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Purity: ≥98%
LY3000328 is a novel, potent and selective Cathepsin S (Cat S) inhibitor with IC50s of 7.7 and 1.67 nM for hCat S and mCat S, respectively. It has excellent in vitro potency and selectivity against other cysteine proteases. LY3000328 is currently in clinical trials. Cathepsin S (Cat S) plays an important role in many pathological conditions, including abdominal aortic aneurysm (AAA). Inhibition of Cat S may provide a new treatment for AAA.
| Targets |
Human Cathepsin S (hCat S) (IC50 = 7.70 ± 5.85 nM, n = 11) [1]
Mouse Cathepsin S (mCat S) (IC50 = 1.67 ± 1.17 nM, n = 9) [1] |
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| ln Vitro |
Excellent in vitro potency and selectivity are maintained by LY 3000328. LY 3000328 demonstrates low in vitro metabolism in mouse, rat, dog, and human liver microsomes (after 30 min incubation at 4 μM <20%); excellent permeability (MDCK AB>4%); and low in vitro CYP450 inhibition (CYP3A4, CYP2D6, and CYP2C9 at 10 μM <15%). Merely 6% displacement of [3H]-astemizole was seen in experiments employing HEK293 membrane preparations at a dosage of 100 μM for LY 3000328, suggesting a poor potential for hERG blocking [1]. A strong and selective inhibitor of cathepsin S (CatS) is LY 3000328. The inhibition of CatS activity in plasma is 50% of the maximum activity when the concentration of LY 3000328 in plasma is roughly 60 ng/mL [2].
LY3000328 is a potent and selective noncovalent inhibitor of human Cathepsin S (hCat S) with an IC50 of 7.70 ± 5.85 nM (n=11). [1] It also potently inhibits mouse Cathepsin S (mCat S) with an IC50 of 1.67 ± 1.17 nM (n=9). [1] LY3000328 exhibits very high selectivity against other cysteine proteases such as Cathepsin L, K, B, and V in enzyme inhibition assays. [1] The compound is inactive against rat Cathepsin S (rCat S) due to a G137C mutation in rCat S that reduces the size of the S2 pocket, rejecting the binding of inhibitors with larger P2 groups like LY3000328. [1] X-ray protein crystallography of the Cat S/LY3000328 complex reveals that the compound binds to the S2 and S3 subsites of the enzyme without forming any covalent interaction with the active site Cys25 residue. The closest non-hydrogen atom of the inhibitor is 4.3 Å away from the sulfur of Cys25. [1] Key interactions include: a hydrogen bond (2.8 Å) between the benzamide NH of LY3000328 and the carbonyl oxygen of Gly69; a hydrogen bond (3.1 Å) between the carbamate carbonyl oxygen and the NH of Gly69; a hydrogen bond (2.9 Å) between the carbamate NH and the backbone carbonyl of Asn163. The piperazine moiety may have a cation-π interaction with Phe211. [1] |
| ln Vivo |
LY 3000328's effectiveness was investigated using a mouse model of abdominal aortic aneurysm (AAA). In this model, peritoneal surface application of CaCl2 induces inflammation. The findings suggest that this model's disease state features are comparable to those of human AAA. At 1, 3, 10, and 30 mg/kg, LY 3000328 showed a dose-responsive decrease in aortic diameter. Aortic diameter was lowered by 58% at the lowest dosage of LY 3000328—1 mg/kg—83% at 3 mg/kg, and 87% at 10 mg/kg. Good drug disposal properties of LY 3000328 were indicated by the dose-dependent increase in exposure (AUC) of both compounds [1].
In a mouse CaCl2-induced abdominal aortic aneurysm (AAA) model, oral administration of LY3000328 (5) twice daily (BID) for 28 days resulted in a dose-dependent reduction of aortic diameter. At doses of 1, 3, and 10 mg/kg, aortic diameter was reduced by 58%, 83%, and 87%, respectively. [1] The in vivo efficacy (percent reduction in aortic diameter on a per dose basis) of LY3000328 was superior to its analog compound 9, which may be driven by the improved pharmacokinetic properties of LY3000328. [1] |
| Enzyme Assay |
Test compounds were evaluated for their inhibitory activity against human Cathepsin S (hCat S) and mouse Cathepsin S (mCat S) enzymes. The assays measured the concentration of compound required to inhibit 50% of the enzyme activity (IC50). The specific buffer conditions, substrate, and detection method are not detailed in the main text. [1]
Selectivity profiling was conducted against other cysteine proteases including Cathepsin L, K, B, and V using similar enzyme inhibition assays. The results indicated that LY3000328 had very high selectivity over these related enzymes. [1] |
| Animal Protocol |
In Vivo Efficacy Study in AAA Model: A mouse model of abdominal aortic aneurysm (AAA) was induced by applying CaCl2 to the albumenal surface. Mice were treated orally with LY3000328 (5) at doses of 1, 3, 10, and 30 mg/kg, administered twice daily (BID), for a duration of 28 days. Aortic diameter was measured to assess treatment efficacy. [1]
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| ADME/Pharmacokinetics |
In preclinical pharmacokinetic assessments in dogs, LY3000328 exhibited high oral bioavailability (F > 75%), low clearance (CL < 4 mL/min/kg), low volume of distribution (Vd < 1 L/kg), and a relatively short half-life. [1] In rats, LY3000328 was cleared 3-fold less than its analog compound 9, while exposure was 3-fold higher. [1] In mice, rats, dogs, and human liver microsomes, LY3000328 was metabolized at a low rate in vitro (<20% after 30 min of incubation at 4 µM). [1] The compound showed good permeability in the MDCK cell monolayer permeation assay (AB > 4%). [1]
LY3000328 showed low inhibitory activity against major CYP450 enzymes (CYP3A4, CYP2D6, CYP2C9) in vitro, with an inhibition rate of less than 15% at a concentration of 10 µM. [1] In mouse pharmacodynamic studies, the exposure (AUC) of LY3000328 increased in a dose-dependent manner. [1] |
| Toxicity/Toxicokinetics |
In tests performed on HEK293 cell membrane preparations, 100 µM LY3000328 replaced only 6% of the [³H]-astemizole binding, indicating a low potential for blocking hERG channels. [1]
After incubation in human or rat plasma for up to 30 minutes, the loss of the parent compound LY3000328 was not statistically significant, indicating good plasma stability. [1] |
| References | |
| Additional Infomation |
LY3000328 (compound 5) is a novel non-peptide, non-covalent cathepsin S inhibitor. It does not contain an electrophilic center and does not form a covalent bond with the active site cysteine (Cys25), which distinguishes it from many earlier cathepsin S inhibitors. [1]
It was discovered through medium-throughput focusing cassette screening and optimized using structure-based drug design with X-ray crystallography of the inhibitor-enzyme complex. [1] The primary therapeutic indication explored in this study is abdominal aortic aneurysm (AAA). [1] Based on its in vitro potency, selectivity, good drug distribution characteristics, and in vivo efficacy in the AAA model, LY3000328 was selected as a candidate for clinical development. [1] LY3000328 has completed a Phase I single-dose escalation study in healthy subjects. [1] |
| Molecular Formula |
C24H28N2O5.H2O
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|---|---|
| Molecular Weight |
442.505
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| Exact Mass |
484.212
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| CAS # |
1373215-15-6
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| PubChem CID |
67475270
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| Appearance |
White to off-white solid powder
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| Density |
1.4±0.1 g/cm3
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| Boiling Point |
701.6±60.0 °C at 760 mmHg
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| Flash Point |
378.1±32.9 °C
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| Vapour Pressure |
0.0±2.2 mmHg at 25°C
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| Index of Refraction |
1.639
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| LogP |
1.44
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
35
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| Complexity |
739
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| Defined Atom Stereocenter Count |
2
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| SMILES |
CNC(=O)O[C@H]1COC2=C([C@@H]1NC(=O)C3=CC=C(C=C3)F)C=C(C=C2)N4CCN(CC4)C5COC5
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| InChi Key |
NDEBZCZEAVMSQF-GOTSBHOMSA-N
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| InChi Code |
InChI=1S/C25H29FN4O5/c1-27-25(32)35-22-15-34-21-7-6-18(29-8-10-30(11-9-29)19-13-33-14-19)12-20(21)23(22)28-24(31)16-2-4-17(26)5-3-16/h2-7,12,19,22-23H,8-11,13-15H2,1H3,(H,27,32)(H,28,31)/t22-,23-/m0/s1
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| Chemical Name |
(3R,4S)-4-(4-fluorobenzamido)-6-(4-(oxetan-3-yl)piperazin-1-yl)chroman-3-yl methylcarbamate
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| Synonyms |
LY3000328; LY-3000328; LY 3000328
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ≥ 50 mg/mL (~103.19 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.16 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 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (5.16 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 25.0 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (5.16 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.2598 mL | 11.2992 mL | 22.5984 mL | |
| 5 mM | 0.4520 mL | 2.2598 mL | 4.5197 mL | |
| 10 mM | 0.2260 mL | 1.1299 mL | 2.2598 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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