caspase-2/3

Several caspases have been implicated in the pathogenesis of Huntington's disease (HD); however, existing caspase inhibitors lack the selectivity required to investigate the specific involvement of individual caspases in the neuronal cell death associated with HD. In order to explore the potential role played by caspase-2, the potent but non-selective canonical AAc-VDVAD-CHO caspase-2 inhibitor 1 was rationally modified at the P(2) residue in an attempt to decrease its activity against caspase-3. With the aid of structural information on the caspase-2, and -3 active sites and molecular modeling, a 3-(S)-substituted-l-proline along with four additional scaffold variants were selected as P(2) elements for their predicted ability to clash sterically with a residue of the caspase-3 S(2) pocket. These elements were then incorporated by solid-phase synthesis into pentapeptide aldehydes 33a-v. Proline-based compound 33h bearing a bulky 3-(S)-substituent displayed advantageous characteristics in biochemical and cellular assays with 20- to 60-fold increased selectivity for caspase-2 and ∼200-fold decreased caspase-3 potency compared to the reference inhibitor 1. Further optimization of this prototype compound may lead to the discovery of valuable pharmacological tools for the study of caspase-2 mediated cell death, particularly as it relates to HD. [1]

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The molecular basis for the substrate specificity of human caspase-3 has been investigated using peptide analog inhibitors and substrates that vary at the P2, P3, and P5 positions. Crystal structures were determined of caspase-3 complexes with the substrate analogs at resolutions of 1.7 A to 2.3 A. Differences in the interactions of caspase-3 with the analogs are consistent with the Ki values of 1.3 nM, 6.5 nM, and 12.4 nM for Ac-DEVD-Cho, AAc-VDVAD-CHO and Ac-DMQD-Cho, respectively, and relative kcat/Km values of 100%, 37% and 17% for the corresponding peptide substrates. The bound peptide analogs show very similar interactions for the main-chain atoms and the conserved P1 Asp and P4 Asp, while interactions vary for P2 and P3. P2 lies in a hydrophobic S2 groove, consistent with the weaker inhibition of Ac-DMQD-Cho with polar P2 Gln. S3 is a surface hydrophilic site with favorable polar interactions with P3 Glu in Ac-DEVD-Cho. Ac-DMQD-Cho and AAc-VDVAD-CHO have hydrophobic P3 residues that are not optimal in the polar S3 site, consistent with their weaker inhibition. A hydrophobic S5 site was identified for caspase-3, where the side-chains of Phe250 and Phe252 interact with P5 Val of AAc-VDVAD-CHO, and enclose the substrate-binding site by conformational change. The kinetic importance of hydrophobic P5 residues was confirmed by more efficient hydrolysis of caspase-3 substrates Ac-VDVAD-pNA and Ac-LDVAD-pNA compared with Ac-DVAD-pNA. In contrast, caspase-7 showed less efficient hydrolysis of the substrates with P5 Val or Leu compared with Ac-DVAD-pNA. Caspase-3 and caspase-2 share similar hydrophobic S5 sites, while caspases 1, 7, 8 and 9 do not have structurally equivalent hydrophobic residues; these caspases are likely to differ in their selectivity for the P5 position of substrates. The distinct selectivity for P5 will help define the particular substrates and signaling pathways associated with each caspase. [2]

The crystal structures of caspase-3 in complex with the three substrate analog inhibitors Ac-DEVD-Cho, Ac-DMQD-Cho and AAc-VDVAD-CHO were determined. The crystallographic statistics are summarized in Table 1. The crystal structures were refined to resolutions of 1.7–2.3 Å and R-factors of 18.4–21.0. The structures of caspase-3/DMQD and caspase-3/VDVAD have not been reported, while our structure of caspase-3/DEVD was determined at a significantly higher resolution of 1.7 Å compared to 2.5 Å for...

[1]. Exploiting differences in caspase-2 and -3 S₂ subsites for selectivity: structure-based design, solid-phase synthesis and in vitro activity of novel substrate-based caspase-2 inhibitors. Bioorg Med Chem. 2011 Oct 1;19(19):5833-51.

[2]. Structural and kinetic analysis of caspase-3 reveals role for s5 binding site in substrate recognition. J Mol Biol. 2006 Jul 14;360(3):654-66.

In order to improve our knowledge of the molecular basis for substrate specificity, the crystal structures were determined of complexes of recombinant human caspase-3 with the peptide analogs Ac-DEVD-Cho, Ac-DMQD-Cho and Ac-VDVAD-Cho. The complex with Ac-DEVD-Cho was obtained at the significantly higher resolution of 1.70 Å compared to 2.5 Å for the previously reported structure.13 These complexes explore the structural basis of specificity for positions P2 and P3 in the peptide substrates, and examine the effect of adding the P5 residue. The observed caspase-3 interactions with inhibitors were analyzed together with kinetic data for peptide substrates and inhibitors of similar sequences. These new structures with peptide analog inhibitors will help in the design of new caspase-3 inhibitors as potential therapeutic agents for neurodegenerative diseases.[2]

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Further substantiating these findings is the efficacy of broad-spectrum peptide-based caspase inhibitors in preventing HTT cleavage and reducing toxicity in vitro7(b), 10 and in HD animal models.11 However, the lack of selective caspase inhibitors has hampered further progress in understanding the specific involvement of individual caspases in mHTT-induced apoptosis or mHTT cleavage; consequently, we set out to develop these selective inhibitors. Selective Csp-1,12 Csp-312(a), 13 and Csp-713a inhibitors have been disclosed; however, for Csp-2 and Csp-6, the two leading caspase targets for HD, there are no reports to our knowledge of selective inhibitors. In order to be able to rapidly test our inhibitor design strategy, we decided to focus our effort initially on the development of Csp-2 inhibitors with reduced activity for Csp-3. We report here the successful application of structure-based design to the development of potent inhibitors of Csp-2 with improved selectivity. We discuss the rationale for targeting the S2 pocket and choosing proline-based templates as the P2 element. Synthesis of these key P2 scaffolds and their incorporation into pentapeptide aldehydes via solid-phase chemistry are presented along with a detailed analysis of the biochemical and cellular selectivity of these new inhibitors.[1]

C23H37N5O10

543.57

543.254

194022-51-0

9850347

Ac-Val-Asp-Val-Ala-Asp-al; Ac-Val-Asp-Val-Ala-Asp-CHO; N-acetyl-L-valyl-L-alpha-aspartyl-L-valyl-L-alanyl-L-aspart-1-al

Ac-VDVAD-CHO; VDVAD

Typically exists as solid at room temperature

1.3±0.1 g/cm3

996.6±65.0 °C at 760 mmHg

556.5±34.3 °C

0.0±0.6 mmHg at 25°C

1.520

0.42

7

10

16

38

920

5

CC([C@H](NC(C)=O)C(N[C@H](C(N[C@H](C(N[C@H](C(N[C@H](C=O)CC(O)=O)=O)C)=O)C(C)C)=O)CC(O)=O)=O)C

OOGHGBCRVSBUHH-GOYXDOSHSA-N

InChI=1S/C23H37N5O10/c1-10(2)18(25-13(6)30)23(38)27-15(8-17(33)34)21(36)28-19(11(3)4)22(37)24-12(5)20(35)26-14(9-29)7-16(31)32/h9-12,14-15,18-19H,7-8H2,1-6H3,(H,24,37)(H,25,30)(H,26,35)(H,27,38)(H,28,36)(H,31,32)(H,33,34)/t12-,14-,15-,18-,19-/m0/s1

(3S)-3-[[(2S)-2-acetamido-3-methylbutanoyl]amino]-4-[[(2S)-1-[[(2S)-1-[[(2S)-1-carboxy-3-oxopropan-2-yl]amino]-1-oxopropan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-4-oxobutanoic acid

Ac-VDVAD-CHO; 194022-51-0; Ac-Val-Asp-Val-Ala-Asp-aldehyde (pseudo acid); (3S)-3-[[(2S)-2-acetamido-3-methylbutanoyl]amino]-4-[[(2S)-1-[[(2S)-1-[[(2S)-1-carboxy-3-oxopropan-2-yl]amino]-1-oxopropan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-4-oxobutanoic acid; Ac-VDVAD-CHO (trifluoroacetate salt); MFCD01862574; SCHEMBL20257125; DTXSID80431757;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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