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Synthetic routes to potent steroidal and nonsteroidal sulfamate-based active site-directed inhibitors of the enzyme steroid sulfatase, a topical target in the treatment of postmenopausal women with hormone-dependent breast cancer, are described. Novel compounds were examined for estrone sulfatase (E1-STS) inhibition in intact MCF-7 breast cancer cells and placental microsomes. Reaction of the sodium salt of estrone with sulfamoyl chloride gave estrone 3-O-sulfamate (EMATE, 2) which inhibits E1-STS activity potently (> 99% at 0.1 microM in intact MCF-7 cells, IC50 = 65 pM) in a time- and concentration-dependent manner, suggesting that EMATE is an active site-directed inhibitor. EMATE is also active in vivo orally. 5,6,7,8-Tetrahydronaphthalene 2-O-sulfamate (7) and its N-methylated derivatives (8 and 9) were synthesized, and 7 inhibits the E1-STS activity in intact MCF-7 cells by 79% at 10 microM. 4-Methylcoumarin 7-O-sulfamate (COUMATE) and its derivatives (14, 16, and 18) were prepared to extend this series of nonsteroidal inhibitors, and COUMATE reduces the E1-STS activity in placental microsomes by > 90% at 10 microM. Although the orally active COUMATE is less potent than EMATE as an active site-directed inhibitor, it has the important advantage of being nonestrogenic. Analogues (20, 22, 24, 26, 27, 31, 33, 39, and 44) of COUMATE were synthesized to study its structure-activity relationships, and sulfamates of tetralones (46 and 48) and indanones (49, 51, and 53) were also prepared. While most of these compounds were found to inhibit E1-STS activity less effectively than COUMATE, one analogue, 3,4-dimethylcoumarin 3-O-sulfamate (24), was found to be some 12-fold more potent than COUMATE as an E1-STS inhibitor in intact MCF-7 cells (IC50 = 30 nM for 24, cf. 380 nM for COUMATE). Hence, highly potent sulfamate-based inhibitors of steroid sulfatase, such as EMATE, COUMATE, and 24, possess therapeutic potential and will allow the importance of estrogen formation in breast tumors via the E1-STS pathway to be assessed. A pharmacophore for active site-directed sulfatase inhibition is proposed.

Steroid sulfatases are responsible for the hydrolysis of 3beta-hydroxy steroid sulfates, such as cholesterol and pregnenolone sulfate, and have an important role in regulating the synthesis of estrogenic steroids, from estrone sulfate and dehydroepiandrosterone sulfate, in endocrine-dependent tumors. Although little is known about the mechanism by which the sulfate group is removed from a steroid nucleus, an active site-directed sulfatase inhibitor has been developed. This inhibitor, estrone-3-O-sulfamate (EMATE), was synthesized by treating the sodium salt of estrone with sulfamoyl chloride. This compound inhibited not only estrone sulfatase but also dehydroepiandrosterone sulfatase activity in placental microsomes and in intact MCF-7 breast cancer cells. Pretreatment of MCF-7 cells or placental microsomes with EMATE, followed by extensive washing or dialysis indicated irreversible inhibition. This was confirmed by showing that EMATE inhibited estrone sulfatase activity in placental microsomes in a time-, concentration-, and pH-dependent manner. The enzyme is protected from inactivation by estrone sulfate, which is also consistent with active site-directed inhibition. EMATE is proposed to inactivate estrone sulfatase by irreversible sulfamoylation of the enzyme. Maximum enzyme activity was detected at pH 8.6, and the maximum rate of enzyme inactivation by EMATE also occurred at this pH. The pKa values of the enzymatic reaction and pKa of inactivation were 7.2 and 9.8, providing evidence that two active site residues are being modified by EMATE. As the phenolic pKa of tyrosine (9.7) and the pKa of histidine will allow the roles that (6.8) are similar to the pKa values of inactivation, these amino acid residues may play a role in the catalytic mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Organ-on-chips are miniaturised devices aiming at replacing animal models for drug discovery, toxicology and studies of complex biological phenomena. The field of Organ-On-Chip has grown exponentially, and has led to the formation of companies providing commercial Organ-On-Chip devices. Yet, it may be surprising to learn that the majority of these commercial devices are made from Polydimethylsiloxane (PDMS), a silicone elastomer that is widely used in microfluidic prototyping, but which has been proven difficult to use in industrial settings and poses a number of challenges to experimentalists, including leaching of uncured oligomers and uncontrolled adsorption of small compounds. To alleviate these problems, we propose a new substrate for organ-on-chip devices: Polylactic Acid (PLA). PLA is a material derived from renewable resources, and compatible with high volume production technologies, such as microinjection moulding. PLA can be formed into sheets and prototyped into desired devices in the research lab. In this article we uncover the suitability of Polylactic acid as a substrate material for Microfluidic cell culture and Organ-on-a-chip applications. Surface properties, biocompatibility, small molecule adsorption and optical properties of PLA are investigated and compared with PDMS and other reference polymers.

polylactic acid sheets for the rapid prototyping of sustainable, single-use, disposable medical micro-components', ACS Sustainable Chemistry & Engineering.

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Peptide nucleic acid (PNA) analogues of DNA have attracted interest as potential pharmacological regulators of gene expression since they have the capacity to invade duplex DNA forming Watson−Crick base paired PNA:DNA heteroduplexes. Unfortunately, strand invasion is limited to homopurine and homopyrimidine sequences and there is the need to explore further PNA analogues for the purpose of expanding the strand invasion alphabet. Accordingly, we have designed a true peptide mimic of DNA (designated α-PNA) involving novel l-α-amino acids, with side chains comprising the four DNA bases attached via an ethylene linkage, interspaced with glycine. The four base-containing amino acids have been synthesized from N-Boc-l-homoserine, via alkylation of the appropriate base with the key intermediate (S)-2-(N-Boc-amino)-4-bromobutyric acid methyl ester followed by hydrolysis. These amino acids have been incorporated into α-PNA oligomers using both solution and solid phase methods.

The chemical nature of the non-tryptophan (non-Trp) fluorescence of porcine and human eye lens proteins was identified by Mass Spectrometry (MS) and Fluorescence Steady-State and Lifetime spectroscopy as post-translational modifications (PTM) of Trp and Arg amino acid residues. Fluorescence intensity profiles measured along the optical axis of human eye lenses with age-related nuclear cataract showed increasing concentration of fluorescent PTM towards the lens centre in accord with the increased optical density in the lens nucleolus. Significant differences between fluorescence lifetimes of “free” Trp derivatives hydroxytryptophan (OH-Trp), N-formylkynurenine (NFK), kynurenine (Kyn), hydroxykynurenine (OH-Kyn) and their residues were observed. Notably, the lifetime constants of these residues in a model peptide were considerably greater than those of their “free” counterparts. Fluorescence of Trp, its derivatives and argpyrimidine (ArgP) can be excited at the red edge of the Trp absorption band which allows normalisation of the emission spectra of these PTMs to the fluorescence intensity of Trp, to determine semi-quantitatively their concentration. We show that the cumulative fraction of OH-Trp, NFK and ArgP emission dominates the total fluorescence spectrum in both emulsified post-surgical human cataract protein samples, as well as in whole lenses and that this correlates strongly with cataract grade and age.