The maleimide motif is widely used for the selective chemical modification of cysteine residues in proteins. Despite widespread utilization, there are some potential limitations, including the ...irreversible nature of the reaction and, hence, the modification and the number of attachment positions. We conceived of a new class of maleimide which would address some of these limitations and provide new opportunities for protein modification. We report herein the use of mono- and dibromomaleimides for reversible cysteine modification and illustrate this on the SH2 domain of the Grb2 adaptor protein (L111C). After initial modification of a protein with a bromo- or dibromomaleimide, it is possible to add an equivalent of a second thiol to give further bioconjugation, demonstrating that bromomaleimides offer opportunities for up to three points of attachment. The resultant protein−maleimide products can be cleaved to regenerate the unmodified protein by addition of a phosphine or a large excess of a thiol. Furthermore, dibromomaleimide can insert into a disulfide bond, forming a maleimide bridge, and this is illustrated on the peptide hormone somatostatin. Fluorescein-labeled dibromomaleimide is synthesized and inserted into the disulfide to construct a fluorescent somatostatin analogue. These results highlight the significant potential for this new class of reagents in protein modification.
The rapidly increasing interest in the synthesis of antibody–drug conjugates as powerful targeted anticancer agents demonstrates the growing appreciation of the power of antibodies and antibody ...fragments as highly selective targeting moieties. This targeting ability is of particular interest in the area of photodynamic therapy, as the applicability of current clinical photosensitizers is limited by their relatively poor accumulation in target tissue in comparison to healthy tissue. Although synthesis of porphyrin–antibody conjugates has been previously demonstrated, existing work in this area has been hindered by the limitations of conventional antibody conjugation methods. This work describes the attachment of azide-functionalized, water-soluble porphyrins to a tratuzumab Fab fragment via a novel conjugation methodology. This method allows for the synthesis of a homogeneous product without the loss of structural stability associated with conventional methods of disulfide modification. Biological evaluation of the synthesized conjugates demonstrates excellent selectivity for a HER2 positive cell line over the control, with no dark toxicity observed in either case.
Although recent methods for the engineering of antibody-drug conjugates (ADCs) have gone some way to addressing the challenging issues of ADC construction, significant hurdles still remain. There is ...clear demand for the construction of novel ADC platforms that offer greater stability, homogeneity and flexibility. Here we describe a significant step towards a platform for next-generation antibody-based therapeutics by providing constructs that combine site-specific modification, exceptional versatility and high stability, with retention of antibody binding and structure post-modification. The relevance of the work in a biological context is also demonstrated in a cytotoxicity assay and a cell internalization study with HER2-positive and -negative breast cancer cell lines.
Preterm-labour-associated preterm birth is a common cause of perinatal mortality and morbidity in twin pregnancy. We aimed to test the hypothesis that the Arabin pessary would reduce ...preterm-labour-associated preterm birth by 40% or greater in women with a twin pregnancy and a short cervix.
We conducted an open-label randomised controlled trial in 57 hospital antenatal clinics in the UK and Europe. From 1 April 2015 to 14 February 2019, 2,228 women with a twin pregnancy underwent cervical length screening between 18 weeks 0 days and 20 weeks 6 days of gestation. In total, 503 women with cervical length ≤ 35 mm were randomly assigned to pessary in addition to standard care (n = 250, mean age 32.4 years, mean cervical length 29 mm, with pessary inserted in 230 women 92.0%) or standard care alone (n = 253, mean age 32.7 years, mean cervical length 30 mm). The pessary was inserted before 21 completed weeks of gestation and removed at between 35 and 36 weeks or before birth if earlier. The primary obstetric outcome, spontaneous onset of labour and birth before 34 weeks 0 days of gestation, was present in 46/250 (18.4%) in the pessary group compared to 52/253 (20.6%) following standard care alone (adjusted odds ratio aOR 0.87 95% CI 0.55-1.38, p = 0.54). The primary neonatal outcome-a composite of any of stillbirth, neonatal death, periventricular leukomalacia, early respiratory morbidity, intraventricular haemorrhage, necrotising enterocolitis, or proven sepsis, from birth to 28 days after the expected date of delivery-was present in 67/500 infants (13.4%) in the pessary group compared to 76/506 (15.0%) following standard care alone (aOR 0.86 95% CI 0.54-1.36, p = 0.50). The positive and negative likelihood ratios of a short cervix (≤35 mm) to predict preterm birth before 34 weeks were 2.14 and 0.83, respectively. A meta-analysis of data from existing publications (4 studies, 313 women) and from STOPPIT-2 indicated that a cervical pessary does not reduce preterm birth before 34 weeks in women with a short cervix (risk ratio 0.74 95% CI 0.50-1.11, p = 0.15). No women died in either arm of the study; 4.4% of babies in the Arabin pessary group and 5.5% of babies in the standard treatment group died in utero or in the neonatal period (p = 0.53). Study limitations include lack of power to exclude a smaller than 40% reduction in preterm labour associated preterm birth, and to be conclusive about subgroup analyses.
These results led us to reject our hypothesis that the Arabin pessary would reduce the risk of the primary outcome by 40%. Smaller treatment effects cannot be ruled out.
ISRCTN Registry ISRCTN 02235181. ClinicalTrials.gov NCT02235181.
Herein we report the use of next generation maleimides (NGMs) for the construction of a potent antibody-drug conjugate (ADC) via functional disulfide bridging. The linker has excellent stability in ...blood serum and the ADC, armed with monomethyl auristatin E (MMAE), shows excellent potency and cancer cell selectivity in vitro.
Disulfide bridging offers a convenient approach to generate site-selective antibody conjugates from native antibodies. To optimise the reagents available to achieve this strategy, we describe here ...the use of dibromomaleimides designed to undergo accelerated post-conjugation hydrolysis. Conjugation and hydrolysis, which serve to 'lock' the conjugates as robustly stable maleamic acids, is achieved in just over 1 h. This dramatic acceleration is also shown to infer significant improvements in homogeneity, as demonstrated by mass spectrometry analysis.
The introduction of non-natural entities into proteins by chemical modification has numerous applications in fundamental biological science and for the development and manipulation of peptide and ...protein therapeutics. The reduction of native disulfide bonds provides a convenient method to access two nucleophilic cysteine residues that can serve as ideal attachment points for such chemical modification. The optimum bioconjugation strategy utilizing these cysteine residues should include the reconstruction of a bridge to mimic the role of the disulfide bond, maintaining structure and stability of the protein. Furthermore, the bridging chemical modification should be as rapid as possible to prevent problems associated with protein unfolding, aggregation, or disulfide scrambling. This study reports on an in situ disulfide reduction-bridging strategy that ensures rapid sequestration of the free cysteine residues in a bridge, using dithiomaleimides. This approach is then used to PEGylate the peptide hormone somatostatin and retention of biological activity is demonstrated.
In this communication we describe a novel acid-cleavable linker strategy for antibody-drug conjugation. Functional disulfide bridging of the single interchain disulfide bond of a trastuzumab Fab ...fragment yields a homogeneous antibody-drug conjugate bearing a thiomaleamic acid linker. This linker is stable at physiological pH and temperature, but quantitatively cleaves at lysosomal pH to release the drug payload.
A new ω-transaminase (ω-TA) from
Chromobacterium violaceum DSM30191 has been recruited from the pool of fully sequenced genomes, using the
V. fluvialis JS17 ω-TA sequence Shin JS, Kim BG. Biosci ...Biotechnol Biochem 2001;65:1782–8; Shin JS, Yun H, Jang JW, Park I, Kim BG. Appl Microbiol Biotechnol 2003;61:463–71 in a BLASTP search. The protein sequence of the
C. violaceum enzyme exhibits 38% sequence identity to the
V. fluvialis enzyme. The gene was functionally expressed in
Escherichia coli BL21 by using the pET-expression system, making up 63% of the protein in the clarified cell extract (1.2
U/mg). As with the
V. fluvialis TA, the
C. violaceum enzyme shows (
S)-enantioselective TA activity and converts (
S)-α-methylbenzylamine and pyruvate almost completely to acetophenone and
l-Ala. Glyoxylate proved to be the best acceptor and aliphatic and aromatic aldehydes were both readily converted. Importantly we show here for the first time that an ω-TA can be used for the amination of aromatic 2-keto-1,3-diols. An LC-ESI-MS method was developed to quantitatively detect aromatic aminodiol formation. Using clarified cell extract, we were able to convert 20
mM of racemic 1,3-dihydroxy-1-phenylpropan-2-one to 2-amino-1-phenylpropane-1,3-diol with 60% conversion in 24
h.
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