Cyclopentadiene as a Multifunctional Reagent for Normal- and Inverse-Electron Demand Diels-Alder Bioconjugation.

Optimizing the Diels-Alder (DA) reaction for aqueous coupling has resulted in practical methods to link molecules such as drugs and diagnostic agents to proteins. Both normal electron demand (NED) and inverse electron demand (IED) DA coupling schemes have been employed, but neither mechanism entails a common multipurpose reactive group. This report focuses on expanding the bioconjugation toolbox for cyclopentadiene through the identification of reactive groups that couple through NED or IED mechanisms in aqueous solution. Dienophiles and tetrazine derivatives were screened for reactivity and selectivity toward antibodies bearing cyclopentadiene amino acids to yield bioconjugates. Twelve NED dienophiles and four tetrazine-based IED substrates were identified as capable of practical biocoupling. Furthermore, tetrazine ligation to cyclopentadiene occurred at a rate of 3.3 ± 0.5 M-1 s-1 and was capable of bioorthogonal transformations, as evidenced by the selective protein labeling in serum. Finally, an antibody-drug conjugate (ADC)-bearing monomethyl auristatin E was prepared via tetrazine conjugation to cyclopentadiene. The resulting ADC was stable and demonstrated potent activity in vitro. These findings expand the utility of cyclopentadiene as a tool to couple entities to proteins via dual DA addition mechanisms.

Metabolite-Based Modification of Poly(l-lysine) for Improved Gene Delivery.

Synthetic gene delivery systems employ multiple functions to enable safe and effective transport of DNA to target cells. Here, we describe metabolite-based poly(l-lysine) (PLL) modifiers that improve transfection by imparting both pH buffering and nanoparticle stabilization functions within a single molecular unit. PLL modifiers were based on morpholine (M), morpholine and niacin (MN), or thiomorpholine (TM). PLL modification with (MN) or (TM) imparted buffering function over the pH range of 5-7 both in solution and live cells and enhanced the stability of PLL DNA nanoparticles, which exhibited higher resistance to polyanion exchange and prolonged blood circulation. These properties translated into increased transfection efficiency in vitro coupled with reduced toxicity compared to unmodified PLL and PLL(M). Furthermore, PEG-PLL(MN) DNA nanoparticles transfected muscle tissue in vivo for >45 days following intramuscular injection. These polymer modifiers demonstrate the successful design of multifunctional units that improve transfection of synthetic gene delivery systems while maintaining biocompatibility.

A threonine turnstile defines a dynamic amphiphilic binding motif in the AAA ATPase p97 allosteric binding site.

The turnstile motion of two neighboring threonines sets up a dynamic side chain interplay that can accommodate both polar and apolar ligands in a small molecule allosteric protein binding site. A computational model based on SAR data and both X-ray and cryo-EM structures of the AAA ATPase p97 was used to analyze the effects of paired threonines at the inhibitor site. Specifically, the Thr side chain hydroxyl groups form a hydrogen bonding network that readily accommodates small, highly polar ligand substituents. Conversely, diametric rotation of the χ1 torsion by 150-180° orients the side chain ß-methyl groups into the binding cleft, creating a hydrophobic pocket that can accommodate small, apolar substituents. This motif was found to be critical for rationalizing the affinities of a structurally focused set of inhibitors of p97 covering a > 2000-fold variation in potencies, with a preference for either small-highly polar or small-apolar groups. The threonine turnstile motif was further validated by a PDB search that identified analogous binding modes in ligand interactions in PKB, as well as by an analysis of NMR structures demonstrating additional gear-like interactions between adjacent Thr pairs. Combined, these data suggest that the threonine turnstile motif may be a general feature of interest in protein binding pockets.

Total Synthesis and Biological Evaluation of Tubulysin Analogues.

We report a second-generation synthesis of the exceedingly potent antimitotic agent N14-desacetoxytubulysin H (1) as well as the preparation of nine analogues of this lead structure. Highlights of our synthetic efforts include an efficient late-stage functionalization that allows for the preparation of new side-chain- and backbone-modified analogues. We also discovered C-terminal modifications that preserve the exquisite biological activity of acid 1 and offer the opportunity for effective conjugation to cell type-targeting moieties. All analogues had antiproliferative activities in the high picomolar to low nanomolar range and caused apoptosis and mitotic arrest as measured in a high content nuclear morphology assay. The ten synthetic agents described herein spanned a range of almost 4 orders of magnitude in biological activity and illustrate the continued potential to discover extraordinarily potent antiproliferative compounds based on natural product leads.

Optimization of pyrazole-containing 1,2,4-triazolo-[3,4-b]thiadiazines, a new class of STAT3 pathway inhibitors.

Structure-activity relationship studies of a 1,2,4-triazolo-[3,4-b]thiadiazine scaffold, identified in an HTS campaign for selective STAT3 pathway inhibitors, determined that a pyrazole group and specific aryl substitution on the thiadiazine were necessary for activity. Improvements in potency and metabolic stability were accomplished by the introduction of an α-methyl group on the thiadiazine. Optimized compounds exhibited anti-proliferative activity, reduction of phosphorylated STAT3 levels and effects on STAT3 target genes. These compounds represent a starting point for further drug discovery efforts targeting the STAT3 pathway.

Design and Evaluation of ZD06519, a Novel Camptothecin Payload for Antibody Drug Conjugates.

In recent years, the field of antibody drug conjugates (ADC) has seen a resurgence, largely driven by the clinical benefit observed in patients treated with ADCs incorporating camptothecin-based topoisomerase I inhibitor payloads. Herein, we present the development of a novel camptothecin ZD06519 (FD1), which has been specifically designed for its application as an ADC payload. A panel of camptothecin analogs with different substituents at the C-7 and C-10 positions of the camptothecin core was prepared and tested in vitro. Selected compounds spanning a range of potency and hydrophilicity were elaborated into drug-linkers, conjugated to trastuzumab, and evaluated in vitro and in vivo. ZD06519 was selected on the basis of its favorable properties as a free molecule and as an antibody conjugate, which include moderate free payload potency (∼1 nmol/L), low hydrophobicity, strong bystander activity, robust plasma stability, and high-monomeric ADC content. When conjugated to different antibodies using a clinically validated MC-GGFG-based linker, ZD06519 demonstrated impressive efficacy in multiple cell line-derived xenograft models and noteworthy tolerability in healthy mice, rats, and non-human primates.

Engineered Ultrasmall Nanoparticle Drug-Immune Conjugates with "Hit and Run" Tumor Delivery to Eradicate Gastric Cancer.

Despite advances by recently approved antibody-drug conjugates in treating advanced gastric cancer patients, substantial limitations remain. Here, several key obstacles are overcome by developing a first-in-class ultrasmall (sub-8-nanometer (nm)) anti-human epidermal growth factor receptor 2 (HER2)-targeting drug-immune conjugate nanoparticle therapy. This multivalent fluorescent core-shell silica nanoparticle bears multiple anti-HER2 single-chain variable fragments (scFv), topoisomerase inhibitors, and deferoxamine moieties. Most surprisingly, drawing upon its favorable physicochemical, pharmacokinetic, clearance, and target-specific dual-modality imaging properties in a "hit and run" approach, this conjugate eradicated HER2-expressing gastric tumors without any evidence of tumor regrowth, while exhibiting a wide therapeutic index. Therapeutic response mechanisms are accompanied by the activation of functional markers, as well as pathway-specific inhibition. Results highlight the potential clinical utility of this molecularly engineered particle drug-immune conjugate and underscore the versatility of the base platform as a carrier for conjugating an array of other immune products and payloads.

Cyclic RGD peptidomimetics containing bifunctional diketopiperazine scaffolds as new potent integrin ligands.

The synthesis of eight bifunctional diketopiperazine (DKP) scaffolds is described; these were formally derived from 2,3-diaminopropionic acid and aspartic acid (DKP-1-DKP-7) or glutamic acid (DKP-8) and feature an amine and a carboxylic acid functional group. The scaffolds differ in the configuration at the two stereocenters and the substitution at the diketopiperazinic nitrogen atoms. The bifunctional diketopiperazines were introduced into eight cyclic peptidomimetics containing the Arg-Gly-Asp (RGD) sequence. The resulting RGD peptidomimetics were screened for their ability to inhibit biotinylated vitronectin binding to the purified integrins α(v)ß(3) and α(v)ß(5), which are involved in tumor angiogenesis. Nanomolar IC(50) values were obtained for the RGD peptidomimetics derived from trans DKP scaffolds (DKP-2-DKP-8). Conformational studies of the cyclic RGD peptidomimetics by (1)H NMR spectroscopy experiments (VT-NMR and NOESY spectroscopy) in aqueous solution and Monte Carlo/Stochastic Dynamics (MC/SD) simulations revealed that the highest affinity ligands display well-defined preferred conformations featuring intramolecular hydrogen-bonded turn motifs and an extended arrangement of the RGD sequence [Cß(Arg)-Cß(Asp) average distance ≥8.8 Å]. Docking studies were performed, starting from the representative conformations obtained from the MC/SD simulations and taking as a reference model the crystal structure of the extracellular segment of integrin α(v)ß(3) complexed with the cyclic pentapeptide, Cilengitide. The highest affinity ligands produced top-ranked poses conserving all the important interactions of the X-ray complex.

The therapeutic window of antibody drug conjugates: A dogma in need of revision.

Despite a prevailing dogma wherein antibody drug conjugates (ADCs) increase the maximum tolerated dose of potent cytotoxin payloads while lowering the minimum effective dose, mounting clinical evidence argues that the tolerated doses of ADCs are not significantly different from those of related small molecules. Nonetheless, when dosed at or near the maximum tolerated dose, certain ADCs demonstrate improved efficacy. Understanding the challenges and opportunities for this class of biotherapeutics will help improve the design of next-generation ADCs.

Chemoselective synthesis of sialic acid 1,7-lactones.

The chemoselective synthesis of the 1,7-lactones of N-acetylneuraminic acid, N-glycolylneuraminic acid, and 3-deoxy-d-glycero-d-galacto-nononic acid is accomplished in two steps: a simple treatment of the corresponding free sialic acid with benzyloxycarbonyl chloride and a successive hydrogenolysis of the formed 2-benzyloxycarbonyl 1,7-lactone. The instability of the 1,7-lactones to protic solvents has been also evidenced together with the rationalization of the mechanism of their formation under acylation conditions. The results permit to dispose of authentic 1,7-sialolactones to be used as reference standards and of a procedure useful for the preparation of their isotopologues to be used as inner standards in improved analytical procedures for the gas liquid chromatography-mass spectrometry (GLC-MS) analysis of 1,7-sialolactones in biological media.

PTPRT epigenetic silencing defines lung cancer with STAT3 activation and can direct STAT3 targeted therapies.

Signal Transducers and Activators of Transcription-3 (STAT3), a potent oncogenic transcription factor, is constitutively activated in lung cancer, but mutations in pathway genes are infrequent. Protein Tyrosine Phosphatase Receptor-T (PTPRT) is an endogenous inhibitor of STAT3 and PTPRT loss-of-function represents one potential mechanism of STAT3 hyperactivation as observed in other malignancies. We determined the role of PTPRT promoter methylation and sensitivity to STAT3 pathway inhibitors in non-small cell lung cancer (NSCLC). TCGA and Pittsburgh lung cancer cohort methylation data revealed hypermethylation of PTPRT associated with diminished mRNA expression in a subset of NSCLC patients. We report frequent hypermethylation of the PTPRT promoter which correlates with transcriptional silencing of PTPRT and increased STAT3 phosphorylation (Y705) as determined by methylation-specific PCR (MSP) and real time quantitative reverse transcription (RT)-PCR in NSCLC cell lines. Silencing of PTPRT using siRNA in H520 lung cancer cell line resulted in increased pSTAT3Tyr705 and upregulation of STAT3 target genes such as Cyclin D1 and Bcl-XL expression. We show this association of PRPRT methylation with upregulation of the STAT3 target genes Cyclin D1 and Bcl-XL in patient derived lung tumour samples. We further demonstrate that PTPRT promoter methylation associated with different levels of pSTAT3Ty705 in lung cancer cell lines had selective sensitivity to STAT3 pathway small molecule inhibitors (SID 864,669 and SID 4,248,543). Our data strongly suggest that silencing of PTPRT by promoter hypermethylation is an important mechanism of STAT3 hyperactivation and targeting STAT3 may be an effective approach for the development of new lung cancer therapeutics.

Combination of a binaphthol-derived phosphite and a C1-symmetric phosphinamine generates heteroleptic catalysts in Rh- and Pd-mediated reactions.

Heteroleptic complexes, formed selectively by using a 1 : 1 combination of a sigma-donor and a pi-acceptor ligand, are involved in Rh- and Pd-catalysed reactions.

The first synthesis of N-acetylneuraminic acid 1,7-lactone.

N-Acetylneuraminic acid is transformed into its until now unavailable and rather unwieldy 1,7-lactone, via the manageable 2-benzyloxycarbonyl N-acetylneuraminic acid 1,7-lactone which generates the free lactone in quantitative yield by hydrogenolysis.

Optimization of Phenyl Indole Inhibitors of the AAA+ ATPase p97.

Optimization of the side-chain of a phenyl indole scaffold identified from a high-throughput screening campaign for inhibitors of the AAA+ ATPase p97 is reported. The addition of an N-alkyl piperazine led to high potency of this series in a biochemical assay, activity in cell-based assays, and excellent pharmaceutical properties. Molecular modeling based on a subsequently obtained cryo-EM structure of p97 in complex with a phenyl indole was used to rationalize the potency of these allosteric inhibitors.

Elucidation of the opening of the epoxidic ring of the 3beta-acetoxy-14alpha,15alpha-epoxy-5alpha-cholest-8-en-7-one by methanol, using NMR techniques assisted by a conformational study through theoretical calculations.

This paper demonstrates that the crystallization of 3beta-acetoxy-14alpha,15alpha-epoxy-5alpha-cholest-8-en-7-one from methanol affords the 3beta-acetoxy-9alpha-methoxy-15alpha-hydroxycholest-8(14)-en-7-one. The structure of this steroid, which shows an apparently anomalous UV absorption maximum, is determined by high field NMR experiments, supporting the coupling constant values assignments and the NOE contacts by a conformational study through theoretical calculations at the B3LYP/6-31G* level. The computational study also justifies the observed UV absorption of the steroid, thus demonstrating the usefulness of computer chemistry in providing support for the identification of unknown compounds.

Toxicity, pharmacokinetics and metabolism of a novel inhibitor of IL-6-induced STAT3 activation.

PURPOSE: The oncogenic transcription factor signal transducer and activator of transcription 3 (STAT3) promotes gene transcription involved in cancer, and its activation by IL-6 is found in head and neck squamous cell carcinoma. Four triazolothiadizine STAT3 pathway inhibitors were evaluated to prioritize a single compound for in vivo examination. METHODS: Metabolic stability in mouse liver microsome incubation was used to evaluate four triazolothiadizine analogues, and UPCDC-10205 was administered to mice IV as single or multiple doses to evaluate toxicity. Single-dose pharmacokinetics (PK), bioavailability and metabolism were studied after IV 4 mg/kg, PO 4 mg/kg, or PO 30 mg/kg suspension in 1% carboxymethyl cellulose. Mice were euthanized between 5 min to 24 h after dosing, and plasma and tissues were analyzed by LC-MS. Non-compartmental PK parameters were determined. RESULTS: Of the four triazolothiadizine analogues evaluated, UPCDC-10205 was metabolically most stable. The maximum soluble dose of 4 mg/kg in 10% Solutol™ was not toxic to mice after single and multiple doses. PK analysis showed extensive tissue distribution and rapid plasma clearance. Bioavailability was ~5%. A direct glucuronide conjugate was identified as the major metabolite which was recapitulated in vitro. CONCLUSIONS: Rapid clearance of UPCDC-10205 was thought to be the result of phase II metabolism despite its favorable stability in a phase I in vitro metabolic stability assay. The direct glucuronidation explains why microsomal stability (reflective of phase I metabolism) did not translate to in vivo metabolic stability. UPCDC-10205 did not demonstrate appropriate exposure to support efficacy studies in the current formulation.

Structure-Activity Study of Bioisosteric Trifluoromethyl and Pentafluorosulfanyl Indole Inhibitors of the AAA ATPase p97.

Exploratory SAR studies of a new phenyl indole chemotype for p97 inhibition revealed C-5 indole substituent effects in the ADPGlo assay that did not fully correlate with either electronic or steric factors. A focused series of methoxy-, trifluoromethoxy-, methyl-, trifluoromethyl-, pentafluorosulfanyl-, and nitro-analogues was found to exhibit IC50s from low nanomolar to double-digit micromolar. Surprisingly, we found that the trifluoromethoxy-analogue was biochemically a better match of the trifluoromethyl-substituted lead structure than a pentafluorosulfanyl-analogue. Moreover, in spite of their almost equivalent strongly electron-depleting effect on the indole core, pentafluorosulfanyl- and nitro-derivatives were found to exhibit a 430-fold difference in p97 inhibitory activities. Conversely, the electronically divergent C-5 methyl- and nitro-analogues both showed low nanomolar activities.