Adenylosuccinate synthetase (also known as adenosylsuccinate synthetase), which converts inosine monophosphate (IMP) to adenosine monophosphate (AMP) in the de novo purine biosynthesis pathway. No IC50, Ki, or EC50 values are provided for the enzyme inhibition in this study. [1]

L-Alanosine, an antibiotic from Streptomyces alanosinicus, blocks the common de novo purine biosynthesis pathway and, thereby, inhibits tumor cells with MTAP deficiency. Normal cells escape the detrimental effects of L-alanosine due to their proficiency in the MTAP salvage pathway. The present analysis was undertaken to gain insights into the molecular architecture of tumor cells that determines the response to L-alanosine apart from the MTAP gene. Analysis of cell doubling times and IC(50) values for L-alanosine showed that slowly growing cell lines were more resistant to L-alanosine than rapidly growing ones. Mining the database of the National Cancer Institute (N.C.I.), for the mRNA expression of 9706 genes in 60 cell lines by means of Kendall's tau-test, false discovery rate calculation, and hierarchical cluster analysis pointed to 11 genes or expressed sequence tags whose mRNA expression correlated with the IC(50) values for L-alanosine. Furthermore, we tested L-alanosine for cross-resistance in multidrug-resistant cell lines which overexpress selectively either the P-glycoprotein/MDR1 (CEM/ADR5000), MRP1 (HL-60/AR), or BCRP (MDA-MB-231-BCRP) genes. None of the multidrug-resistant cell lines was cross-resistant to L-alanosine indicating that L-alanosine may be suitable to treat multidrug-resistant, refractory tumors in the clinic. Finally, the IC(50) values for L-alanosine of the 60 cell lines were correlated to the p53 mutational status and expression of p53 downstream genes. We found that p53 mutated cell lines were more resistant to L-alanosine than p53 wild type cell lines.[1]

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The antitumor action of L-alanosine (NSC 153553) was investigated in murine leukemia P388 (P388/S), P388 resistant to adriamycin (P388/ADR), P388 resistant to vincristine (P388/VCR) and leukemia L5178Y sensitive to L-asparaginase (L5178Y/S). L-alanosine demonstrated good antineoplastic activity against P388/S and P388/ADR, whereas it showed better anticancer activity against P388/VCR and L5178Y/S at the various dose levels employed.[2]

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L-Alanosine exhibited cytotoxic activity against a panel of 60 human tumor cell lines from the N.C.I., with IC50 values determined by sulforhodamine B assay. Cell lines with shorter doubling times were more sensitive to L-Alanosine than slowly growing ones (P = 0.0000246, R = 0.35961). [1]
L-Alanosine was not cross-resistant in multidrug-resistant cell lines overexpressing P-glycoprotein/MDR1 (CEM/ADR5000), MRP1 (HL-60/AR), or BCRP (MDA-MB-231-BCRP clone 23). [1]
Cell lines with wild-type p53 were more sensitive to L-Alanosine than those with mutated p53 (P = 0.01733). Induction of p53 downstream genes GADD45 and MDM2 after γ-irradiation inversely correlated with IC50 values for L-Alanosine (P = 0.00314 and 0.02236, respectively). [1]

Administration of 50 microCi of DL-[1-14C]alanosine along with unlabeled L-alanosine (500 mg/kg) to BD2F1 mice bearing s.c. nodules of Leukemia L5178Y/AR resulted in the accumulation in tumors of a material with properties compatible with those of L-alanosyl-5-amino-4-imidazolecarboxylic acid ribonucleotide.[3]

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L-Alanosine showed good antineoplastic activity against P388/S (sensitive murine leukemia) and P388/ADR (adriamycin-resistant subline), with %ILS values up to 74% and 63% respectively at 400 mg/kg. It showed better anticancer activity against P388/VCR (vincristine-resistant subline) and L5178Y/S (L-asparaginase-sensitive lymphoma), with %ILS values up to 127% and 108% respectively. [2]

The growth inhibition assay was used to evaluate the in vitro response to L-Alanosine. Aliquots of 5 × 10⁵ cells/mL were seeded in culture medium, and drugs were added immediately at different concentrations. Cells were counted up to 10 days after seeding depending on the growth rate. Cell numbers were determined in eight independent determinations. The resulting growth curves represent the net outcome of cell proliferation and cell death. [1]
The sulforhodamine B assay was used to determine IC50 values for L-Alanosine in 60 N.C.I. cell lines. [1]

BDF₁ male mice weighing 18–22 g were used. Tumor cells (1 × 10⁶ cells/mouse) were transplanted intraperitoneally. L-Alanosine sodium salt was dissolved in sterile distilled water and administered intraperitoneally at doses ranging from 51.84 to 400 mg/kg, using an intermittent schedule on days 1, 5, and 9 after tumor transplantation. The treatment volume was 0.01 ml/g body weight. Animals were divided into groups of 6. Median survival time and percentage increase in life-span (%ILS) were calculated. [2]

[1]. Identification of gene expression profiles predicting tumor cell response to L-alanosine. Biochem Pharmacol. 2003 Aug 15;66(4):613-21.

[2]. Antitumor effect of L-alanosine (NSC 153553) on sensitive and resistant sublines of murine leukemias. Tumori. 1984 Aug 31;70(4):317-20.

[3]. Identification of the antimetabolite of L-alanosine, L-alanosyl-5-amino-4-imidazolecarboxylic acid ribonucleotide, in tumors and assessment of its inhibition of adenylosuccinate synthetase. Cancer Res. 1980 Dec;40(12):4390-7.

Streptomyces alanosinicus is an amino acid analog and antibiotic with antimetabolite and potential antitumor activity. L-Streptomycin is an amino acid analog and antibiotic derived from Streptomyces alanosinicus, possessing antimetabolite and potential antitumor activity. L-Streptomycin inhibits adenylate succinate synthase, an enzyme that converts inosine monophosphate (IMP) to adenylate succinate, an intermediate product of purine metabolism. Methylthioadenosine phosphorylase (MTAP) deficiency enhances the inhibitory effect of L-Streptomycin on de novo purine synthesis. The clinical application of this drug may be limited by its toxicity. MTAP is a key enzyme in the adenine and methionine rescue pathways.
Drug Indications
It has been investigated for the treatment of brain cancer and cancer/tumor (not specified).

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Mechanism of Action L-alanine inhibits adenosylsuccinate synthase, which converts inosine monophosphate (IMP) into adenosylsuccinate, an intermediate in purine metabolism. MTAP deficiency enhances the blocking effect of L-alanine-induced de novo purine synthesis.

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L-alanine is an antibiotic isolated from Streptomyces alanosisicus. Its metabolite, L-alanine-5-amino-4-imidazolium carboxylate ribonucleotide, inhibits adenosylsuccinate synthase, thereby blocking the de novo purine synthesis pathway. [1] L-alanine selectively targets MTAP-deficient tumor cells that lack the salvage pathway for adenine synthesis. Normal cells are unaffected because their MTAP pathway functions normally. [1] Microarray analysis of 9706 genes from 60 cell lines identified 11 genes/ESTs whose mRNA expression was associated with L-alanine sensitivity/resistance, including MTHFD2, which is involved in the de novo purine synthesis pathway. [1] L-alanine is not a substrate of MDR transporters (MDR1, MRP1, BCRP), suggesting that it may be effective against multidrug-resistant tumors. [1]

C3H7N3O4

149.10538

149.043

C, 24.17; H, 4.73; N, 28.18; O, 42.92

5854-93-3

135409347

Soluble in water

1.8±0.1 g/cm3

366.9±52.0 °C at 760 mmHg

190ºC (dec.)

175.7±30.7 °C

0.0±1.9 mmHg at 25°C

1.616

-0.56

3

6

3

10

156

1

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

MLFKVJCWGUZWNV-REOHCLBHSA-N

InChI=1S/C3H7N3O4/c4-2(3(7)8)1-6(10)5-9/h2,10H,1,4H2,(H,7,8)/t2-/m0/s1

(S)-2-amino-3-(hydroxy(nitroso)amino)propanoic acid

L-Alanosine; NSC-153353; NSC153353; NSC 153353; SDX102; SDX-102; SDX 102

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product is not stable in solution, please use freshly prepared working solution for optimal results.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~50 mg/mL (~335.32 mM)
H2O : ~15 mg/mL (~100.60 mM)

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.)