator eIF2α shows elatol's anti-lymphoma activities are not dependent on ATF4 induction. In addition, an inhibitor of the eIF2α kinase PERK and other evidence demonstrate elatol does not produce an unfolded protein response, but the reason for ATF4 induction is not yet clear and additional eIF2α kinases are under investigation. The novel mechanism of inhibition of ATP hydrolysis may be driving these unique observations compared to other eIF4A inhibitors, but unexpected off targets are also under evaluation. In sum we report a new natural inhibitor of protein translation with potent anti-lymphoma activities. Elatol provides a novel starting point for development of eIF4A inhibitors in cancer therapeutics. DisclosuresNo relevant conflicts of interest to declare.

Structural insights into novel mechanisms of inhibition of the major β-carbonic anhydrase CafB from the pathogenic fungus Aspergillus fumigatus

In fungi the β-class of carbonic anhydrases (β-CAs) are zinc metalloenzymes that are essential for growth, survival, differentiation, and virulence. Aspergillus fumigatus is the most important pathogen responsible for invasive aspergillosis and possesses two major β-CAs, CafA and CafB. Recently we reported the biochemical characterization and 1.8 Å crystal structure of CafA. Here, we report a crystallographic analysis of CafB revealing the mechanism of enzyme catalysis and establish the relationship of this enzyme to other β-CAs. While CafA has a typical open conformation, CafB, when exposed to acidic pH and/or an oxidative environment, has a novel type of active site in which a disulfide bond is formed between two zinc-ligating cysteines, expelling the zinc ion and stabilizing the inactive form of the enzyme. Based on the structural data, we generated an oxidation-resistant mutant (Y159A) of CafB. The crystal structure of the mutant under reducing conditions retains a catalytic zinc at the expected position, tetrahedrally coordinated by three residues (C57, H113 and C116) and an aspartic acid (D59), and replacing the zinc-bound water molecule in the closed form. Furthermore, the active site of CafB crystals grown under zinc-limiting conditions has a novel conformation in which the solvent-exposed catalytic cysteine (C116) is flipped out of the metal coordination sphere, facilitating release of the zinc ion. Taken together, our results suggest that A. fumigatus use sophisticated activity-inhibiting strategies to enhance its survival during infection.

Abstract 2572: Compound C inhibits metabolic programming of ovarian cancer

Abstract High grade serous ovarian carcinoma (HGSC) exhibits elevated energy demands to sustain rapid proliferation, survival and invasiveness. We have shown that Compound C, also known as Dorsomorphin, inhibits ovarian cancer growth, invasiveness and survival as well as interactions with mesothelial cells and macrophages. However, the effect of Compound C on metabolic programming has not been reported. The purpose of this study is to determine whether Compound C inhibits the malignant phenotypes of ovarian cancer through suppression of energy production. Therefore, we performed comprehensive metabolic profiling of ovarian cancer cells treated with Compound C. Our findings indicate that Compound C significantly inhibits ATP production in the ovarian cancer cell lines: OVCAR3, SKOV3 and IGROV1. Compound C inhibited basal and maximal mitochondrial respiration as well as the non-mitochondrial oxygen consumption in these three cell lines. Compound C had no effect on basal, induced or compensatory glycolysis. However, it significantly decreased the ratio of mitochondrial oxygen consumption rate to glycolytic proton efflux rate (mitoOCR/glycoPER), indicating a direct inhibitory effect of Compound C on mitochondrial electron transport chain and a bioenergetic shift from oxidative phosphorylation towards glycolysis. These results demonstrate for the first time, the direct inhibitory effect of Compound C on mitochondrial function as a novel mechanism of inhibition of the malignant phenotype of ovarian cancer cells. Citation Format: Alia Dawlat Ghoneum, Daniela Gonzalez, Neveen Said. Compound C inhibits metabolic programming of ovarian cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2572.

A novel mechanism of inhibition by phenylthiourea on PvdP, a tyrosinase synthesizing pyoverdine of Pseudomonas aeruginosa

The biosynthesis of pyoverdine, the major siderophore of Pseudomonas aeruginosa, is a well-organized process involving a discrete number of enzyme-catalyzed steps. The final step of this process involves the PvdP tyrosinase, which converts ferribactin into pyoverdine. Thus, inhibition of the PvdP tyrosinase activity provides an attractive strategy to interfere with siderophore synthesis to manage P. aeruginosa infections. Here, we report phenylthiourea as a non-competitive inhibitor of PvdP for which we solved a crystal structure in complex with PvdP. The crystal structure indicates that phenylthiourea binds to an allosteric binding site and thereby interferes with its tyrosinase activity. We further provide proofs that PvdP tyrosinase inhibition by phenylthiourea requires the C-terminal lid region. This provides opportunities to develop inhibitors that target the allosteric site, which seems to be confined to fluorescent pseudomonads, and not the tyrosinase active site. Furthermore, increases the chances to identify PvdP inhibitors that selectively interfere with siderophore synthesis.

Inhibition of Drug-Resistant Candida glabrata by Killer Toxins Produced by Saccharomyces cerevisiae

Combatting the spread of drug-resistant microbes requires new antifungal compounds with novel mechanisms of inhibition. Our lab investigates natural, proteinaceous toxins that are coded by double stranded RNA satellites found within Saccharomyces cerevisiae. Commonly known as “killer yeasts”, toxin-producing strains of S. cerevisiae have been found to inhibit the growth of many important fungal pathogens. To assess the value of killer yeasts for medical application we tested over 6,000 interactions between killer yeasts and various important fungal pathogens. To determine the susceptibility of fungi to killer toxins, we competed killer yeasts against a dilute lawn of a fungal pathogen; an active toxin produces a zone of growth inhibition in the lawn around the killer yeast. We determined that Candida glabratais broadly susceptible to killer toxins, while other Candida species showed little or no susceptibility. Of the 90 strains of S. cerevisiae tested against C. glabrata seven were capable of inhibiting all clinical strains of drug-resistant C. glabrata available from the Center for Disease Control and the U.S. Department of Agriculture. In our evaluation of these toxins as potential antifungal therapeutics we confirmed their inhibitory capability against C. glabrata under physiological conditions. Based on the results of these tests and prior knowledge about killer yeasts, we believe that these toxins show a potential as antifungal therapeutic precursors to treat recalcitrant infections caused by C. glabrata.

Discovery of Novel Integrase Inhibitors Acting outside the Active Site Through High-Throughput Screening.

Currently, an increasing number of drugs are becoming available to clinics for the treatment of HIV infection. Even if this targeted therapy is highly effective at suppressing viral replication, caregivers are facing growing therapeutic failures in patients, due to resistance with or without treatment adherence concerns. Accordingly, it is important to continue to discover small molecules that have a novel mechanism of inhibition. In this work, HIV integrase inhibitors were selected by high-throughput screening. Chemical structure comparisons enabled the identification of stilbene disulfonic acids as a potential new chemotype. Biochemical characterization of the lead compound stilbenavir (NSC34931) and a few derivatives was performed. Stilbene disulfonic acid derivatives exhibit low to sub-micromolar antiviral activity, and they inhibit integrase through DNA-binding inhibition. They probably bind to the C-terminal domain of integrase, in the cavity normally occupied by the noncleaved strand of the viral DNA substrate. Because of this original mode of action compared to active site strand transfer inhibitors, they do not exhibit cross-resistance to the three main resistance pathways to integrase inhibitors (G140S-Q148H, N155H, and Y143R). Further structure–activity optimization should enable the development of more active and less toxic derivatives with potential clinical relevance.

Alteration of Prion Strain Emergence by Nonhost Factors.

Prions can persist in the environment for extended periods of time after adsorption to surfaces, including soils, feeding troughs, or fences. Prion strain- and soil-specific differences in prion adsorption, infectivity, and response to inactivation may be involved in strain maintenance or emergence of new strains in a population. Extensive proteinase K (PK) digestion of Hyper (HY) and Drowsy (DY) PrPSc resulted in a greater reduction in the level of DY PrPSc than of HY PrPSc Use of the PK-digested material in protein misfolding cyclic amplification strain interference (PMCAsi) resulted in earlier emergence of HY PrPSc than of undigested controls. This result established that strain-specific alteration of the starting ratios of conversion-competent HY and DY PrPSc can alter strain emergence. We next investigated whether environmentally relevant factors such as surface binding and weathering could alter strain emergence. Adsorption of HY and DY PrPSc to silty clay loam (SCL), both separately and combined, resulted in DY interfering with the emergence of HY in PMCAsi in a manner similar to that seen with unbound controls. Similarly, repeated cycles of wetting and drying of SCL-bound HY and DY PrPSc did not alter the emergence of HY PrPSc compared to untreated controls. Importantly, these data indicate that prion strain interference can occur when prions are bound to surfaces. Interestingly, we found that drying of adsorbed brain homogenate on SCL could restore its ability to interfere with the emergence of HY, suggesting a novel strain interference mechanism. Overall, these data provide evidence that the emergence of a strain from a mixture can be influenced by nonhost factors.IMPORTANCE The prion strain, surface type, and matrix containing PrPSc can influence PrPSc surface adsorption. The cumulative effect of these factors can result in strain- and soil-specific differences in prion bioavailability. Environmental weathering processes can result in decreases in PrPSc conversion efficiency and infectivity. Little is known about how incomplete inactivation of surface-bound PrPSc affects transmission and prion strain emergence. Here, we show that strain interference occurs with soil-bound prions and that altering the ratios of prion strains by strain-specific inactivation can affect strain emergence. Additionally, we identify a novel mechanism of inhibition of prion conversion by environmental treatment-induced changes at the soil-protein interface altering strain emergence. These novel findings suggest that environmental factors can influence strain emergence of surface-bound prions.

Patrolling monocytes scavenge endothelial-adherent sickle RBCs: a novel mechanism of inhibition of vaso-occlusion in SCD

AbstractPainful vaso-occlusive crisis (VOC) is the most common complication of sickle cell disease (SCD). Increasing evidence suggests that vaso-occlusion is initiated by increased adherence of sickle red blood cells (RBCs) to the vascular endothelium. Thus, the mechanisms that remove endothelial-attached sickle RBCs from the microvasculature are expected to be critical for optimal blood flow and prevention of VOC in SCD. We hypothesized that patrolling monocytes (PMos), which protect against vascular damage by scavenging cellular debris, could remove endothelial-adherent sickle RBCs and ameliorate VOC in SCD. We detected RBC (GPA+)-engulfed material in circulating PMos of patients with SCD, and their frequency was further increased during acute crisis. RBC uptake by PMos was specific to endothelial-attached sickle, but not control, RBCs and occurred mostly through ICAM-1, CD11a, and CD18. Heme oxygenase 1 induction, by counteracting the cytotoxic effects of engulfed RBC breakdown products, increased PMo viability. In addition, transfusions, by lowering sickle RBC uptake, improved PMo survival. Selective depletion of PMos in Townes sickle mice exacerbated vascular stasis and tissue damage, whereas treatment with muramyl dipeptide (NOD2 ligand), which increases PMo mass, reduced stasis and SCD associated organ damage. Altogether, these data demonstrate a novel mechanism for removal of endothelial attached sickle RBCs mediated by PMos that can protect against VOC pathogenesis, further supporting PMos as a promising therapeutic target in SCD VOC.

Pharmacological and Genetic Inhibition of Caveolin-1 Promotes Epithelialization and Wound Closure.

Chronic wounds-including diabetic foot ulcers, venous leg ulcers, and pressure ulcers-represent a major health problem that demands an urgent solution and new therapies. Despite major burden to patients, health care professionals, and health care systems worldwide, there are no efficacious therapies approved for treatment of chronic wounds. One of the major obstacles in achieving wound closure in patients is the lack of epithelial migration. Here, we used multiple pre-clinical wound models to show that Caveolin-1 (Cav1) impedes healing and that targeting Cav1 accelerates wound closure. We found that Cav1 expression is significantly upregulated in wound edge biopsies of patients with non-healing wounds, confirming its healing-inhibitory role. Conversely, Cav1 was absent from the migrating epithelium and is downregulated in acutely healing wounds. Specifically, Cav1 interacted with membranous glucocorticoid receptor (mbGR) and epidermal growth factor receptor (EGFR) in a glucocorticoid-dependent manner to inhibit cutaneous healing. However, pharmacological disruption of caveolae by MβCD or CRISPR/Cas9-mediated Cav1 knockdown resulted in disruption of Cav1-mbGR and Cav1-EGFR complexes and promoted epithelialization and wound healing. Our data reveal a novel mechanism of inhibition of epithelialization and wound closure, providing a rationale for pharmacological targeting of Cav1 as potential therapy for patients with non-healing chronic wounds.

Sequential Cyclophosphamide and Trametinib Improve Clinical Graft Versus Host Disease and Survival in Murine Hematopoietic Stem Cell Transplant

Background Ongoing acute graft versus host disease (aGVHD) after allogeneic stem cell transplantation (alloSCT) causes significant morbidity and mortality with no standard therapy except corticosteroids, which are broadly immunosuppressive and toxic. Cyclophosphamide (Cy) given day 3 and day 4 is effective for prophylaxis of aGVHD but has proven ineffective when given later as in ongoing aGVHD in both murine and human studies. We previously demonstrated that the MEK inhibitor trametinib (Tram) is effective for aGVHD prophylaxis in murine models of aGVHD through the novel mechanism of inhibition of naive T cells (CD44−CD62L+). Our hypothesis is that ongoing aGVHD could be better inhibited by sequential Cy and post-Cy Tram inhibition (to prevent naive T cells from re-initiating alloreactivity) without compromising graft-versus-malignancy (GVM) responses. Methods Mice were administered sequential Cy and Tram post SCT starting day 12. Efficacy of treatment was assessed in a murine model of MHC-mismatched alloSCT. Overall survival was assessed and clinical GVHD was scored three times weekly. Peripheral blood, spleen, and lymph node cells phenotypes were determined by flow cytometry. GVM was also assessed with A20 luciferase/YFP (A20luc/YFP) lymphoma cells imaged with in vivo imaging system (IVIS). Results Overall survival following sequential Cy and Tram was identical to that seen with T cell depleted bone marrow alone and significantly superior to treatment using either single-agent Cy or Tram (p Conclusions Combination cyclophosphamide and trametinib demonstrates superior overall survival and limits GVHD severity without affecting regulatory T cells or GVM in murine allogeneic stem cell models. Utilizing Cy and Tram are readily translatable and support the notion that clinical approaches can be developed in the future.

Synergistic inactivation of AXL: a (cross)road to cure ovarian cancer?

Inhibition of the receptor tyrosine kinase AXL, a key molecular driver of ovarian cancer, has recently been highlighted as promising therapeutic strategy. In this issue of EMBO Reports , Antony et al [1] have identified a novel mechanism of inhibition of AXL, wherein the GPI‐anchored tumour suppressor OPCML sequesters AXL into specialised plasma membrane domains where the phosphatase PTPRG is located, therefore facilitating AXL dephosphorylation. This attenuation of AXL signalling has translational implications for the design of synergistic therapies, to target the kinase for this aggressive malignancy.

Abstract 5445: A novel mechanism of inhibition of ovarian cancer growth by Compound C, dorsomorphin

Abstract High grade serous ovarian carcinoma (HGSC) is the most lethal gynecologic malignancy with a five-year survival rate of 15%. As a highly aggressive cancer, HGSC has elevated energy demands to ensure its survival and proliferation in its unique peritoneal environment. Compound C or Dorsomophin is of particular interest as it is the only available cell permeable molecule that works selectively and competitively to inhibit Adenosine Monophosphate Protein-activated protein kinase or AMPK, a sensitive sensor of ATP levels. It has long been established that Compound C has a role as a BMP1 inhibitor during embryogenesis and as an inhibitor of AMPK activity in different contexts. However, the mechanism by which Compound C exerts anti-tumorigenic effects remains unknown. The purpose of this study is to understand how Compound C suppresses growth of HGSC. Our preliminary studies indicate that Compound C inhibits ovarian cancer cell proliferation and survival via a novel mechanism not only through inhibition of AMPK but also through the inhibition of upstream PI3K-Akt as well as TGFβ1/ TGFβ1/2 receptors/Smad 3,4 activation. These findings elucidate a novel mechanism of inhibition of ovarian cancer by Compound C, suggesting its potential use as a novel therapeutic agent inhibiting oncogenic and metabolic pathways. Citation Format: Alia Ghoneum, Hesham M. Afify, Neveen Said. A novel mechanism of inhibition of ovarian cancer growth by Compound C, dorsomorphin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5445.