In cultured rat hippocampal slices, 1 µM CA-074 reduced K+-stimulated MMP-associated fluorescence, whereas its effect under basal conditions was not significant. This extracellular signaling readout used a broad-MMP substrate and does not establish direct MMP inhibition or an in vivo treatment effect. [2]
pH-dependent inhibition of recombinant human cathepsin B
Biochemical assay · Recombinant human cathepsin B; Z-Phe-Arg-AMC fluorogenic assay at pH 4.6, 5.5 and 7.2 [1]
CA-074 potency decreased as assay pH increased. The methyl ester comparator was much less potent and did not show the same pH preference.
Literature-Reported Treatment Conditions
- Treatment concentration
- CA-074 concentration series: 4096–0.5 nM (2-fold dilution) or 144–0.5 nM (1.5-fold dilution), as reported in shared kinetic methods.
- Exposure duration
- No inhibitor preincubation; 30-min assay at 25°C.
- Method
- 0.04 ng/µL cathepsin B and 40 µM Z-Phe-Arg-AMC; fluorescence at 360/460 nm; shared methods specify quadruplicate assays.
- Vehicle
- 2% (v/v) DMSO in vehicle control.
- Reported limits
- The methods give two concentration-series designs without allocating each series to each plotted pH curve. IC50 values have no uncertainty attached in the figure/table.
Methods and source locations
Compound reported in the paper: CA-074; Compound label in the paper: CA-074 (free carboxyl form)
Compound-level evidence; no inference of Adooq lot identity, purity or performance.
Figure 2a–b and caption; Table 1 IC50 row; Methods: Inhibitors, enzymes, peptides, and reagents; Cathepsin B activity assay; Inhibition of cathepsin B by CA-074 and CA-074Me in kinetic studies
Cathepsin B activity at pH 4.6: Z-Phe-Arg-AMC activity relative to no-inhibitor vehicle control. — Figure 2a.ii; Table 1, CA-074 IC50 row, pH 4.6
Cathepsin B activity at pH 5.5: Same reported fluorogenic assay at pH 5.5. — Figure 2a.ii; Table 1, CA-074 IC50 row, pH 5.5
Cathepsin B activity at pH 7.2: Same reported fluorogenic assay at pH 7.2. — Figure 2a.ii; Table 1, CA-074 IC50 row, pH 7.2
CA-074Me comparator inhibition of cathepsin B: Methyl ester comparator assayed separately; these are not CA-074 potencies. — Figure 2b.ii; Table 1, CA-074Me IC50 row
Persistent inhibition after dilution
Biochemical assay · Recombinant human cathepsin B at pH 4.6, 5.5 and 7.2; dilution followed by fluorogenic activity monitoring [1]
CA-074-treated enzyme did not recover activity after dilution, whereas the no-inhibitor control generated product. This supports an irreversible inhibition mechanism under the tested conditions.
Literature-Reported Treatment Conditions
- Treatment concentration
- Preincubation: 58 nM at pH 4.6, 440 nM at pH 5.5, 7230 nM at pH 7.2 (reported as 10× IC50).
- Exposure duration
- 30-min preincubation, then dilution and time-course monitoring through 120 min in Figure 3.
- Method
- 100-fold dilution to 0.1× IC50, followed by addition of Z-Phe-Arg-AMC; activity compared with no-inhibitor control.
- Vehicle
- DMSO control identified in Figure 3.
- Reported limits
- Figure 3 does not attach a separate replicate count or statistical test to the dilution experiment; shared general assays are described as quadruplicate. The 58 nM value reflects a less-rounded IC50 than the 6 nM summary.
Methods and source locations
Compound reported in the paper: CA-074; Compound label in the paper: CA-074 (free carboxyl form)
Compound-level evidence; no inference of Adooq lot identity, purity or performance.
Figure 3a–c and caption; Results: Irreversible mechanism of CA-074 inhibition at acidic and neutral pH conditions; Methods: Irreversible inhibition mechanism and Cathepsin B activity assay
Cathepsin B activity following 100-fold dilution: Dilution challenge followed by Z-Phe-Arg-AMC fluorescence time courses. — Figure 3a–c, caption and corresponding Results subsection
Activity-dependent modulation of MMP signaling in hippocampal slices
Cell-based study · Cultured hippocampal slices from male Wistar rats; extracellular broad-MMP fluorogenic readout under basal or 30 mM K+ conditions [2]
CA-074 reduced K+-stimulated MMP-associated fluorescence but had no significant effect under basal conditions. This is an ex vivo tissue assay, not administration to living rats and not a direct cathepsin B potency measurement.
Literature-Reported Treatment Conditions
- Treatment concentration
- 1 µM CA-074.
- Exposure duration
- 2-h substrate incubation; supplement describes 37°C and 5% CO2 for this assay.
- Method
- Cultured slices incubated with 5 µM PEPDAB0502; fluorescence in supernatant measured at 480/520 nm after blank subtraction.
- Vehicle
- CA-074 dissolved in DMSO; final DMSO fraction not specified in the read assay/drug sections.
- Co-treatment
- Basal and 30 mM K+ conditions compared separately with and without CA-074.
- Reported limits
- No product catalog number or lot validation provided for CA-074 in the read drug list. The general 10-min drug application note is for imaging and is not assigned to this 2-h plate-reader assay. Number of donor rats and donor independence of slices are not established by the reported n.
Methods and source locations
Compound reported in the paper: CA-074; Compound label in the paper: CA-074 (free carboxyl form)
Compound-level evidence; no inference of Adooq lot identity, purity or performance.
Results: Cathepsin B Release Activates MMP-9, first paragraph; Figure 6F and caption; Experimental Procedures: Hippocampal Tissue Preparation, Cathepsin B and MMP Assays, Statistical Analysis; Supplemental Experimental Procedures pp. 18–19: MMP activity assay and Drugs
Basal MMP-associated supernatant fluorescence with/without CA-074: Broad-MMP fluorogenic substrate PEPDAB0502, 2-h slice incubation; no significant basal treatment effect. — Results: Cathepsin B Release Activates MMP-9, first paragraph; Figure 6F; Statistical Analysis
K+-stimulated MMP-associated fluorescence with/without CA-074: Same broad-MMP fluorogenic assay under 30 mM K+ stimulation; ANOVA/post hoc Bonferroni comparisons. — Results: Cathepsin B Release Activates MMP-9, first paragraph; Figure 6F and caption; Supplemental MMP activity assay; Statistical Analysis
