The application of mechanical force to induce the formation and cleavage of covalent bonds is a rapidly developing field within organic chemistry which has particular value in reducing or eliminating ...solvent usage, enhancing reaction rates and also in enabling the preparation of products which are otherwise inaccessible under solution-phase conditions. Mechanochemistry has also found recent attention in materials chemistry and API formulation during which rearrangement of non-covalent interactions give rise to functional products. However, this has been known to nucleic acids science almost since its inception in the late nineteenth century when Miescher exploited grinding to facilitate disaggregation of DNA from tightly bound proteins through selective denaturation of the latter. Despite the wide application of ball milling to amino acid chemistry, there have been limited reports of mechanochemical transformations involving nucleoside or nucleotide substrates on preparative scales. A survey of these reactions is provided, the majority of which have used a mixer ball mill and display an almost universal requirement for liquid to be present within the grinding vessel. Mechanochemistry of charged nucleotide substrates, in particular, provides considerable benefits both in terms of efficiency (reducing total processing times from weeks to hours) and by minimising exposure to aqueous conditions, access to previously elusive materials. In the absence of large quantities of solvent and heating, side-reactions can be reduced or eliminated. The central contribution of mechanochemistry (and specifically, ball milling) to the isolation of biologically active materials derived from nuclei by grinding will also be outlined. Finally non-covalent associative processes involving nucleic acids and related materials using mechanochemistry will be described: specifically, solid solutions, cocrystals, polymorph transitions, carbon nanotube dissolution and inclusion complex formation.
In response to pheromones, yeast cells activate a MAPK pathway to direct processes important for mating, including gene induction, cell-cycle arrest, and polarized cell growth. Although a variety of ...assays have been able to elucidate signaling activities at multiple steps in the pathway, measurements of MAPK activity during the pheromone response have remained elusive, and our understanding of single-cell signaling behavior is incomplete. Using a yeast-optimized FRET-based mammalian Erk-activity reporter to monitor Fus3 and Kss1 activity in live yeast cells, we demonstrate that overall mating MAPK activity exhibits distinct temporal dynamics, rapid reversibility, and a graded dose dependence around the K
D of the receptor, where phenotypic transitions occur. The complex dose response was found to be largely a consequence of two feedbacks involving cyclin-mediated scaffold phosphorylation and Fus3 autoregulation. Distinct cell cycle-dependent response patterns comprised a large portion of the cell-to-cell variability at each dose, constituting the major source of extrinsic noise in coupling activity to downstream gene-expression responses. Additionally, we found diverse spatial MAPK activity patterns to emerge over time in cells undergoing default, gradient, and true mating responses. Furthermore, ramping up and rapid loss of activity were closely associated with zygote formation in mating-cell pairs, supporting a role for elevated MAPK activity in successful cell fusion and morphogenic reorganization. Altogether, these findings present a detailed view of spatiotemporal MAPK activity during the pheromone response, elucidating its role in mediating complex long-term developmental fates in a unicellular differentiation system.
The relationship between the operational parameters and the performance of gas–liquid plasma reactors for water treatment and other applications is complex and often affects the plasma and the bulk ...liquid physicochemical processes simultaneously. This work investigates the effects of discharge frequency between 40 and 120 Hz on degradation of six organic compounds (solutes): phenol, pyrazole, rhodamine B, perfluorooctanoic acid (PFOA), 2-(2-Aminoethoxy) ethanol (DGA), and caffeine in a rail-plane gas–liquid plasma reactor without external mixing. To explain the differences in the measured degradation rates, the bulk liquid mass transport (i.e., flow fields) was visualized and quantified using particle image velocimetry (PIV) and subsequently coupled with interfacial reaction kinetics. Results show that the discharge frequency controls the compound degradation via multiple mechanisms that include variation in reactive species production and hydrodynamic effects based on charged particle transport (ionic wind) and surface tension (Marangoni flow). Moreover, two qualitatively different degradation regimes are identified: the mass transport limited regime that is characterized by an exponential decay of the solute concentration with time and is independent of the discharge frequency and the reactive species production limited regime that is characterized by a linear decay of the solute concentration with respect to both time and discharge frequency. A model has been developed that predicts the shapes of the experimentally measured concentration profiles by assessing the relative magnitudes of transport and reaction (kinetic) fluxes. The results of this study in which a solute’s degradation kinetic profile identifies the mechanism(s) inhibiting its degradation (i.e., transport vs. kinetics) have significant implications for the design of chemical reactors in water treatment applications.
Novel sulfur and selenium substituted 5',5'-linked dinucleoside pyrophate analogues were prepared in a vibration ball mill from the corresponding persilylated monophosphate. The chemical hydrolysis ...of pyrophosphorochalcogenolate-linked dimers was studied over a wide pH-range. The effect of the chalcogeno-substitution on the reactivity of dinucleoside pyrophosphates was surprisingly modest, and the chemical stability is promising considering the potential therapeutic or diagnostic applications. The chemical stability of the precursor phosphorochalcogenolate monoesters was also investigated. Hydrolytic desilylation of these materials was effected in aqueous buffer at pH 3, 7 or 11 and resulted in phosphorus-chalcogen bond scission which was monitored using
P NMR. The rate of dephosphorylation was dependent upon both the nature of the chalcogen and the pH. The integrity of the P-S bond in the corresponding phosphorothiolate was maintained at high pH but rapidly degraded at pH 3. In contrast, P-Se bond cleavage of the phosphoroselenolate monoester was rapid and the rate increased with alkalinity. The results obtained in kinetic experiments provide insight on the reactivity of the novel pyrophosphates studied as well as of other types of thiosubstituted biological phosphates. At the same time, these results also provide evidence for possible formation of unexpectedly reactive intermediates as the chalcogen-substituted analogues are metabolised.
After significant effort over the last 30 years, antibody-drug conjugates (ADC) have recently gained momentum as a therapeutic modality, and nine ADCs have been approved by the FDA to date, with ...additional ADCs in late stages of development. Here, we introduce dolaflexin, a novel ADC technology that overcomes key limitations of the most common ADC platforms with two key features: a higher drug-to-antibody ratio and a novel auristatin with a controlled bystander effect. The novel, cell permeable payload, auristatin F-hydroxypropylamide, undergoes metabolic conversion to the highly potent, but less cell permeable auristatin F to balance the bystander effect through drug trapping within target cells. We conducted studies in mice, rats, and cynomolgus monkeys to complement
characterization and contrasted the performance of dolaflexin with regard to antitumor activity, pharmacokinetic properties, and safety in comparison with the ADC platform utilized in the approved ADC ado-trastuzumab emtansine (T-DM1). A HER2-targeted dolaflexin ADC was shown to have a much lower threshold of antigen expression for potent cell killing
, was effective
in tumors with low HER2 expression, and induced tumor regressions in a xenograft model that is resistant to T-DM1.
Recent studies in colorectal cancer patients (CRC) have shown that increased resistance to thymidylate synthase (TS) inhibitors such as 5-fluorouracil (5-FU), reduce the efficacy of standard of care ...(SoC) treatment regimens. The nucleotide pool cleanser dUTPase is highly expressed in CRC and is an attractive target for potentiating anticancer activity of chemotherapy. The purpose of the current work was to investigate the activity of P1, P4-di(2′,5′-dideoxy-5′-selenouridinyl)-tetraphosphate (P4-SedU2), a selenium-modified symmetrically capped dinucleoside with prodrug capabilities that is specifically activated by dUTPase. Using mechanochemistry, P4-SedU2 and the corresponding selenothymidine analogue P4-SeT2 were prepared with a yield of 19% and 30% respectively. The phosphate functionality facilitated complexation with the amphipathic cell-penetrating peptide RALA to produce nanoparticles (NPs). These NPs were designed to deliver P4-SedU2 intracellularly and thereby maximise in vivo activity. The NPs demonstrated effective anti-cancer activity and selectivity in the HCT116 CRC cell line, a cell line that overexpresses dUTPase; compared to HT29 CRC cells and NCTC-929 fibroblast cells which have reduced levels of dUTPase expression. In vivo studies in BALB/c SCID mice revealed no significant toxicity with respect to weight or organ histology. Pharmacokinetic analysis of blood serum showed that RALA facilitates effective delivery and rapid internalisation into surrounding tissues with NPs eliciting lower plasma Cmax than the equivalent injection of free P4-SedU2, translating the in vitro findings. Tumour growth delay studies have demonstrated significant inhibition of growth dynamics with the tumour doubling time extended by >2weeks. These studies demonstrate the functionality and action of a new pro-drug nucleotide for CRC.
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Target selection for antibody-drug conjugates (ADC) frequently focuses on identifying antigens with differential expression in tumor and normal tissue, to mitigate the risk of on-target toxicity. ...However, this strategy restricts the possible target space. SLC34A2/NaPi2b is a sodium phosphate transporter expressed in a variety of human tumors including lung and ovarian carcinoma, as well as the normal tissues from which these tumors arise. Previous clinical trials with a NaPi2b targeting MMAE-ADCs have shown objective durable responses. However, the protein-based biomarker assay developed for use in that study was unable to discern a statistically significant relationship between NaPi2b protein expression and the probability of response. XMT-1536 is a NaPi2b targeting ADC comprised of a unique humanized antibody conjugated with 10-15 auristatin F- hydroxypropylamide (AF-HPA) payload molecules via the Dolaflexin platform. AF-HPA is a cell-permeable, antimitotic compound that is slowly metabolized intratumorally to an active, very low-permeable metabolite, auristatin F (AF), resulting in controlled bystander killing. We describe the preclinical
and
antitumor effects of XMT-1536 in models of ovarian and lung adenocarcinoma. Pharmacokinetic analysis showed approximately proportional increases in exposure in rat and monkey. Systemic free AF-HPA and AF concentrations were observed to be low in all animal species. Finally, we describe a unique IHC reagent, generated from a chimeric construct of the therapeutic antibody, that was used to derive a target expression and efficacy relationship in a series of ovarian primary xenograft cancer models.
In this work, particle image velocimetry has been used to visualize and quantify plasma-induced flow fields in liquid water. Experiments were performed in a rod-plane plasma reactor with a thin wire ...electrode suspended above the surface of the liquid in argon gas and a grounded plate immersed in the liquid. The velocity field has been quantified for two types of solutions: (1) aqueous NaCl solutions of varying solution conductivities and discharge frequencies and (2) aqueous NaCl solutions containing varying concentrations of the following four organic compounds: rhodamine B dye, caffeine, fluoxetine, and perfluorooctanoic acid. Results show that in neat water and aqueous caffeine solutions, the liquid is “pulled” along by the interaction of the gas molecules with the liquid molecules at the free surface and thus the direction of the liquid flow is in the direction of the gas phase flow (i.e., away from the discharge location). However, the flow was reversed (i.e., towards the discharge) for those solutions with strong surfactants such as perfluorooctanoic acid. The magnitude of the reversal depended on the initial concentration of the compound and for some compounds as time progressed the reversed flow pattern weakened and then reverted to a normal flow pattern. To determine the most likely cause of these flow reversals, a simple numerical model of the velocity field was developed to estimate relative contributions of various flow inducing mechanisms. The model indicates that in the presence of surfactants, Marangoni stresses are responsible for inducing the flow in the liquid.
Oligodeoxynucleotides incorporating internucleotide phosphoroselenolate linkages have been prepared under solid-phase synthesis conditions using dimer phosphoramidites. These dimers were constructed ...following the high yielding Michaelis-Arbuzov (M-A) reaction of nucleoside
H
-phosphonate derivatives with 5′-deoxythymidine-5′-selenocyanate and subsequent phosphitylation. Efficient coupling of the dimer phosphoramidites to solid-supported substrates was observed under both manual and automated conditions and required only minor modifications to the standard DNA synthesis cycle. In a further demonstration of the utility of M-A chemistry, the support-bound selenonucleoside was reacted with an
H
-phosphonate and then chain extended using phosphoramidite chemistry. Following initial unmasking of methyl-protected phosphoroselenolate diesters, pure oligodeoxynucleotides were isolated using standard deprotection and purification procedures and subsequently characterised by mass spectrometry and circular dichroism. The CD spectra of both modified and native duplexes derived from self-complementary sequences with A-form, B-form or mixed conformational preferences were essentially superimposable. These sequences were also used to study the effect of the modification upon duplex stability which showed context-dependent destabilisation (−0.4 to −3.1 °C per phosphoroselenolate) when introduced at the 5′-termini of A-form or mixed duplexes or at juxtaposed central loci within a B-form duplex (−1.0 °C per modification). As found with other nucleic acids incorporating selenium, expeditious crystallisation of a modified decanucleotide A-form duplex was observed and the structure solved to a resolution of 1.45 Å. The DNA structure adjacent to the modification was not significantly perturbed. The phosphoroselenolate linkage was found to impart resistance to nuclease activity.
Stable selenium-modified DNA which maintains the native tertiary structure has been prepared under automated conditions enabling SAD X-ray crystallography.
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