Cdk5/p25 (IC50 = 1 nM); CDK5/p35 (IC50 = 1 nM); Cdk1/cyclin B (IC50 = 2 nM); cdk2/cyclin A (IC50 = 2 nM); CDK2/cyclinE (IC50 = 5 nM); CDK9/cyclinT1 (IC50 = 15 nM); CDK3/Cyclin E (IC50 = 42 nM); cdk6/cyclin D3 (IC50 = 187 nM); CDK7/Cyclin H/MAT1 (IC50 = 3564 nM)
LCQ195 potently inhibits CDK 1, 2 and 5 activity and also modestly targets CDK3 and 9. These distinct patterns of inhibition of CDKs correlate with different patterns of activity against MM cells. [1]
EC50 values with seliciclib for most MM cell lines are >10 μM compared to ~1 μM or lower for similar cell lines treated with LCQ195. [1]
Signaling through the Rb pathway is reduced by LCQ195, but remains largely unaffected by flavopiridol. In addition, the anti-MM activity of LCQ195 does not involve the release of cytochrome c and is not suppressed by the over-expression of bcl-2. LCQ195, therefore, appears to operate through mitochondrial-independent mechanisms of apoptosis.[1]

LCQ195 potently inhibits CDK 1, 2 and 5 activity and also modestly targets CDK3 and 9. These distinct patterns of inhibition of CDKs correlate with different patterns of activity against MM cells. [1]
EC50 values with seliciclib for most MM cell lines are >10 μM compared to ~1 μM or lower for similar cell lines treated with LCQ195. [1]
Signaling through the Rb pathway is reduced by LCQ195, but remains largely unaffected by flavopiridol. In addition, the anti-MM activity of LCQ195 does not involve the release of cytochrome c and is not suppressed by the over-expression of bcl-2. LCQ195, therefore, appears to operate through mitochondrial-independent mechanisms of apoptosis.[1]

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NVP-LCQ-195 inhibited the viability of a panel of human multiple myeloma (MM) cell lines in vitro with EC50 values around 1 µM or lower for many cell lines (e.g., S6B45: 0.761 µM, OPM2: <1 µM, KMS-12-BM: 0.737 µM, U266: 0.817 µM). Some cell lines showed higher EC50 values (e.g., Dox40: 2.810 µM). [1]
NVP-LCQ-195 reduced the viability of purified primary CD138+ tumor cells from MM patients in a dose-dependent manner (0-4 µM for 48 hours). [1]
Cell death commitment assays showed that exposure of sensitive MM cell lines (e.g., OPM-2, MM.1S) to NVP-LCQ-195 (0.5 µM) for as little as 8-16 hours was sufficient to commit cells to death upon subsequent incubation in drug-free medium. [1]
NVP-LCQ-195 exhibited selective cytotoxicity towards MM cells compared to non-malignant cells. The calculated EC50 values were >4 µM for non-malignant HS-5 bone marrow stromal cells (BMSCs) and immortalized THLE-3 hepatocytes. PHA-stimulated peripheral blood mononuclear cells (PBMCs) from healthy donors showed reduced sensitivity compared to most MM cell lines. [1]
NVP-LCQ-195 (at concentrations up to 2 µM) overcame the proliferative and protective effects on MM.1S cells conferred by interleukin-6 (IL-6, 10 ng/mL), insulin-like growth factor-1 (IGF-1, 50 ng/mL), or co-culture with HS-5 stromal cells. [1]
Treatment of MM.1S cells with NVP-LCQ-195 (2 µM) induced cell cycle arrest, characterized by an early increase in the percentage of cells in S and G2/M phases (within 4-16 hours), followed by an increase in the sub-G1 population (indicative of cell death) at later time points (48 hours). [1]
NVP-LCQ-195 (2 µM) induced apoptosis in MM.1S cells, as evidenced by time-dependent increases in Annexin V-positive cells (early apoptotic: Annexin V+/PI-; late apoptotic/dead: Annexin V+/PI+). [1]
Immunoblotting analysis revealed that NVP-LCQ-195 treatment (2 µM) led to cleavage of caspase-3, caspase-8, and PARP, but not caspase-9. It also caused a pronounced decrease in Rb phosphorylation, decreased levels of CDK2, CDK5, cyclin B1, cyclin D2, cyclin E2, cdc25, E2F1, and increased levels of p53, p21, p27, Puma, and Noxa. No changes in Bcl-2, Bad, or Bax protein levels were observed. [1]
Cytochrome c release was not detected in MM.1S cells treated with NVP-LCQ-195 (2 µM) for up to 16 hours. Overexpression of Bcl-2 in MM.1S cells did not significantly affect their sensitivity to NVP-LCQ-195, suggesting a mitochondrial-independent mechanism of apoptosis. [1]
NVP-LCQ-195 demonstrated synergistic in vitro anti-MM activity when combined with dexamethasone (Dex) across multiple dose combinations (Combination Index, CI < 1 for most conditions). No significant synergy or antagonism was observed with bortezomib or doxorubicin. [1]
Gene expression profiling of MM.1S cells treated with NVP-LCQ-195 (2 µM) showed suppression of transcripts associated with oncogenesis (e.g., MYC, IRF4, XBP-1), drug resistance (e.g., HIF1A), growth factor signaling, and anti-apoptosis (e.g., XIAP, survivin), while inducing some pro-apoptotic genes (e.g., TRAIL-R2/DR5). [1]
Pathway analysis indicated that NVP-LCQ-195 treatment decreased the amplitude of transcriptional signatures related to the activity of key transcription factors (MYC, HIF-1α, IRF4, NF-κB), signaling pathways (Notch, Ras, Akt, Hedgehog), the ubiquitin-proteasome system, ribosome biogenesis, cancer stem cell self-renewal, and high-risk MM. [1]
A transcriptional index derived from early (2-hour) gene expression changes induced by NVP-LCQ-195 in MM.1S cells was applied to clinical datasets. Bortezomib-treated MM patients whose tumors had high baseline expression of genes suppressed by NVP-LCQ-195 had significantly shorter progression-free survival (PFS) and overall survival (OS) compared to patients with low expression of these genes. [1]

Kinases involved in cell cycle were screened using the Invitrogen Z’-LYTE™ technology. Selected kinases were in the presence of 500 nM of LCQ195 (in 1% DMSO) and 100 μM ATP.[1]

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In vitro kinase activity assays were performed using a fluorescence resonance energy transfer (FRET)-based peptide phosphorylation detection platform. Selected kinases were incubated in the presence of 500 nM NVP-LCQ-195 (in 1% DMSO) and 100 µM ATP. The assay involves the transfer of the gamma-phosphate of ATP to a tyrosine, serine, or threonine residue on a synthetic FRET-peptide. A secondary reaction uses a site-specific protease that cleaves only non-phosphorylated FRET-peptides. The ratio of donor to acceptor fluorophore emission after donor excitation is used to quantitate kinase activity, with inhibition indicated by a shift in this ratio. [1]
A separate kinase profiling platform involved incubating various recombinant CDK/cyclin complexes with a 5-log dose titration of NVP-LCQ-195 (0.001-10 µM) and ATP. Kinase activity was measured, and dose-response curves were generated to calculate IC50 values for each kinase. [1]

The minimum exposure of MM cells to LCQ195 that is required to commit them to death was evaluated by incubating cells in 24-well plates with LCQ195 (2 μM) for 1-24 hrs.[1]
The MM cell line MM.1S was treated with 2 μM LCQ195 for 0-48 hrs. [1]
MM.1S cells were treated with 2 μM LCQ195 or with DMSO as a control for 2, 4, 8, 16, and 24 hrs; total RNA was then extracted and purified, cDNA synthesized and cRNA labeled prior to hybridization to the HT-U133 A and B Arrays. [1]

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Cell Viability (MTT) Assay: MM cell lines or primary cells were plated in multi-well plates. NVP-LCQ-195 (dissolved in DMSO) was added at indicated concentrations (typically 0-4 µM). After 48 or 72 hours of incubation, MTT reagent was added for the final 4 hours. The resulting formazan crystals were dissolved using an acidified organic solvent, and the optical absorbance was measured spectrophotometrically at 570/630 nm. Cell survival was expressed as a percentage relative to vehicle-treated controls. [1]
Cell Death Commitment Assay: MM cells were incubated with a fixed concentration of NVP-LCQ-195 (e.g., 0.5 or 2 µM) for varying durations (1-24 hours). Cells were then washed with drug-free medium to remove residual compound and incubated in drug-free medium for an additional period (e.g., 3 days) to allow commitment to death to manifest. Survival was quantified using the MTT assay. [1]
Flow Cytometry for Cell Cycle Analysis: Treated and control cells were harvested, fixed in 70% ethanol, treated with RNase, and stained with propidium iodide (PI). DNA content was analyzed using a flow cytometer to determine the distribution of cells in G0/G1, S, and G2/M phases, as well as the sub-G1 (apoptotic) population. [1]
Flow Cytometry for Apoptosis (Annexin V/PI Staining): Cells were harvested, washed, and resuspended in a binding buffer. They were then stained with fluorescein isothiocyanate-conjugated Annexin V and PI according to the kit instructions. The stained cells were analyzed by flow cytometry to distinguish live (Annexin V-/PI-), early apoptotic (Annexin V+/PI-), late apoptotic/necrotic (Annexin V+/PI+), and dead (Annexin V-/PI+) cells. [1]
Cytochrome C Release Assay: Treated cells were collected, washed, and permeabilized using a dedicated buffer. Cells were fixed, washed, and stained with an antibody specific for cytochrome c that remains associated with mitochondria unless released. The intracellular fluorescence signal was measured by flow cytometry to assess cytochrome c release. [1]
Western Blotting: Treated cells were lysed in a buffer containing detergents and protease/phosphatase inhibitors. Protein concentration was determined. Equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis, transferred to a membrane, and blocked. Membranes were incubated with specific primary antibodies overnight, followed by incubation with horseradish peroxidase-conjugated secondary antibodies. Protein bands were visualized using enhanced chemiluminescence. [1]
Stromal Co-culture Assay: Bone marrow stromal cells (e.g., HS-5) were plated and allowed to adhere overnight. Luciferase-expressing MM cells were then added to the wells and treated with NVP-LCQ-195. After incubation, luciferin substrate was added, and the resulting bioluminescence signal, proportional to viable MM cell number, was measured using a luminometer. [1]
Gene Expression Profiling: MM cells were treated with NVP-LCQ-195 or vehicle for various durations. Total RNA was extracted, purified, and used for cDNA synthesis, followed by cRNA labeling and hybridization to oligonucleotide microarrays (e.g., Affymetrix HT-U133 A&B arrays). Data were analyzed to identify differentially expressed genes. [1]

This study showed that, at in vitro tested concentrations, NVP-LCQ-195 was more cytotoxic to multiple myeloma (MM) cells than to non-malignant cells (HS-5 stromal cells, THLE-3 hepatocytes, and PHA-stimulated PBMCs). However, specific toxicity data from in vivo studies (e.g., LD50, organ toxicity) were not provided. [1]

[1]. Molecular and cellular effects of multi-targeted cyclin-dependent kinase inhibition in myeloma: biological and clinical implications. Br J Haematol. 2011 Feb;152(4):420-32.

NVP-LCQ-195 (also known as AT9311) is a small molecule non-chiral heterocyclic kinase inhibitor discovered by Astex Therapeutics and developed in collaboration with Novartis. [1] It is a multi-target CDK inhibitor with a unique activity spectrum, primarily targeting CDK1, CDK2, and CDK5, with activity against CDK3 and CDK9, but weaker activity against CDK6 and CDK7. This activity spectrum differs from other CDK inhibitors such as flavopiridol, seliciclib, and SNS-032. [1] Its mechanism of action against multiple myeloma (MM) involves cell cycle arrest (S phase and G2/M phase), leading to caspase-8 and caspase-3-dependent but mitochondrial-independent apoptosis. It regulates key oncogenic pathways and transcription factors that are crucial for MM cell survival and invasiveness. [1]
This study highlights that specific transcriptional changes induced by NVP-LCQ-195 are associated with poor clinical outcomes in patients treated with bortezomib, suggesting that it may target biologically aggressive disease subgroups. This approach of linking drug-induced molecular characteristics with clinical data is considered a strategy for evaluating multi-target drugs. [1]

C17H19CL2N5O4S

460.3349

459.053

C, 44.36; H, 4.16; Cl, 15.40; N, 15.21; O, 13.90; S, 6.96

902156-99-4

902156-99-4

11655534

White to off-white solid powder

3.916

3

6

5

29

701

0

O=C(C1C(Cl)=CC=CC=1Cl)NC1C(C(NC2CCN(S(C)(=O)=O)CC2)=O)=NNC=1

CCUXEBOOTMDSAM-UHFFFAOYSA-N

InChI=1S/C17H19Cl2N5O4S/c1-29(27,28)24-7-5-10(6-8-24)21-17(26)15-13(9-20-23-15)22-16(25)14-11(18)3-2-4-12(14)19/h2-4,9-10H,5-8H2,1H3,(H,20,23)(H,21,26)(H,22,25)

4-[(2,6-dichlorobenzoyl)amino]-N-(1-methylsulfonylpiperidin-4-yl)-1H-pyrazole-5-carboxamide

NVP-LCQ-195; NVP-LCQ195; NVP-LCQ 195; LCQ 195; LCQ-195; LCQ195; AT9311; AT-9311; AT 9311

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: 66.7~92 mg/mL (144.8~199.9 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.43 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.43 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 25.0 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.)