A synthetically lethal nanomedicine delivering novel inhibitors of polynucleotide kinase 3'-phosphatase (PNKP) for targeted therapy of PTEN-deficient colorectal cancer.

Phosphatase and TENsin homolog deleted on chromosome 10 (PTEN) is a major tumor-suppressor protein that is lost in up to 75% of aggressive colorectal cancers (CRC). The co-depletion of PTEN and a DNA repair protein, polynucleotide kinase 3'-phosphatase (PNKP), has been shown to lead to synthetic lethality in several cancer types including CRC. This finding inspired the development of novel PNKP inhibitors as potential new drugs against PTEN-deficient CRC. Here, we report on the in vitro and in vivo evaluation of a nano-encapsulated potent, but poorly water-soluble lead PNKP inhibitor, A83B4C63, as a new targeted therapeutic for PTEN-deficient CRC. Our data confirmed the binding of A83B4C63, as free or nanoparticle (NP) formulation, to intracellular PNKP using the cellular thermal shift assay (CETSA), in vitro and in vivo. Dose escalating toxicity studies in healthy CD-1 mice, based on measurement of animal weight changes and biochemical blood analysis, revealed the safety of both free and nano-encapsulated A83B4C63, at assessed doses of ≤50 mg/kg. Nano-carriers of A83B4C63 effectively inhibited the growth of HCT116/PTEN-/- xenografts in NIH-III nude mice following intravenous (IV) administration, but not that of wild-type HCT116/PTEN+/+ xenografts. This was in contrast to IV administration of A83B4C63 solubilized with the aid of Cremophor EL: Ethanol (CE), which led to similar tumor growth to that of formulation excipients (NP or CE without drug) or 5% dextrose. This observation was attributed to the higher levels of A83B4C63 delivered to tumor tissue by its NP formulation. Our data provide evidence for the success of NPs of A83B4C63, as novel synthetically lethal nano-therapeutics in the treatment of PTEN-deficient CRC. This research also highlights the potential of successful application of nanomedicine in the drug development process.

Lewis Acid-Mediated Cyclization of Allenyl Aryl Ketones.

The cyclization of a series of nonheterocyclic allenyl aryl ketones was examined using boron trifluoride etherate and indium triflate to mediate the reaction. Yields with BF3 were low in most instances due mainly to competitive destruction of the substrates. With In(OTf)3, there was less decomposition, and the yields of the cyclized product were much higher, but only for substrates with electron-donating substituents. Cyclization did not occur without those substituents. A computational study using the ωB97X-D/6-311+G(2d,p)//ωB97X-D/6-31+G(d,p) method confirmed better stability of the σ-complexed substrate by indium(III) and that meta-substituents on the phenyl ring of the substrate significantly influenced the activation barrier of the cyclization, whereas the effect of para-substituents was almost negligible. The computational results supported the idea that the cyclization is a 4π-electrocyclization and not a 5-endo-dig ring closure as had been proposed in the literature.

Nanoencapsulation of Novel Inhibitors of PNKP for Selective Sensitization to Ionizing Radiation and Irinotecan and Induction of Synthetic Lethality.

There is increasing interest in developing and applying DNA repair inhibitors in cancer treatment to augment the efficacy of radiation and conventional genotoxic chemotherapy. However, targeting the inhibitor is required to avoid reducing the repair capacity of normal tissue. The aim of this study was to develop nanodelivery systems for the encapsulation of novel imidopiperidine-based inhibitors of the DNA 3'-phosphatase activity of polynucleotide kinase/phosphatase (PNKP), a DNA repair enzyme that plays a critical role in rejoining DNA single- and double-strand breaks. For this purpose, newly identified hit compounds with potent PNKP inhibitory activity, imidopiperidines A12B4C50 and A83B4C63 were encapsulated in polymeric micelles of different poly(ethylene oxide)- b-poly(ε-caprolactone) (PEO- b-PCL)-based structures. Our results showed efficient loading of A12B4C50 and A83B4C63 in PEO- b-PCLs with pendent carboxyl and benzyl carboxylate groups, respectively, and relatively slow release over 24 h. Both free and encapsulated inhibitors were able to sensitize HCT116 cells to radiation and the topoisomerase I poison, irinotecan. In addition, the encapsulated inhibitors were capable of inducing synthetic lethalilty in phosphatase and tensin homologue (PTEN)-deficient cells. We also established the validity of the peptide GE11 as a suitable ligand for active targeted delivery of nanoencapsulated drugs to colorectal cancer cells overexpressing epidermal growth factor receptor (EGFR). Our results show the potential of nanoencapsulated inhibitors of PNKP as either mono or combined therapeutic agents for colorectal cancer.

Scope and Mechanism of a True Organocatalytic Beckmann Rearrangement with a Boronic Acid/Perfluoropinacol System under Ambient Conditions.

Catalytic activation of hydroxyl functionalities is of great interest for the production of pharmaceuticals and commodity chemicals. Here, 2-alkoxycarbonyl- and 2-phenoxycarbonyl-phenylboronic acid were identified as efficient catalysts for the direct and chemoselective activation of oxime N-OH bonds in the Beckmann rearrangement. This classical organic reaction provides a unique approach to prepare functionalized amide products that may be difficult to access using traditional amide coupling between carboxylic acids and amines. Using only 5 mol % of boronic acid catalyst and perfluoropinacol as an additive in a polar solvent mixture, the operationally simple protocol features mild conditions, a broad substrate scope, and a high functional group tolerance. A wide variety of diaryl, aryl-alkyl, heteroaryl-alkyl, and dialkyl oximes react under ambient conditions to afford high yields of amide products. Free alcohols, amides, carboxyesters, and many other functionalities are compatible with the reaction conditions. Investigations of the catalytic cycle revealed a novel boron-induced oxime transesterification providing an acyl oxime intermediate involved in a fully catalytic nonself-propagating Beckmann rearrangement mechanism. The acyl oxime intermediate was prepared independently and was subjected to the reaction conditions. It was found to be self-sufficient; it reacts rapidly, unimolecularly without the need for free oxime. A series of control experiments and 18O labeling studies support a true catalytic pathway involving an ionic transition structure with an active and essential role for the boronyl moiety in both steps of transesterification and rearrangement. According to 11B NMR spectroscopic studies, the additive perfluoropinacol provides a transient, electrophilic boronic ester that is thought to serve as an internal Lewis acid to activate the ortho-carboxyester and accelerate the initial, rate-limiting step of transesterification between the precatalyst and the oxime substrate.

Nazarov reactions intercepted by (4 + 3) cycloadditions with oxygen-substituted dienes.

The oxyallyl cation intermediate from the Lewis acid mediated Nazarov reaction of an allenyl vinyl ketone was intercepted by acyclic, 2-silyloxy-substituted butadienes by highly regioselective (4 + 3) cycloadditions. Stereoselectivity was often modest, but in some instances steric interactions were responsible for high selectivity. The results are consistent with concerted (4 + 3) cycloadditions. In many instances, the (4 + 3) products were susceptible to fragmentation or rearrangement in the presence of the Lewis acid.

Torquoselectivity in the Nazarov reactions of allenyl vinyl ketones.

Nazarov reactions mediated by BF3-etherate of a series of carbon-substituted allenyl vinyl ketones provided intermediates in which substituents on the termini of the allenes had rotated away from the vinyl moieties, and these intermediates were trapped by (4 + 3)-cyclizations. A computational examination of the torquoselectivity of these Nazarov reactions confirmed a kinetic preference for the observed isomers and pointed to steric interactions and the degree of allene deformation as significant factors in determining the torquoselectivity. The study also suggested that the high proportion of one geometrical isomer in the Nazarov products might also be due to some preferential trapping of the major Nazarov intermediate.

Susceptibility of inbred mice to Rickettsia parkeri.

Rickettsia parkeri, a member of the spotted fever group Rickettsia, is the causative agent of American boutonneuse fever in humans. Despite the increased recognition of human cases, limited information is available regarding the infection of invertebrate and vertebrate hosts for this emerging tick-borne disease. Toward the development of a viable transmission model and to further characterize the pathology associated with R. parkeri infection, inbred mouse strains (A/J, BALB/c, C3H/HeJ, and C3H/HeN) were intravenously and intradermally inoculated with 10(5) low-passage-number R. parkeri (Portsmouth strain), and infection, gross pathology, and histopathology were scored. Additionally, a quantitative real-time PCR (qPCR) was performed to estimate rickettsial load in heart, lung, spleen, and liver tissues of infected mice at 19 days postinoculation. Of the A/J, BALB/c, and C3H/HeN mice, none displayed universal pathology consistent with sustained infection. Compared to age-matched control mice, the intravenously inoculated C3H/HeJ mice exhibited marked facial edema and marked splenomegaly upon gross examination, while the intradermally inoculated mice developed characteristic eschar-like lesions. The C3H/HeJ mice also exhibited the greatest concentrations of rickettsial DNA from heart, lung, liver, and spleen samples when examined by qPCR. The similarity of the pathology of human disease and sustained infection suggests that the C3H/HeJ strain of mice is a promising candidate for subsequent experiments to examine the tick transmission, dissemination, and pathology of R. parkeri rickettsiosis.