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The periplasmic oligopeptide binding protein, OppA, acts as the initial receptor for the uptake of peptides by the oligopeptide permease (Opp) in Gram-negative bacteria. Opp will handle peptides between two and five amino acid residues regardless of their sequence. The crystal structures of a series of OppA-peptide complexes have revealed an enclosed but versatile peptide binding pocket and have illustrated how tri- and tetrapeptide ligands are accommodated. Here, the crystal structures of (i) OppA complexed with a dipeptide (lysyllysine) and (ii) unliganded OppA have been solved using X-ray data extending to 1.8 and 2.4 A spacing, respectively. In the dipeptide complex, the alpha-amino group of the ligand is anchored through an ion pair interaction with Asp419, as observed in complexes with longer peptides. However, its alpha-carboxylate group forms water-mediated interactions with the guanidinium groups of Arg404 and Arg413 rather than the direct salt bridges to Arg413 and His371 observed in the tripeptide and tetrapeptide complexes, respectively. Isothermal titration calorimetric measurements of the binding of lysine-containing peptides of different lengths to OppA show that the dipeptide, KK, is bound with approximately 60-fold lower affinity than related tri- and tetrapeptides (KKK and KKKA, respectively). These data are discussed with reference to the calculated enthalpic and entropic contributions to ligand binding and the structures of the OppA peptide complexes. In the unliganded molecule, domain III has rotated as a rigid body through 26 degrees away from domains I and II, exposing the ligand binding site. The water structure in the binding cleft shows similarities to that in the various OppA-peptide complexes.

Breast, ovarian and endometrial cancers cause significant morbidity and mortality. Despite the presence of existing screening, diagnostic and treatment modalities, they continue to pose considerable unsolved challenges. Overdiagnosis is a growing problem in breast cancer screening and neither screening nor early diagnosis of ovarian or endometrial cancer is currently possible. Moreover, treatment of the diversity of these cancers presenting in the clinic is not sufficiently personalized at present. Recent technological advances, including reduced representation bisulfite sequencing, methylation arrays, digital PCR, next-generation sequencing and advanced statistical data analysis, enable the analysis of methylation patterns in cell-free tumor DNA in serum/plasma. Ongoing work is bringing these methods together for the analysis of samples from large clinical trials, which have been collected well in advance of cancer diagnosis. These efforts pave the way for the development of a noninvasive method that would enable us to overcome existing challenges to personalized medicine.

Toca 511 (vocimagene amiretrorepvec), a nonlytic, amphotropic retroviral replicating vector (RRV), encodes and delivers a functionally optimized yeast cytosine deaminase (CD) gene to tumors. In orthotopic glioma models treated with Toca 511 and 5-fluorocytosine (5-FC) the CD enzyme within infected cells converts 5-FC to 5-fluorouracil (5-FU), resulting in tumor killing. Toca 511, delivered locally either by intratumoral injection or by injection into the resection bed, in combination with subsequent oral extended-release 5-FC (Toca FC), is under clinical investigation in patients with recurrent high-grade glioma (HGG). If feasible, intravenous administration of vectors is less invasive, can easily be repeated if desired, and may be applicable to other tumor types. Here, we present preclinical data that support the development of an intravenous administration protocol. First we show that intravenous administration of Toca 511 in a preclinical model did not lead to widespread or uncontrolled replication of the RVV. No, or low, viral DNA was found in the blood and most of the tissues examined 180 days after Toca 511 administration. We also show that RRV administered intravenously leads to efficient infection and spread of the vector carrying the green fluorescent protein (GFP)-encoding gene (Toca GFP) through tumors in both immune-competent and immune-compromised animal models. However, initial vector localization within the tumor appeared to depend on the mode of administration. Long-term survival was observed in immune-competent mice when Toca 511 was administered intravenously or intracranially in combination with 5-FC treatment, and this combination was well tolerated in the preclinical models. Enhanced survival could also be achieved in animals with preexisting immune response to vector, supporting the potential for repeated administration. On the basis of these and other supporting data, a clinical trial investigating intravenous administration of Toca 511 in patients with recurrent HGG is currently open and enrolling.