Peter Ruminski

Targeting of αv integrin identifies a core molecular pathway that regulates fibrosis in several organs.

Myofibroblasts are the major source of extracellular matrix components that accumulate during tissue fibrosis, and hepatic stellate cells (HSCs) are believed to be the major source of myofibroblasts in the liver. To date, robust systems to genetically manipulate these cells have not been developed. We report that Cre under control of the promoter of Pdgfrb (Pdgfrb-Cre) inactivates loxP-flanked genes in mouse HSCs with high efficiency. We used this system to delete the gene encoding αv integrin subunit because various αv-containing integrins have been suggested as central mediators of fibrosis in multiple organs. Such depletion protected mice from carbon tetrachloride–induced hepatic fibrosis, whereas global loss of β3, β5 or β6 integrins or conditional loss of β8 integrins in HSCs did not. We also found that Pdgfrb-Cre effectively targeted myofibroblasts in multiple organs, and depletion of the αv integrin subunit using this system was protective in other models of organ fibrosis, including pulmonary and renal fibrosis. Pharmacological blockade of αv-containing integrins by a small molecule (CWHM 12) attenuated both liver and lung fibrosis, including in a therapeutic manner. These data identify a core pathway that regulates fibrosis and suggest that pharmacological targeting of all αv integrins may have clinical utility in the treatment of patients with a broad range of fibrotic diseases.

Synthesis of new verapamil analogues and their evaluation in combination with rifampicin against Mycobacterium tuberculosis and molecular docking studies in the binding site of efflux protein Rv1258c

New verapamil analogues were synthesized and their inhibitory activities against Mycobacterium tuberculosis H37Rv determined in vitro alone and in combination with rifampicin (RIF). Some analogues showed comparable activity to verapamil and exhibited better synergies with RIF. Molecular docking studies of the binding sites of Rv1258c, a M. tuberculosis efflux protein previously implicated in intrinsic resistance to RIF, suggested a potential rationale for the superior synergistic interactions observed with some analogues.

Inhibitors of Arg-Gly-Asp-Binding Integrins Reduce Development of Pancreatic Fibrosis in Mice

Background & Aims Pancreatic stellate cells (PSCs) regulate the development of chronic pancreatitis (CP) and are activated by the cytokine transforming growth factor β (TGFB). Integrins of the αv family promote TGFB signaling in mice, probably by interacting with the Arg-Gly-Asp (RGD) sequence of the TGFB latency-associated peptide, which frees TGFB to bind its cellular receptors. However, little is known about the role of integrins in the development of CP. We investigated the effects of small-molecule integrin inhibitors in a mouse model of CP. Methods We induced CP in C57BL/6 female mice by repeated cerulein administration. An active RGD peptidomimetic compound (Center for World Health and Medicine [CWHM]-12) was delivered by continuous infusion, starting 3 days before or 5 days after cerulein administration began. Pancreata were collected and parenchymal atrophy, fibrosis, and activation of PSCs were assessed by histologic, gene, and protein expression analyses. We measured CWHM-12 effects on activation of TGFB in co-culture assays in which rat PSC cells (large T immortalized cells [LTC-14]) activate expression of a TGFB-sensitive promoter in reporter cells. Results Pancreatic tissues of mice expressed messenger RNAs encoding subunits of RGD-binding integrins. Cerulein administration increased expression of these integrins, altered pancreatic cell morphology, and induced fibrosis. The integrin inhibitor CWHM-12 decreased acinar cell atrophy and loss, and substantially reduced fibrosis, activation of PSCs, and expression of genes regulated by TGFB. CWHM-12 also reduced established fibrosis in mice and blocked activation of TGFB in cultured cells. Conclusions Based on studies of a mouse model of CP and cultured PSCs, integrins that bind RGD sequences activate PSCs and promote the development of pancreatic fibrogenesis in mice. Small-molecule antagonists of this interaction might be developed for treatment of pancreatic fibrotic diseases.

αv integrins on mesenchymal cells regulate skeletal and cardiac muscle fibrosis

Mesenchymal cells expressing platelet-derived growth factor receptor beta (PDGFRβ) are known to be important in fibrosis of organs such as the liver and kidney. Here we show that PDGFRβ+ cells contribute to skeletal muscle and cardiac fibrosis via a mechanism that depends on αv integrins. Mice in which αv integrin is depleted in PDGFRβ+ cells are protected from cardiotoxin and laceration-induced skeletal muscle fibrosis and angiotensin II-induced cardiac fibrosis. In addition, a small-molecule inhibitor of αv integrins attenuates fibrosis, even when pre-established, in both skeletal and cardiac muscle, and improves skeletal muscle function. αv integrin blockade also reduces TGFβ activation in primary human skeletal muscle and cardiac PDGFRβ+ cells, suggesting that αv integrin inhibitors may be effective for the treatment and prevention of a broad range of muscle fibroses.The mechanisms underlying tissue fibrosis are unclear. The authors show that mesenchymal cells expressing PDGFRβ mediate fibrosis in skeletal muscle and heart via a mechanism involving αvxa0integrin, and that inhibitors of αvxa0integrins attenuate fibrotic responses in mice.

Evaluation of spiropiperidine hydantoins as a novel class of antimalarial agents.

Given the rise of parasite resistance to all currently used antimalarial drugs, the identification of novel chemotypes with unique mechanisms of action is of paramount importance. Since Plasmodium expresses a number of aspartic proteases necessary for its survival, we have mined antimalarial datasets for drug-like aspartic protease inhibitors. This effort led to the identification of spiropiperidine hydantoins, bearing similarity to known inhibitors of the human aspartic protease β-secretase (BACE), as new leads for antimalarial drug discovery. Spiropiperidine hydantoins have a dynamic structure-activity relationship profile with positions identified as being tolerant of a variety of substitution patterns as well as a key piperidine N-benzyl phenol pharmacophore. Lead compounds 4e (CWHM-123) and 12k (CWHM-505) are potent antimalarials with IC50 values against Plasmodium falciparum 3D7 of 0.310 μM and 0.099 μM, respectively, and the former features equivalent potency on the chloroquine-resistant Dd2 strain. Remarkably, these compounds do not inhibit human aspartic proteases BACE, cathepsins D and E, or Plasmodium plasmepsins II and IV despite their similarity to known BACE inhibitors. Although the current leads suffer from poor metabolic stability, they do fit into a drug-like chemical property space and provide a new class of potent antimalarial agents for further study.

Role of αv integrins on perivascular mesenchymal cells in regulation of skeletal and cardiac muscle fibrosis

Abstract Background Muscle fibrosis is a major global health-care burden. Although cells expressing platelet-derived growth factor receptor β (PDGFRβ) are important in liver fibrosis, their contribution to skeletal and cardiac muscle fibrogenesis remains unclear. Using PDGFRβ-Cre mice, we aimed to identify a pathway driving both skeletal and cardiac muscle fibrosis, focusing on αv integrins and their activation of transforming growth factor β (TGFβ), a central mediator of fibrosis. Methods The contribution of PDGFRβ+ cells to muscle fibrosis was assessed in fluorescent reporter (mTmG) mice under the control of PDGFRβ-Cre. Itgavflox/flox;PDGFRβ-Cre mice were used to investigate whether loss of αv integrins influences fibrosis development after cardiotoxin-induced muscle injury and in an angiotensin II model of cardiac fibrosis. A small-molecule inhibitor of αv integrins (CWHM12) and control enantiomer (CWHM96) were used to determine whether pharmacological blockade of αv integrins could attenuate fibrosis. Findings PDGFRβ-Cre effectively targeted quiescent PDGFRβ+ cells and activated myofibroblasts in both skeletal and cardiac muscle. αv integrin depletion on PDGFRβ+ cells protected mice from cardiotoxin-induced skeletal muscle fibrosis and angiotensin II-induced cardiac fibrosis. In addition, CWHM12 attenuated fibrosis, even when pre-established, in both skeletal and cardiac muscle. αv integrin blockade also reduced TGFβ activation in primary human skeletal muscle and cardiac PDGFRβ+ cells, further highlighting the potential clinical utility of small-molecule αv integrin inhibition in the treatment and prevention of a broad range of muscle fibroses. Interpretation We have demonstrated that PDGFRβ-Cre labels profibrotic cells in skeletal muscle and that depletion of αv integrins in these cells using this genetic strategy reduces skeletal muscle fibrosis. Most importantly from a treatment standpoint, we have shown that pharmacological inhibition of αv integrins with a small-molecule inhibitor might have utility in the prevention and treatment of established skeletal muscle fibrosis. Funding Wellcome Trust, British Heart Foundation, Royal College of Surgeons of Edinburgh.

Reversed Isoniazids: Design, synthesis and evaluation against Mycobacterium tuberculosis

Novel reversed isoniazid (RINH) agents were synthesized by covalently linking isoniazid with various efflux pump inhibitor (EPI) cores and their structural motifs. These RINH agents were then evaluated for anti-mycobacterial activity against sensitive, isoniazid mono-resistant and MDR clinical isolates of M. tuberculosis and a selected number of compounds were also tested ex vivo for intracellular activity as well as in the ethidium bromide (EB) assay for efflux pump inhibition efficacy. The potency of some compounds against various strains of M. tuberculosis (4a-c, 7 and 8; H37Rv-MIC99 ≤1.25u202fµM, R5401-MIC99 ≤2.5u202fµM, X_61-MIC99 ≤5u202fµM) demonstrated the potential of the reversed anti-TB agent strategy towards the development of novel anti-mycobacterial agents to address the rapidly growing issue of resistance. Further, macrophage activity with >90% inhibition by 1a-c and 3b (MIC90 ≤13.42u202fµM) and inhibition of EB efflux demonstrated by these compounds are encouraging.

Pharmacologic Comparison of Clinical Neutral Endopeptidase Inhibitors in a Rat Model of Acute Secretory Diarrhea.

Racecadotril (acetorphan) is a neutral endopeptidase (NEP) inhibitor with known antidiarrheal activity in animals and humans; however, in humans, it suffers from shortcomings that might be improved with newer drugs in this class that have progressed to the clinic for nonenteric disease indications. To identify potentially superior NEP inhibitors with immediate clinical utility for diarrhea treatment, we compared their efficacy and pharmacologic properties in a rat intestinal hypersecretion model. Racecadotril and seven other clinical-stage inhibitors of NEP were obtained or synthesized. Enzyme potency and specificity were compared using purified peptidases. Compounds were orally administered to rats before administration of castor oil to induce diarrhea. Stool weight was recorded over 4 hours. To assess other pharmacologic properties, select compounds were orally administered to normal or castor oil–treated rats, blood and tissue samples collected at multiple time points, and active compound concentrations determined by mass spectroscopy. NEP enzyme activity was measured in tissue homogenates. Three previously untested clinical NEP inhibitors delayed diarrhea onset and reduced total stool output, with little or no effect on intestinal motility assessed by the charcoal meal test. Each was shown to be a potent, highly specific inhibitor of NEP. Each exhibited greater suppression of NEP activity in intestinal and nonintestinal tissues than did racecadotril and sustained this inhibition longer. These results suggest that newer clinical-stage NEP inhibitors originally developed for other indications may be directly repositioned for treatment of acute secretory diarrhea and offer advantages over racecadotril, such as less frequent dosing and potentially improved efficacy.

OC-002 Selective Alpha V Integrin Deletion Identifies a Core, Targetable Molecular Pathway that Regulates Fibrosis Across Solid Organs

Introduction Myofibroblasts are the major source of extracellular matrix components that accumulate during tissue fibrosis, and hepatic stellate cells (HSCs) are the major source of myofibroblasts in the liver. To date, robust systems to genetically manipulate these cells have not existed. The paucity of tools that allow reliable, specific inactivation of genes in myofibroblasts in vivo has greatly hindered progress in understanding the underlying biology of fibrotic diseases. Methods Mouse models of organ fibrosis: Chronic carbon tetrachloride injection (liver fibrosis), intratracheal bleomycin instillation (lung fibrosis) and unilateral ureteric obstruction (kidney fibrosis). Fluorescent reporter mice: mTmG (Td tomato/EGFP) and Ai14 (Rosa-CAG-LSL-tdTomato-WPRE) mice were crossed with PDGFRβ(platelet derived growth factor beta)-Cre mice. Integrin knockout mice: Itgavflox/flox, itgb8flox/flox and Itgb6-/- mice were maintained on C57BL/6 background and itgb3-/- and Itgb5-/- mice were maintained on a 129/svJae background. Fluorescent cell sorting: Td Tomato positive cells from Ai14; PDGFRβ-Cre mice were sorted using a FACSAria. Results We report that PDGFRβ (platelet derived growth factor receptor beta) Cre inactivates genes in murine HSCs with high efficiency. We used this system to delete the integrin αv subunit because of the suggested role of multiple αv integrins as central mediators of fibrosis in multiple organs. Deletion of the αv integrin subunit in HSCs protected mice from CCl4-induced hepatic fibrosis, whereas global loss of αvβ3, αvβ5 or αvβ6 or conditional loss of αvβ8 on HSCs did not. PDGFRβ-Cre effectively targeted myofibroblasts in multiple organs, and deletion of αv integrins using this system was also protective in bleomycin-induced pulmonary fibrosis and renal fibrosis induced by unilateral ureteric obstruction. Critically, pharmacological blockade of αv integrins by a novel small molecule (CWHM 12) attenuated both liver and lung fibrosis, even when administered after fibrosis was established. Conclusion These data identify a core cellular and molecular pathway that regulates fibrosis, and suggest that pharmacological targeting of all αv integrins may have clinical utility in the treatment of patients with a broad range of fibrotic diseases. Disclosure of Interest N. Henderson: None Declared, T. Arnold: None Declared, Y. Katamura: None Declared, M. Giacomini: None Declared, J. Rodriguez: None Declared, J. McCarty: None Declared, P. Ruminski Shareholder of: Antegrin Therapeutics, LLC., D. Griggs Shareholder of: Antegrin Therapeutics, LLC., J. Maher: None Declared, J. Iredale: None Declared, A. Lacy-Hulbert: None Declared, R. Adams: None Declared, D. Sheppard: None Declared.