Overexpression of penicillin V acylase from Streptomyces lavendulae and elucidation of its catalytic residues.

The pva gene from Streptomyces lavendulae ATCC 13664, encoding a novel penicillin V acylase (SlPVA), has been isolated and characterized. The gene encodes an inactive precursor protein containing a secretion signal peptide that is activated by two internal autoproteolytic cleavages that release a 25-amino-acid linker peptide and two large domains of 18.79 kDa (alpha-subunit) and 60.09 kDA (beta-subunit). Based on sequence alignments and the three-dimensional model of SlPVA, the enzyme contains a hydrophobicpocket involved in catalytic activity, including Serbeta1, Hisbeta23, Valbeta70, and Asnbeta272, which were confirmed by site-directed mutagenesis studies. The heterologous expression of pva in S. lividans led to the production of an extracellularly homogeneous heterodimeric enzyme at a 5-fold higher concentration (959 IU/liter) than in the original host and in a considerably shorter time. According to the catalytic properties of SlPVA, the enzyme must be classified as a new member of the Ntn-hydrolase superfamily, which belongs to a novel subfamily of acylases that recognize substrates with long hydrophobic acyl chains and have biotechnological applications in semisynthetic antifungal production.

Continuous treatment with abemaciclib leads to sustained and efficient inhibition of breast cancer cell proliferation.

Abemaciclib is an oral, selective cyclin-dependent kinase 4 & 6 inhibitor (CDK4 & 6i), approved for hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) advanced breast cancer (ABC) as monotherapy for endocrine refractory disease, and with endocrine therapy (ET) for initial treatment and after progression on ET. Abemaciclib has also shown clinical activity in combination with ET in patients with high risk early BC (EBC). Here, we examined the preclinical attributes of abemaciclib and other CDK4 & 6i using biochemical and cell-based assays. In vitro, abemaciclib preferentially inhibited CDK4 kinase activity versus CDK6, resulting in inhibition of cell proliferation in a panel of BC cell lines with higher average potency than palbociclib or ribociclib. Abemaciclib showed activity regardless of HER2 amplification and phosphatidylinositol 3-kinase (PI3KCA) gene mutation status. In human bone marrow progenitor cells, abemaciclib showed lower impact on myeloid maturation than other CDK4 & 6i when tested at unbound concentrations similar to those observed in clinical trials. Continuous abemaciclib treatment provided profound inhibition of cell proliferation, and triggered senescence and apoptosis. These preclinical results support the unique efficacy and safety profile of abemaciclib observed in clinical trials.

Preclinical characterization of abemaciclib in hormone receptor positive breast cancer.

Abemaciclib is an ATP-competitive, reversible kinase inhibitor selective for CDK4 and CDK6 that has shown antitumor activity as a single agent in hormone receptor positive (HR+) metastatic breast cancer in clinical trials. Here, we examined the mechanistic effects of abemaciclib treatment using in vitro and in vivo breast cancer models. Treatment of estrogen receptor positive (ER+) breast cancer cells with abemaciclib alone led to a decrease in phosphorylation of Rb, arrest at G1, and a decrease in cell proliferation. Moreover, abemaciclib exposure led to durable inhibition of pRb, TopoIIα expression and DNA synthesis, which were maintained after drug removal. Treatment of ER+ breast cancer cells also led to a senescence response as indicated by accumulation of ß-galactosidase, formation of senescence-associated heterochromatin foci, and a decrease in FOXM1 positive cells. Continuous exposure to abemaciclib altered breast cancer cell metabolism and induced apoptosis. In a xenograft model of ER+ breast cancer, abemaciclib monotherapy caused regression of tumor growth. Overall these data indicate that abemaciclib is a CDK4 and CDK6 inhibitor that, as a single agent, blocks breast cancer cell progression, and upon longer treatment can lead to sustained antitumor effects through the induction of senescence, apoptosis, and alteration of cellular metabolism.

Optimization of culture medium and conditions for penicillin acylase production by Streptomyces lavendulae ATCC 13664.

The culture medium for Streptomyces lavendulae ATCC 13664 was optimized on a shake-flask scale by using a statistical factorial design for enhanced production of penicillin acylase. This extracellular enzyme recently has been reported to be a penicillin K acylase, presenting also high hydrolytic activity against penicillin V and other natural aliphatic penicillins such as penicillin K, penicillin F, and penicillin dihydroF. The factorial design indicated that the main factors that positively affect penicillin acylase production by S. lavendulae were the concentration of yeast extract and the presence of oligoelements in the fermentation medium, whereas the presence of olive oil in the medium had no effect on enzyme production. An initial concentration of 2.5% (w/v) yeast extract and 3 microg/mL of CuSO4 x 5H2O was found to be best for acylase production. In such optimized culture medium, fermentation of the microorganism yielded 289 IU/L of enzyme in 72 h when employing a volume medium/volume flask ratio of 0.4 and a 300-rpm shaking speed. The presence of copper, alone and in combination with other metals, stimulated biomass as well as penicillin acylase production. The time course of penicillin acylase production was also studied in the optimized medium and conditions. Enzyme production showed catabolite repression by different carbon sources such as glucose, lactose, citrate, glycerol, and glycine.

Multiparametric cell-based assay for the evaluation of transcription inhibition by high-content imaging.

Loss of normal cell cycle regulation is a hallmark of human cancer. Cyclin-dependent kinases (CDKs) are key regulators of the cell cycle and have been actively pursued as promising therapeutic targets. Likewise, members of the CDK family are functionally related to transcriptional modulation, a molecular pathway suitable for therapeutic intervention. We used a set of 2500 compounds in the U2OS cell line to evaluate its effect in the cell division process. Interestingly, out of this analysis, we identified a subpopulation of compounds that are able to inhibit RNA polymerase activity, thus interfering with gene transcription processes. After this finding, we developed, validated, and fully automated a multiparameter high-content imaging (HCI) assay to measure phosphorylation of the RNA polymerase II carboxyl terminal domain (pCTD). Simultaneously, we measured both the DNA content and cell proliferation index in the treated cells. The linear regression analysis comparing the IC50 for pCTD and the 4N EC50 for DNA content or IC50 for cell proliferation showed an excellent agreement (r (2) = 0.84 and r (2) = 0.94, respectively). Our results confirm that this method allows discriminating between cell cycle and transcription inhibition and confirms HCI as a powerful technology for the identification of compounds with an effective and selective pathway phenotype.

Substrate specificity of penicillin acylase from Streptomyces lavendulae.

The kinetic parameters of several substrates of penicillin acylase from Streptomyces lavendulae have been determined. The enzyme hydrolyses phenoxymethyl penicillin (penicillin V) and other penicillins with aliphatic acyl-chains such as penicillin F, dihydroF, and K. The best substrate was penicillin K (octanoyl penicillin) with a k(cat)/K(m) of 165.3 mM(-1) s(-1). The enzyme hydrolyses also chromogenic substrates as NIPOAB (2-nitro-5-phenoxyacetamido benzoic acid), NIHAB (2-nitro-5-hexanoylamido benzoic acid) or NIOAB (2-nitro-5-octanoylamido benzoic acid), however failed to hydrolyse phenylacetil penicillin (penicillin G) or NIPAB (2-nitro-5-phenylacetamido benzoic acid) and penicillins with polar substituents in the acyl moiety. These results suggest that the structure of the acyl moiety of the substrate is more determinant than the amino moiety for enzyme specificity. The enzyme was inhibited by several organic acids and the extent of inhibition changed with the hydrophobicity of the acid. The best inhibitor was octanoic acid with a K(i) of 0.8 mM. All the results, taking together, point to an active site highly hydrophobic for this penicillin acylase from Streptomyces lavendulae.