Plasminogen activator inhibitor type 1 (PAI-1) – binds to latent PAI-1 with KD of 0.29 μM (ITC at 35°C) and 0.19 μM (SPR at 25°C); no measurable binding to active PAI-1 [1]

In a chromogenic assay, AZ3976 inhibited PAI-1 with an IC50 of 26 μM and showed no effect on tPA alone at 100 μM [1].

In a human plasma clot lysis assay with added PAI-1, AZ3976 exhibited profibrinolytic activity with an IC50 of 16 μM [1].

At 100 μM, AZ3976 did not inhibit PAI-1 in the presence of equimolar concentration of vitronectin (VN) in the chromogenic assay [1].

AZ3976 showed no inhibitory effect on rat PAI-1 at 100 μM in a chromogenic assay [1].

High throughput screening chromogenic assay: Compounds were pre-dispensed in 384-well plates. 10 μL of 48 nM PAI-1 (final 12 nM) in assay buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 0.01% Tween 20, 0.1% BSA) was added and incubated for 15 min at room temperature. Then 20 μL of 16 nM tPA (final 8 nM) was added and incubated for another 20 min. Subsequently, 10 μL of chromogenic substrate Pefachrome tPA (final 250 μM) was added. Residual tPA activity was determined from absorbance at 405 nm measured at 0 and 25 min. Percent effect was calculated relative to diaplasin (max effect) and DMSO (min effect). IC50 was calculated by non-linear regression using a four-parameter sigmoidal fit [1].

Isothermal titration calorimetry (ITC): Titrations were performed at 35°C. For latent PAI-1, 50 μM glycosylated latent PAI-1 in 50 mM sodium phosphate pH 7.4, 100 mM NaCl, 2% DMSO was titrated with 1 mM AZ3976 using 20 × 1 μL injections. For active PAI-1 preparation (75% specific activity), 45 μM active PAI-1 was titrated with 1 mM AZ3976 using 20 × 0.5 μL injections. Waiting time between injections was 90 s. Data were fitted to a single-site binding model to obtain KD, ΔG, ΔH, -TΔS, and stoichiometry (n) [1].

Surface plasmon resonance (SPR) direct binding: AZ3976 was diluted from 100 mM DMSO stock into PBSTD buffer (50 mM sodium phosphate, 100 mM NaCl pH 7.4, 0.005% Tween 20, 1% DMSO) to concentrations ranging from 20 μM to 27.4 nM (3-fold dilution). The compound was injected over immobilized glycosylated latent or active PAI-1 on a CM5 sensor chip at 25°C with a flow rate of 30 μL/min. Association was monitored for 90 s, dissociation for 4 min. No regeneration was needed. Data were fitted to a 1:1 binding model to determine kon, koff, KD, and Rmax [1].

SPR competition experiments to study latency transition: Active PAI-1 (10 nM) was preincubated with various concentrations of AZ3976 at 20°C for increasing time intervals, then injected over immobilized vitronectin (VN) or captured tPA. The initial binding rate (ΔRU/Δt over first 10 s) was measured as a function of incubation time. The decay of active PAI-1 binding followed a mono-exponential function. The rate constant increased linearly with compound concentration, yielding a second-order rate constant of 14 M⁻¹ s⁻¹ at 20°C [1].

SPR using antibody H4B3 (specific for latent non-glycosylated PAI-1): Non-glycosylated active PAI-1 (10 nM) was incubated with 20 μM AZ3976 for 1 h at 20°C, then injected over captured H4B3. Binding to H4B3 increased dramatically upon compound incubation, correlating with loss of activity toward tPA [1].

X-ray crystallography: Latent PAI-1 was co-crystallized with AZ3976, and the structure was solved at 2.4 Å resolution by molecular replacement using PDB code 1DVN. The ligand was bound in a cavity defined by α-helices A, B, D, E, β-strands 1A and 2A, and connecting loops. Key interactions included hydrogen bonds with Tyr-37, Asp-95 (three bonds), Arg-76, and a π-stacking interaction with Tyr-79 [1].

Human plasma clot lysis assay: Plasma was thawed and incubated at 37°C for ~1 h. Compounds were dispensed in 96-well plates (10-point concentration, 2-fold dilution, highest final concentration 82 μM). 45 μL of assay buffer (10 mM Hepes pH 7.4, 0.15 M NaCl, 33.3 mM CaCl₂) was added. Then 45 μL of plasma containing tPA (final tPA concentration 1.2 nM) and PAI-1 (final PAI-1 concentration 0.65 nM) was added to start the reaction. Fibrin formation was monitored by absorbance at 405 nm at 2-min intervals at 37°C for up to 10 h. Clot lysis time was defined as the time to halve the amplitude on the negative slope minus the time to halve the amplitude on the positive slope. Maximal effect (100%) was the lysis time without added PAI-1, minimal effect (0%) the lysis time without compound. IC50 was calculated by non-linear regression [1].

[1]. Characterization of a small molecule inhibitor of plasminogen activator inhibitor type 1 that accelerates the transition into the latent conformation. J Biol Chem. 2013 Jan 11;288(2):873-85.

AZ3976 is a small molecule PAI-1 inhibitor identified from a high throughput screening campaign. It is a neutral molecule with overall drug-like properties [1].

Mechanism of action: AZ3976 does not bind to active PAI-1 but binds to latent PAI-1 with submicromolar affinity. It accelerates the latency transition of active PAI-1, presumably by binding to a prelatent form of PAI-1 that exists in equilibrium with active PAI-1. The compound increased the rate of conversion from active to latent PAI-1 by approximately 23-fold at 20°C (t₁/₂ decreased from 15 h in buffer to 40 min in the presence of 20 μM AZ3976) [1].

Species specificity: AZ3976 showed no effect on rat PAI-1 at 100 μM in the chromogenic assay, and five residues within 5 Å of the bound ligand differ between human and rat [1].

Vitronectin protection: VN protects PAI-1 from the neutralizing effect of AZ3976; the compound did not inhibit the PAI-1-VN complex in the chromogenic assay nor disrupt the complex in SPR experiments [1].

Binding site: The binding pocket is located between α-helix D and β-strand 2A, approximately 20 Å away from glycosylation sites Asn-209 and Asn-265. This region is referred to as the flexible joint region [1].

Comparison with other inhibitors: Unlike other small molecule PAI-1 inhibitors (e.g., PAI-039, AR-H029953xx), AZ3976 binds preferentially to latent PAI-1 and inhibits by accelerating latency transition [1].

Potential therapeutic implication: Since most circulating PAI-1 in plasma is complexed with VN (which protects from AZ3976), the compound may be more effective against intracellular or platelet-derived PAI-1 (which is not VN-bound). Chronic administration could potentially inactivate PAI-1 stored in platelets before its release [1].

C15H19N5O3

317.34

317.148

1418747-15-5

135566649

White to off-white solid powder

1.42±0.1 g/cm3

490.3±55.0 °C

0.5

2

5

4

23

522

0

CC(C)(C)OC(=O)N1CC(C1)NC2=NC3=C(C=CC=N3)C(=O)N2

CKUDSTVQYFRZJW-UHFFFAOYSA-N

InChI=1S/C15H19N5O3/c1-15(2,3)23-14(22)20-7-9(8-20)17-13-18-11-10(12(21)19-13)5-4-6-16-11/h4-6,9H,7-8H2,1-3H3,(H2,16,17,18,19,21)

1-Boc-3-(4-Oxo-3,4-dihydro-pyrido[2,3-d]pyrimidin-2-ylamino)-azetidine

AZ-3976 AZ 3976 AZ3976

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)

DMSO : ~100 mg/mL (~315.12 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.88 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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 25.0 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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 (Please use freshly prepared in vivo formulations for optimal results.)