αvβ8 (IC50 = 0.2 nM); αvβ3 (IC50 = 0.8 nM); αvβ6 (IC50 = 1.5 nM); αvβ1 (IC50 = 1.8 nM); αvβ5 (IC50 = 61 nM)

Moreover, αvβ5 (IC50=61 nM) and αIIbβ3/α2β1/α10β1 (IC50>5000 nM) are weakly inhibited by CWHM-12 (CWHM 12). CWHM-12 demonstrated strong potency against all five potential β-subunit binding partners (αvβ1, αvβ3, αvβ5, αvβ6, and αvβ8) in in vitro ligand binding assays, with a marginally lower potency against αvβ5 compared to other αv integrants. The efficiency of protein [1].

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CWHM‐12 inhibits cell–ligand interactions mediated by RGD integrins. Multiple pro‐fibrotic pathways are attenuated and repair processes are enhanced by CWHM‐12. [2]
The small‐molecule RGD peptidomimetic compound CWHM‐12 has been shown previously to inhibit cell–ligand interactions mediated by αvβ3, αvβ5, and αvβ6, and the interactions of biochemically purified integrins αvβ1 and αvβ8 with their respective ligands (Henderson et al., 2013). We have now determined the potency of this compound against these and additional RGD‐binding integrins entirely using cell‐based assays (Table 1). These results show particularly strong potency (<1 nM) for αvβ1, αvβ3, and αvβ6, with varying lesser activities for the other tested RGD‐binding integrins. As previously reported, CWHM‐12 has no significant activity (>5 μ M) against integrin αIIbβ3, which is essential for platelet aggregation, nor does it affect ligand binding by non‐RGD‐binding integrins (Henderson et al., 2013).

After three weeks of CCl4 treatment to establish fibrotic disease, mice are given either CWHM-12 (CWHM 12) or a vehicle for the last three weeks of CCl4. Even after fibrotic disease has been proven, CWHM-12 dramatically reduces liver fibrosis. The CWHM-12 treated mice showed protection from CCl4-induced hepatic fibrosis, at least partially because of less TGF-β activation by αv integrins, as shown by digital image quantitation, which significantly reduced p-SMAD3 signaling in the livers of treated mice compared to controls. Additionally, pulmonary fibrosis progression was markedly slowed down by CWHM-12 administration[1].

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Continuous subcutaneous administration of CWHM-12, an RGD integrin antagonist, for 28 days improved kidney function as measured by serum creatinine. CWHM-12 significantly reduced Collagen 1 (Col1a1) mRNA expression and scar collagen deposition in the kidney. Protein and gene expression markers of activated myofibroblasts, a major source of extracellular matrix deposition in kidney fibrosis, were diminished by treatment. RNA sequencing revealed that inhibition of RGD integrins influenced multiple pathways that determine the outcome of the response to injury and of repair processes. A second RGD integrin antagonist, CWHM-680, administered once daily by oral gavage was also effective in ameliorating fibrosis. We conclude that targeting RGD integrins with such small-molecule antagonists is a promising therapeutic approach in fibrotic kidney disease [2].

In vitro integrin functional assays [1]
The effects of CWHM-12 and CWHM 96 on cell adhesion mediated by αvβ3, αvβ5, αvβ6, and α5β1 were measured as previously described with minor modifications50,51. Briefly, stably transfected human 293 cells over-expressing human αvβ3 or αvβ5 were pre-incubated in HBSS buffer containing 200 μM MnCl2 for 30 min at 37 °C with 3-fold dilutions of compound. Each sample was then added to triplicate wells of a 96-well plate which had been coated overnight at 4 °C with a predetermined optimal concentration of purified vitronectin, washed, blocked by 1 hr incubation with BSA, and washed again. Cells were allowed to attach for 30 min at 37 °C, and non-adherent cells were removed by washing. Remaining attached cells were measured by endogenous alkaline phosphatase activity using para-nitrophenyl phosphate and reading absorbance signal at 405 nM. The same procedure was used to measure adhesion of αvβ6-expressing human HT-29 cells to purified human latency associated peptide, and α5β1-expressing human K562 cells to human plasma fibronectin. In all cell-based assays, binding by the expected integrin was verified by testing activity of corresponding isotype-matched positive (function-blocking) and negative control antibodies. Functions of integrins αvβ1, αvβ8, α2β1 and α10β1 were measured using cell-free receptor-ligand interaction assays using purified recombinant human integrins. Ligands used were human fibronectin for αvβ1, human LAP for αvβ8, bovine collagen II for α2β1, and murine laminin I for α10β1. 96-well plates were coated with the predetermined optimal concentration of ligand overnight, washed 3X with TBS+++ (25 mM Tris pH7.4, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1 mM MnCl2, 1mM CaCl2), and blocked with TBS+++/1%BSA. Purified integrin was diluted in TBS+++/0.1%BSA with or without compounds, and the solution added to empty wells of the washed ligand-coated plate according to a standard template, with each sample repeated in triplicate. After incubation for 2 hr at room temperature, the plate was washed 3X with TBS+++. Biotin-labeled antibody against the αv subunit (αvβ1, αvβ8 assays) or β1 subunit (α2β1, α10β1 assays) was applied for 1 hr. The plate was washed 3X with TBS/0.1%BSA. Streptavidin-conjugated horseradish peroxidase was added to the wells, and the plate incubated for 20 min at room temperature. Following a 3X TBS+++ wash, bound integrin was detected using streptavidin-conjugated horseradish peroxidase and TMB substrate with absorbance measured at 650 nm. For assay of αIIbβ3 (IIbIIIa) function, plates were coated with the purified human integrin overnight, washed 3X with TBS+++, and blocked with TBS+++/1%BSA. Alexa Fluor647-labeled purified human fibrinogen was diluted in TBS+++/0.1%BSA with or without compounds, and the solutions were added to the integrin-coated plate. After 2 hr incubation, the plate was washed 3X with TBS+++, and bound ligand was detected by absorbance measured at 640/668nm.

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Potency for the compounds in blocking cell attachment mediated by integrins αvβ3, αvβ5, and αvβ6 was measured as previously described (Henderson et al., 2013). Potency for blocking cell attachment mediated by integrins αvβ1, αvβ8, and α8β1 was measured using modifications of this method which are briefly summarized as follows: To assess CWHM-12 and CWHM‐680 effects on cellular αvβ1 function, we varied its concentration in an assay measuring binding of HEK‐293 cells, which naturally express this integrin (Nagarajan et al., 2007), to the surface of 96‐well plates coated with purified recombinant human TGFβ‐1 latency‐associated peptide. To assess the effect on cellular αvβ8 function, we performed the assay with the same LAP ligand but with HEK‐293 cells which had been stably transfected to overexpress this integrin. To assess the effect on cellular α8β1 function, we performed the assay using HEK‐293 cells, which had been stably transfected to overexpress this integrin and used purified recombinant mouse nephronectin as the immobilized ligand. To assess the effect on cellular α5β1 function, we performed the assay using K562 cells, which naturally express this integrin and used purified human plasma fibronectin as the immobilized ligand. For all assays except α8β1, the optimal ligand concentration was defined as that providing maximum inhibition of the relevant cell binding by known specific function‐neutralizing antibodies while retaining strong binding in the presence of isotype‐matched negative control antibodies. Because no validated α8‐specific neutralizing antibodies are commercially available, optimization of ligand coating was performed by comparison of attachment of the α8β1‐overexpressing cells to the parental nontransfected cells [2].

Adhesion assay [1]
Control and itgavflox/flox;Pdgfrb-Cre HSCs were cultured for 5 days and then seeded into 48 well tissue culture plates precoated with fibronectin, collagen I, collagen IV, laminin and fibrinogen or BSA treated controls (CytoSelect 48-well adhesion assay, ECM array). Cells were allowed to adhere for 90 min, washed × 3 with PBS and stained and eluted as per manufacturer’s instructions. Adhesion is expressed as a % of unwashed cells adhered to 1% poly-L-lysine.

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Migration assay [1]
Control and itgavflox/flox;Pdgfrb-Cre HSCs were cultured for 5 days and then seeded into the upper chambers of 8 um pore size modified Boyden chambers as per manufacturer’s instructions (CytoSelect 24 well cell migration assay). Fetal calf serum (10%) was added to the lower chamber and cells were allowed to migrate for 6h at 37 °C. Cells remaining in the upper chamber were wiped with a cotton tip and cells attached to the underside of the membrane were fixed, stained and eluted as per manufacturer’s instructions. Chemotaxis is expressed as % of an unwiped control.

In vivo CWHM-12 and CWHM 96 studies [1]
For all studies CWHM-12 and CWHM 96 were solubilized in 50% DMSO (in sterile water) and dosed to 100mg/kg/day. Drug or vehicle (50% DMSO) were delivered by implantable ALZET osmotic minipumps. For CCl4-induced fibrosis, pumps were inserted subcutaneously either before the first dose of CCl4 (prophylactic) or after 3 weeks of treatment (therapeutic) and livers were harvested after 6 weeks. For bleomycin-induced fibrosis pumps were inserted 14 days after treatment with bleomycin or saline and lungs were harvested at 28 days (therapeutic only).

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Kidney injury model [2]
Alzet osmotic minipumps were implanted subcutaneously in 8‐ to 10‐week‐old wild‐type male ICR outbred mice (Envigo) one day prior to induction of kidney injury to deliver vehicle (DMSO/H2O 1:1) or CWHM-12 at a dose of 100 mg/kg per day. A single 5 mg/kg dose of aristolochic acid I sodium salt in PBS was administered intraperitoneally to induce kidney injury, and mice were monitored daily for 27 days thereafter. Control (uninjured) mice were injected with an equal volume of PBS. Blood was obtained by maxillary vein puncture on days 0, 5 (peak injury), and 28 (study endpoint). For testing CWHM‐680, oral gavage (100 mg/kg per day) was started one day prior to injection of aristolochic acid I sodium salt and continued once daily until the study endpoint. Blood was obtained at days 0, 7, and 23 (study endpoint). Animals that did not survive until the end of the study were not included in serum creatinine analysis. Serum creatinine was measured by liquid chromatography–mass spectrometry (LC–MS/MS) at the University of Alabama O’Brien Center Bioanalytical Core. CWHM-12 and CWHM‐680 concentrations were measured in plasma samples by liquid chromatography–tandem mass spectrometry (LC/MS/MS) using compound spiked into control plasma as a standard.

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Dissolved in 50% DMSO (in sterile water); 100 mg/kg/day; s.c.

The mTmG (Td tomato/EGFP) and Ai14 (Rosa-CAG-LSL-tdTomato-WPRE) mice are used and crossed with Pdgfrb-Cre mice. Wild type C57/BL6 mice, Itgavflox/flox mice and itgb8flox/flox mice are used.

[1]. Targeting of αv integrin identifies a core molecular pathway that regulates fibrosis in several organs. Nat Med. 2013 Dec;19(12):1617-24.

[2]. Basta J, Robbins L, Stout L, Prinsen MJ, Griggs DW, Rauchman M. Pharmacologic inhibition of RGD-binding integrins ameliorates fibrosis and improves function following kidney injury. Physiol Rep. 2020;8(7):e14329.

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 α(v) integrin subunit because various α(v)-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 α(v) integrin subunit using this system was protective in other models of organ fibrosis, including pulmonary and renal fibrosis. Pharmacological blockade of α(v)-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 α(v) integrins may have clinical utility in the treatment of patients with a broad range of fibrotic diseases. [1]

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Fibrosis is a final common pathway for many causes of progressive chronic kidney disease (CKD). Arginine-glycine-aspartic acid (RGD)-binding integrins are important mediators of the pro-fibrotic response by activating latent TGF-β at sites of injury and by providing myofibroblasts information about the composition and stiffness of the extracellular matrix. Therefore, blockade of RGD-binding integrins may have therapeutic potential for CKD. To test this idea, we used small-molecule peptidomimetics that potently inhibit a subset of RGD-binding integrins in a murine model of kidney fibrosis. Acute kidney injury leading to fibrosis was induced by administration of aristolochic acid. Continuous subcutaneous administration of CWHM-12, an RGD integrin antagonist, for 28 days improved kidney function as measured by serum creatinine. CWHM-12 significantly reduced Collagen 1 (Col1a1) mRNA expression and scar collagen deposition in the kidney. Protein and gene expression markers of activated myofibroblasts, a major source of extracellular matrix deposition in kidney fibrosis, were diminished by treatment. RNA sequencing revealed that inhibition of RGD integrins influenced multiple pathways that determine the outcome of the response to injury and of repair processes. A second RGD integrin antagonist, CWHM-680, administered once daily by oral gavage was also effective in ameliorating fibrosis. We conclude that targeting RGD integrins with such small-molecule antagonists is a promising therapeutic approach in fibrotic kidney disease. [2]

C26H32BRN5O6

590.47

589.153

C, 52.89; H, 5.46; Br, 13.53; N, 11.86; O, 16.26

1564286-55-0

72949858

White to light brown solid powder

1.5±0.1 g/cm3

1.658

2.06

7

7

10

38

881

1

CC(C)(C)C1=CC(=CC(=C1)[C@H](CC(=O)O)NC(=O)CNC(=O)C2=CC(=CC(=C2)O)NC3=NCC(CN3)O)Br

YDHAGPCZRFQPOI-NRFANRHFSA-N

InChI=1S/C26H32BrN5O6/c1-26(2,3)16-4-14(5-17(27)8-16)21(10-23(36)37)32-22(35)13-28-24(38)15-6-18(9-19(33)7-15)31-25-29-11-20(34)12-30-25/h4-9,20-21,33-34H,10-13H2,1-3H3,(H,28,38)(H,32,35)(H,36,37)(H2,29,30,31)/t21-/m0/s1

(3S)-3-(3-bromo-5-tert-butylphenyl)-3-[[2-[[3-hydroxy-5-[(5-hydroxy-1,4,5,6-tetrahydropyrimidin-2-yl)amino]benzoyl]amino]acetyl]amino]propanoic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.08 mg/mL (3.52 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (3.52 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (3.52 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)