A new class of low-barrier molecular rotors, metal trans-dihydrides, is suggested here. To test whether rapid rotation can be achieved, the known complex trans-H2Pt(P(t)Bu3)2 was experimentally studied by (2)H and (195)Pt solid-state NMR spectroscopy (powder pattern changes with temperature) and computationally modeled as a (t)Bu3P-Pt-P(t)Bu3 stator with a spinning H-Pt-H rotator. Whereas the related chloro-hydride complex, trans-H(Cl)Pt(P(t)Bu3)2, does not show rotational behavior at room temperature, the dihydride trans-H2Pt(P(t)Bu3)2 rotates fast on the NMR time scale, even at low temperatures down to at least 75 K. The highest barrier to rotation is estimated to be ∼3 kcal mol(-1), for the roughly 3 Å long rotator in trans-H2Pt(P(t)Bu3)2.
Membrane-embedded negatively charged phospholipids (MENCP) can be used as biomarkers for a range of biological processes, including early detection of apoptosis in animal cells, drug-induced phospholipidosis, and selective detection of bacterial over animal cells. Currently, several technologies for the detection of apoptosis and bacterial cells are based on the recognition of MENCPs, including the AnnexinV stain and PSVue™ probes. As probes, these technologies have limitations, the most significant of which is the need for washing the unbound probe away to achieve optimal signal. In contrast, a turn-on chemosensor selective for MENCP would address this shortcoming, and allow for a more rapid protocol for the detection of apoptosis, bacteria and for other relevant applications. In this work, the aim was to explore whether ProxyPhos chemosensors, previously reported by our group for the detection of proximally phosphorylated peptides and proteins, could be re-purposed for the detection of MENCPs. Six lead ProxyPhos sensors were screened against synthetic vesicles containing biologically relevant negatively charged phospholipids including phosphatidic acid (PA), phosphatidylglycerol (PG), cardiolipin (CL) and phosphatidylserine (PS). Through these screens, ProxyPhos sensors exhibiting high selectivity for the detection of MENCPs over zwitterionic lipids were identified. Particular selectivity was observed for PA and CL. Sensitivity of the lead sensors for MENCPs was suitable for the detection of apoptosis: ProxyPhos detected vesicles containing as little as 2.5% PS and detected camptothecin-induced apoptosis in mammalian cells in flow cytometry experiments. The results suggest that ProxyPhos sensors can be used for the detection of MENCPs in synthetic vesicles and live mammalian cells.
Described here are the development and validation of a novel approach to identify genes encoding drug targets in Streptococcus pneumoniae. The method relies on the use of an ordered genomic library composed of PCR amplicons that were generated under error-prone conditions so as to introduce random mutations into the DNA. Since some of the mutations occur in drug target-encoding genes and subsequently affect the binding of the drug to its respective cellular target, amplicons containing drug targets can be identified as those producing drug-resistant colonies when transformed into S. pneumoniae. Examination of the genetic content of the amplicon giving resistance coupled with bioinformatics and additional genetic approaches could be used to rapidly identify candidate drug target genes. The utility of this approach was verified by using a number of known antibiotics. For drugs with single protein targets, amplicons were identified that rendered S. pneumoniae drug resistant. Assessment of amplicon composition revealed that each of the relevant amplicons contained the gene encoding the known target for the particular drug tested. Fusidic acid-resistant mutants that resulted from the transformation of S. pneumoniae with amplicons containing fusA were further characterized by sequence analysis. A single mutation was found to occur in a region of the S. pneumoniae elongation factor G protein that is analogous to that already implicated in other bacteria as being associated with fusidic acid resistance. Thus, in addition to facilitating the identification of genes encoding drug targets, this method could provide strains that aid future mechanistic studies.
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