Triple-negative breast cancer (TNBC) has limited treatment options and the worst prognosis among all types of breast cancer. We describe two prodrugs, namely, CWB-20145 (1) and its methyl analogue FAN-NM-CH 3 (2) that reduced the size of TNBC-derived tumors. The DNA cross-linking of nitrogen mustard prodrugs 1 and 2 was superior to that of chlorambucil and melphalan once activated in the presence of H 2 O 2 . The cellular toxicity of 1 and 2 was demonstrated in seven human cancer cell lines. The TNBC cell line MDA-MB-468 was particularly sensitive toward 1 and 2. Compound 2 was 10 times more cytotoxic than chlorambucil and 16 times more active than melphalan. An evaluation of the gene expression demonstrated an upregulation of the tumor suppressor genes p53 and p21 supporting a transcriptional mechanism of a reduced tumor growth. Pharmacokinetic studies with 1 showed a rapid conversion of the prodrug. The introduction of a methyl group generated 2 with an increased half-life. An in vivo toxicity study in mice demonstrated that both prodrugs were less toxic than chlorambucil. Compounds 1 and 2 reduced tumor growth with an inhibition rate of more than 90% in athymic nude mice xenografted with MDA-MB-468 cells. Together, the in vivo investigations demonstrated that treatment with 1 and 2 suppressed tumor growth without affecting normal tissues in mice. These phenylboronic acid nitrogen mustard prodrugs represent promising drug candidates for the treatment of TNBC. However, the mechanisms underlying their superior in vivo activity and selectivity as well as the correlation between H 2 O 2 level and in vivo efficacy are not yet fully understood.

Thrombosis leads to myocardial infarction, stroke and other cardiovascular complications. Microbial thrombolytic agents such as urokinase, streptokinase etc. are used to treat complications related to thrombosis. To search for new microbial enzymes as thrombolytics having better efficacy and specificity, Bacillus licheniformis EMS-O-1 mutant strain was cultured in modified urea-molasses media followed by purification using ammonium sulphate precipitation and ultrafiltration through centricon tube of 100 MWCO value. The yield of crude enzyme was 11129.14 U/mg and after purification 40180.46 U/mg. Purification process increased the specific activity of purified enzyme to 12.28 fold with a recovery of 17.79%. The purified enzyme was a serine protease with molecular weight of 25.5 kDa as confirmed by irreversible inhibition of activity with phenylmethylsulfonyl fluoride (PMSF) followed by SDS-PAGE gel image and by LC-MS analyses. In vitro clot lysis assay of the purified enzyme exhibited 38.30% thrombolytic activity. The crude enzymes from the mutant strain EMS-O-1 were found to be stable up to 50 o C and showed maximum stability between pH range 7.5 to 8.5. These findings signify that proteases produced by B. licheniformis mutant have the potential to be developed as a viable thrombolytic agent.

Investigations were performed to find out new microbial enzymes as thrombolytics having better efficacy and specificity. Mutant strain of Bacillus species, B. licheniformis MZK05M9 was cultured in modified urea-glucose media followed by purification using ammonium sulphate precipitation and ultrafiltration through centricon tube of specific MWCO value. The production method yielded 823.42 units/mg of the crude enzyme from mutant strain MZK05M9 and after purification 37695.64 units/mg. The molecular weight of the purified enzyme was estimated as 27.2 kDa and purification increased its specific activity to 16.5 fold with a recovery of 10%. The purified proteases were identified as serine proteases by irreversible inhibition of activity with phenylmethylsulfonyl fluoride (PMSF) and it exhibited 32.84% thrombolytic activity, by in vitro clot lysis assay. Stability studies showed that crude enzyme from mutant strain MZK05M9 remained stable up to a temperature of 45˚C and showed maximum stability at pH range 7.5 to 8.5. Our observation indicates that proteases produced by Bacillus licheniformis mutant have the potential to be developed as a viable thrombolytic agent.

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