Targeting Mutant KRAS in Pancreatic Cancer: Futile or Promising?

Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal cancers with a dismal prognosis for the patient. This is due to limited diagnostic options for the early detection of the disease as well as its rather aggressive nature. Despite major advances in oncologic research in general, the treatment options in the clinic for PDAC have only undergone minor changes in the last decades. One major treatment advance would be the successful targeting of the oncogenic driver KRASmut. In the past, the indirect targeting of KRAS has been exploited, e. g., via upstream inhibition of receptor tyrosine kinases or via downstream MEK or PI3K inhibition. However, the experience gained from clinical trials and from the clinic itself in the treatment of KRASmut cancer entities has dampened the initial euphoria. Lately, with the development of KRASG12C-specific inhibitors, not only the direct but also the indirect targeting of KRASmut has gained momentum again. Though preclinical studies and preliminary early clinical studies of monotherapies have shown promising results, they have been overshadowed by the swift development of resistances resulting in inconsistent responses in patient cohorts. Currently, several different combination therapies for KRASmut cancer are being explored. If they hold the promise they have made in preclinical studies, they might also be suitable treatment options for patients suffering from PDAC.

Development and Clinical Validation of Discriminatory Multitarget Digital Droplet PCR Assays for the Detection of Hot Spot KRAS and NRAS Mutations in Cell-Free DNA.

Detection and quantification of tumor-derived KRAS and NRAS mutations in plasma cell-free DNA (cfDNA) holds great potential for cancer diagnostics and treatment response monitoring. Because of high sensitivity, specificity, robustness, and affordability, digital droplet PCR (ddPCR) is ideally suited for this application but requires discriminatory multiplexing when used as screening assay. We therefore designed, optimized, and clinically validated mutation-specific locked nucleic acid-based ddPCR assays for 14 commonly occurring KRAS and NRAS mutations and assembled these assays into seven discriminatory multitarget screening assays covering two to six single-nucleotide variants each. Limit of detection, limit of blank, and interassay accuracy were determined. Assay performance and suitability for screening in cfDNA were validated with plasma samples from a clinically fully characterized cohort of pancreatic cancer patients and healthy controls. Limits of detection for single-target assays were between 0.0015% and 0.069% variant allele fraction, and between 0.022% and 0.16% for multitarget assays. Dilution linearity and interassay accuracy were excellent throughout (r2 > 0.99). Multitarget assay screening of cfDNA extracted from pancreatic cancer patients with unknown KRAS mutational status correctly identified single-nucleotide variants in 45 of 45 (100%) of tumor-derived cell-free DNA-positive samples. In summary, we herein present and clinically validate generic single-target and discriminatory multitarget ddPCR assays for KRAS and NRAS hot spot mutations with broad applicability for clinical and translational research.

Novel auristatin E-based albumin-binding prodrugs with superior anticancer efficacy in vivo compared to the parent compound.

Auristatins are a class of highly cytotoxic tubulin-disrupting peptides, which have shown limited therapeutic effect as free agents in clinical trials. In our continuing effort to develop acid-sensitive albumin-binding anticancer drugs exploiting circulating serum albumin as the drug carrier, we investigated the highly toxic drug payload auristatin E to assess whether the corresponding albumin-binding prodrugs were a viable option for achieving significant and concomitant tolerable antitumor activity. To achieve our goal, we developed a new aromatic maleimide-bearing linker (Sulf07) which enhanced both water solubility and stability of the prodrugs. In this study, we describe two auristatin E-based albumin-binding drugs, AE-Keto-Sulf07 and AE-Ester-Sulf07, which were designed to release the active compound at the tumor site in a pH-dependent manner. These prodrugs incorporate an acid-sensitive hydrazone bond, formed by the reaction of a carbonyl-containing auristatin E derivative with the hydrazide group of the water-solubilizing maleimide-bearing linker Sulf07. A panel of patient- and cell-derived human tumor xenograft models (melanoma A375, ovarian carcinoma A2780, non-small-cell lung cancer LXFA737 and LXFE937, and head and neck squamous cell carcinomas) were screened with starting tumor volumes in the range of either 130-150 mm3 (small tumors) or 270-380 mm3 (large tumors). Both albumin-binding prodrugs showed compelling anticancer efficacy compared to the parent drug auristatin E, inducing statistically significant long-term partial and/or complete tumor regressions. AE-Keto-Sulf07 displayed very good antitumor response over a wide dose range, 3.0-6.5 mg/kg (5-8 injections, biweekly). AE-Ester-Sulf07 was highly efficacious between 1.9 and 2.4 mg/kg (8 injections, biweekly) or at 3.8 mg/kg (4 injections, weekly), but caused cumulative skin irritation due to scratching and biting. In contrast at its MTD, auristatin E (0.3 mg/kg, 8 injections, biweekly) was only marginally active. In summary, AE-Keto-Sulf07 and AE-Ester-Sulf07 are novel acid-sensitive albumin-binding prodrugs demonstrating tumor regressions in all of the evaluated human tumor xenograft models thus supporting the stratagem that albumin can be used as an effective drug carrier for the highly potent class of auristatins.

Revisiting the thiosemicarbazonecopper(II) reaction with glutathione. Activity against colorectal carcinoma cell lines.

Thiosemicarbazones (TSCs), and their copper derivatives, have been extensively studied mainly due to the potential applications as antitumor compounds. A part of the biological activity of the TSC-CuII complexes rests on their reactivity against cell reductants, as glutathione (GSH). The present paper describes the structure of the [Cu(PTSC)(ONO2)]n compound (1) (HPTSC=pyridine-2-carbaldehyde thiosemicarbazone) and its spectroscopic and magnetic properties. ESI studies performed on the reaction of GSH with 1 and the analogous [{Cu(PTSC*)(ONO2)}2] derivative (2, HPTSC*=pyridine-2-carbaldehyde 4N-methylthiosemicarbazone) show the absence of peaks related with TSC-Cu-GSH species. However GSH-Cu ones are detected, in good agreement with the release of CuI ions after reduction in the experimental conditions. The reactivity of 1 and 2 with cytochrome c and myoglobin and their activities against HT-29 and SW-480 colon carcinoma cell lines are compared with those shown by the free HPTSC and HPTSC* ligands.

From Insect to Man: Photorhabdus Sheds Light on the Emergence of Human Pathogenicity.

Photorhabdus are highly effective insect pathogenic bacteria that exist in a mutualistic relationship with Heterorhabditid nematodes. Unlike other members of the genus, Photorhabdus asymbiotica can also infect humans. Most Photorhabdus cannot replicate above 34°C, limiting their host-range to poikilothermic invertebrates. In contrast, P. asymbiotica must necessarily be able to replicate at 37°C or above. Many well-studied mammalian pathogens use the elevated temperature of their host as a signal to regulate the necessary changes in gene expression required for infection. Here we use RNA-seq, proteomics and phenotype microarrays to examine temperature dependent differences in transcription, translation and phenotype of P. asymbiotica at 28°C versus 37°C, relevant to the insect or human hosts respectively. Our findings reveal relatively few temperature dependant differences in gene expression. There is however a striking difference in metabolism at 37°C, with a significant reduction in the range of carbon and nitrogen sources that otherwise support respiration at 28°C. We propose that the key adaptation that enables P. asymbiotica to infect humans is to aggressively acquire amino acids, peptides and other nutrients from the human host, employing a so called "nutritional virulence" strategy. This would simultaneously cripple the host immune response while providing nutrients sufficient for reproduction. This might explain the severity of ulcerated lesions observed in clinical cases of Photorhabdosis. Furthermore, while P. asymbiotica can invade mammalian cells they must also resist immediate killing by humoral immunity components in serum. We observed an increase in the production of the insect Phenol-oxidase inhibitor Rhabduscin normally deployed to inhibit the melanisation immune cascade. Crucially we demonstrated this molecule also facilitates protection against killing by the alternative human complement pathway.

Optimization of adiponectin-derived peptides for inhibition of cancer cell growth and signaling.

Adiponectin, an adipose tissue-excreted adipokine plays protective roles in metabolic and cardiovascular diseases and exerts anti-cancer activities, partially by interfering with leptin-induced signaling. Previously we identified the active site in the adiponectin protein, and generated both a nanomolar monomeric agonist of the adiponectin receptor (10-mer ADP355) and an antagonist (8-mer ADP400) to modulate various adiponectin receptor-mediated cellular functions. As physiologically circulating adiponectin forms multimeric complexes, we also generated an agonist dimer with improved biodistribution and in vitro efficacy. In the current report, we attempted to optimize the monomeric agonist structure. Neither extension of the peptide up to 14-mer analogs nor reinstallation of native residues in permissible positions enhanced significantly the activity profile. The only substitutions that resulted in 5-10-fold improved agonistic activity were the replacement of turn-forming Gly4 and Tyr7 residues with Pro and Hyp, respectively, yielding the more active native ß-sheet structure. All peptides retained good stability in human serum exhibiting half-lives >2 h. The cellular efficacy and stability rankings among the peptides followed expected structure-activity relationship trends. To investigate whether simultaneous activation of adiponectin pathways and inhibition of leptin-induced signals can result in cytostatic and anti-oncogenic signal transduction processes, we developed a chimera of the leptin receptor antagonist peptide Allo-aca (placed to the N-terminus) and ADP355 (at the C-terminus). The in vitro anti-tumor activity and intracellular signaling of the chimera were dominated by the more active Allo-aca component. The ADP355 part, however, reversed unfavorable in vivo metabolic effects of the leptin receptor antagonist.

A Photorhabdus natural product inhibits insect juvenile hormone epoxide hydrolase.

Simple urea compounds ("phurealipids") have been identified from the entomopathogenic bacterium Photorhabdus luminescens, and their biosynthesis was elucidated. Very similar analogues of these compounds have been previously developed as inhibitors of juvenile hormone epoxide hydrolase (JHEH), a key enzyme in insect development and growth. Phurealipids also inhibit JHEH, and therefore phurealipids might contribute to bacterial virulence.

Insect-specific production of new GameXPeptides in photorhabdus luminescens TTO1, widespread natural products in entomopathogenic bacteria.

Discovery of new natural products by heterologous expression reaches its limits, especially when specific building blocks are missing in the heterologous host or the production medium. Here, we describe the insect-specific production of the new GameXPeptides E-H (5-8) from Photorhabdus luminescens TTO1, which can be produced heterologously from expression of the GameXPeptide synthetase GxpS only upon supplementation of the production media with the missing building blocks, and thus must be regarded as the true natural products under natural conditions.

Xenortide Biosynthesis by Entomopathogenic Xenorhabdus nematophila.

The biosynthesis gene cluster of the xenortides and a new derivative, xenortide D, which is produced in only trace amounts, was identified in Xenorhabdus nematophila. The structure of xenortide D was elucidated using a combination of labeling experiments followed by MS analysis and was confirmed by synthesis. Bioactivity tests revealed a weak activity of tryptamine-carrying xenortides against Plasmodium falciparum and Trypanosoma brucei.

Yeast homologous recombination cloning leading to the novel peptides ambactin and xenolindicin.

Heterologous production of GameXPeptide A (1), as well as of the novel peptide natural products ambactin (2) and xenolindicins A-C (3 a-c), was achieved by using the "overlap extension PCR-yeast homologous recombination" (ExRec) method. ExRec cloning is based on the ability of yeast to assemble overlapping DNA fragments into functional plasmids. Here we used this technique to clone a total of 15 biosynthesis gene clusters from Photorhabdus and Xenorhabdus with sizes of up to 45 kb. The structures of the novel compounds 2 and 3 a, which were produced in Escherichia coli, were elucidated by detailed MS and bioinformatics analysis, and additionally confirmed by their chemical synthesis.

Rapid determination of the amino acid configuration of xenotetrapeptide.

An E. coli strain with deletions in five transaminases (ΔaspC ΔilvE ΔtyrB ΔavtA ΔybfQ) was constructed to be unable to degrade several amino acids. This strain was used as an expression host for the analysis of the amino acid configuration of nonribosomally synthesized peptides, including the novel peptide "xenotetrapeptide" from Xenorhabdus nematophila, by using a combination of labeling experiments and mass spectrometry. Additionally, the number of D-amino acids in the produced peptide was assigned following simple cultivation of the expression strain in D2 O.

Hydroxyproline substitutions stabilize non-glycosylated drosocin against serum proteases without challenging its antibacterial activity.

The increasing incidence of multi- and pan-resistant pathogens demands novel compounds to fight Grampositive and especially Gram-negative bacteria. Among the currently investigated compound classes, antimicrobial peptides (AMPs) inhibiting specific bacterial targets appear especially promising for systemic therapy of infections, although unmodified linear peptides are typically rapidly degraded by serum proteases. Proline-rich AMPs have been heavily investigated in recent years due to their low toxicity and proven in vivo efficacy. Here, we report novel unglycosylated drosocin analogs with extended half-life in mouse serum and improved activity against Gram-negative pathogens Escherichia coli and Klebsiella pneumoniae. Substituting proline (Pro) residues in positions 3, 5, 10, and 14 with trans-4-hydroxy-Lproline ((t)Hyp) improved the antibacterial activity, whereas substitution of Pro-16 reduced the activity. Drosocin analogs with (t)Hyp in positions 3 and 5 were also four to eight times more stable in mouse serum than the unmodified analog. The new compounds were not toxic against human HeLa, HEK293, and HepG2 cell lines and showed no hemolytic activity against human erythrocytes at peptide concentrations of at least 600 µg/mL.

Rhabdopeptides as insect-specific virulence factors from entomopathogenic bacteria.

Six novel linear peptides, named "rhabdopeptides", have been identified in the entomopathogenic bacterium Xenorhabdus nematophila after the discovery of the corresponding rdp gene cluster by using a promoter trap strategy for the detection of insect-inducible genes. The structures of these rhabdopeptides were deduced from labeling experiments combined with detailed MS analysis. Detailed analysis of an rdp mutant revealed that these compounds participate in virulence towards insects and are produced upon bacterial infection of a suitable insect host. Furthermore, two additional rhabdopeptide derivatives produced by Xenorhabdus cabanillasii were isolated, these showed activity against insect hemocytes thereby confirming the virulence of this novel class of compounds.

Controlled systemic release of therapeutic peptides from PEGylated prodrugs by serum proteases.

A novel concept to release peptidic drugs systemically by serum proteases from a PEGylated precursor makes it possible to tune release kinetics to fit the medical needs. Drug release depends on the size of the PEG polymer and the sequence and length of the peptide linker. The antimicrobial activities of the prodrugs were even better than those of the free peptides, whereas direct PEGylation abolished the peptide activity.

Neutral loss fragmentation pattern based screening for arginine-rich natural products in Xenorhabdus and Photorhabdus.

Although sharing a certain degree of structural uniformity, natural product classes exhibit variable functionalities such as different amino acid or acyl residues. During collision induced dissociation, some natural products exhibit a conserved fragmentation pattern close to the precursor ion. The observed fragments result from a shared set of neutral losses, creating a unique fragmentation pattern, which can be used as a fingerprint for members of these natural product classes. The culture supernatants of 69 strains of the entomopathogenic bacteria Photorhabdus and Xenorhabdus were analyzed by MALDI-MS(2), and a database comprising MS(2) data from each strain was established. This database was scanned for concordant fragmentation patterns of different compounds using a customized software, focusing on relative mass differences of the fragment ions to their precursor ion. A novel group of related natural products comprising 25 different arginine-rich peptides from 16 different strains was identified due to its characteristic neutral loss fragmentation pattern, and the structures of eight compounds were elucidated. Two biosynthesis gene clusters encoding nonribosomal peptide synthetases were identified, emphasizing the possibility to identify a group of structurally and biosynthetically related natural products based on their neutral loss fragmentation pattern.

Synthesis of szentiamide, a depsipeptide from entomopathogenic Xenorhabdus szentirmaii with activity against Plasmodium falciparum.

The synthesis of the recently characterized depsipeptide szentiamide (1), which is produced by the entomopathogenic bacterium Xenorhabdus szentirmaii, is described. Whereas no biological activity was previously identified for 1, the material derived from the efficient synthesis enabled additional bioactivity tests leading to the identification of a notable activity against insect cells and Plasmodium falciparum, the causative agent of malaria.