Optimization of 1,2,4-Triazole-Based p97 Inhibitors for the Treatment of Cancer.

The AAA+ ATPase p97 (valosin-containing protein, VCP) is a master regulator of protein homeostasis and therefore represents a novel target for cancer therapy. Starting from a known allosteric inhibitor, NMS-873, we systematically optimized this scaffold, in particular, by applying a benzene-to-acetylene isosteric replacement strategy, specific incorporation of F, and eutomer/distomer identification, which led to compounds that exhibited nanomolar biochemical and cell-based potency. In cellular pharmacodynamic assays, robust effects on biomarkers of p97 inhibition and apoptosis, including increased levels of ubiquitinated proteins, CHOP and cleaved caspase 3, were observed. Compound (R)-29 (UPCDC-30766) represents the most potent allosteric inhibitor of p97 reported to date.

Affinity Capture of p97 with Small-Molecule Ligand Bait Reveals a 3.6 Å Double-Hexamer Cryoelectron Microscopy Structure.

Recent progress in the development of affinity grids for cryoelectron microscopy (cryo-EM) typically employs genetic engineering of the protein sample such as histidine or Spy tagging, immobilized antibody capture, or nonselective immobilization via electrostatic interactions or Schiff base formation. We report a powerful and flexible method for the affinity capture of target proteins for cryo-EM analysis that utilizes small-molecule ligands as bait for concentrating human target proteins directly onto the grid surface for single-particle reconstruction. This approach is demonstrated for human p97, captured using two different small-molecule high-affinity ligands of this AAA+ ATPase. Four electron density maps are revealed, each representing a p97 conformational state captured from solution, including a double-hexamer structure resolved to 3.6 Å. These results demonstrate that the noncovalent capture of protein targets on EM grids modified with high-affinity ligands can enable the structure elucidation of multiple configurational states of the target and potentially inform structure-based drug design campaigns.

Therapeutic and Prophylactic Vaccines to Counteract Fentanyl Use Disorders and Toxicity.

The incidence of fatal overdoses has increased worldwide due to the widespread access to illicit fentanyl and its potent analogues. Vaccines offer a promising strategy to reduce the prevalence of opioid use disorders (OUDs) and to prevent toxicity from accidental and deliberate exposure to fentanyl and its derivatives. This study describes the development and characterization of vaccine formulations consisting of novel fentanyl-based haptens conjugated to carrier proteins. Vaccine efficacy was tested against opioid-induced behavior and toxicity in mice and rats challenged with fentanyl and its analogues. Prophylactic vaccination reduced fentanyl- and sufentanil-induced antinociception, respiratory depression, and bradycardia in mice and rats. Therapeutic vaccination also reduced fentanyl intravenous self-administration in rats. Because of their selectivity, vaccines did not interfere with the pharmacological effects of commonly used anesthetics nor with methadone, naloxone, oxycodone, or heroin. These preclinical data support the translation of vaccines as a viable strategy to counteract fentanyl use disorders and toxicity.

Toward Next-Generation Biohybrid Catalyst Design: Influence of Degree of Polymerization on Enzyme Activity.

Due to their capacity to conduct complex organic transformations, enzymes find extensive use in medical and industrial settings. Unfortunately, enzymes are limited by their poor stability when exposed to harsh non-native conditions. While a host of methods have been developed to stabilize enzymes in non-native conditions, recent research into the synthesis of polymer-enzyme biohybrids using reversible deactivation radical polymerization approaches has demonstrated the potential of increased enzymatic activity in both native and non-native environments. In this manuscript, we utilize the enzyme lipase, as a model system, to explore the impact that modulation of grafted polymer molecular weight has on enzyme activity in both aqueous and organic media. We studied the properties of these hybrids using both solution-phase enzyme activity methods and coarse-grain modeling to assess the impact of polymer grafting density and grafted polymer molecular weight on enzyme activity to gain a deeper insight into this understudied property of the biohybrid system.

Covalent Poly(lactic acid) Nanoparticles for the Sustained Delivery of Naloxone.

The opioid epidemic currently plaguing the United States has been exacerbated by an alarming rise in fatal overdoses as a result of the proliferated abuse of synthetic mu opioid receptor (MOR) agonists, such as fentanyl and its related analogues. Attempts to manage this crisis have focused primarily on widespread distribution of the clinically approved opioid reversal agent naloxone (Narcan); however, due to the intrinsic metabolic lability of naloxone, these measures have demonstrated limited effectiveness against synthetic opioid toxicity. This work reports a novel polymer-based strategy to create a long-acting formulation of naloxone with the potential to address this critical issue by utilizing covalent nanoparticle (cNP) drug delivery technology. Covalently loaded naloxone nanoparticles (Nal-cNPs) were prepared via the naloxone-initiated, ring-opening polymerization (ROP) of l-lactide in the presence of a bifunctional thiourea organocatalyst with subsequent precipitation of the resulting naloxone-poly(l-lactic acid) polymer. This protocol afforded well-defined nanoparticles possessing a drug loading of approximately 7% w/w. The resulting Nal-cNPs demonstrated excellent biocompatibility, while exhibiting sustained linear release kinetics in vitro and blocking the effects of high dose (10 mg/kg) acute morphine for up to 98 h in an in vivo rodent model of neuropathic pain.

In-situ Chemiluminescence-Driven Reversible Addition-Fragmentation Chain-Transfer Photopolymerization.

The power of chemical light generation (chemiluminescence) is used to drive polymerization reactions. A biphasic reaction is developed such that light-generating reactions are confined to the organic phase and photopolymerization occurs in the aqueous phase. Well-defined RAFT-capped polymers are synthesized and the kinetics are shown to be dictated by light generation.

SuFEx-based strategies for the preparation of functional particles and cation exchange resins.

A predictable and reproducible number of sulfuric acid sites have been achieved for cation exchange resins by employing a mild SuFEx-based reagent system to effect the hydrolysis of fluorosulfonated polymer beads. The resultant poly(hydrogen sulfate) beads effectively demonstrate their utility as cation exchange resins in ion-capture experiments. Furthermore, their polyfluorosulfonated precursors have also proven to be suitable substrates for traditional SuFEx-type click reactions in both small molecule and protein immobilization applications.

Fentanyl Initiated Polymers Prepared by ATRP for Targeted Delivery.

The targeted delivery of polymers to neurons is a challenging yet important goal for polymer based drug delivery. We prepared a fentanyl based atom transfer radical polymerization (ATRP) initiator to target the Mu opioid receptor (MOR) for neuronal targeting. We incorporated our recently discovered rigid acrylate linking group into the initiator to retain a high degree of binding to the MOR and grafted random or block copolymers of poly(oligo(ethylene oxide) methacrylate)-block-(glycidyl methacrylate). Trifluoroethanol promoted amine ring opening of the glycidyl methacrylate was used for post-polymerization modification of the fentanyl initiated polymers to attach a near-infrared fluorescent dye (ADS790WS) or to build a targeted siRNA delivery system via modification with secondary amines. We examined the biocompatibility, cellular internalization, and siRNA binding properties of our polymer library in a green fluorescent protein expressing SY SH5Y neuroblastoma cell-line.

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.

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.

Antimicrobial benzodiazepine-based short cationic peptidomimetics.

Antimicrobial peptides (AMPs) appear to be good candidates for the development of new antibiotic drugs. We describe here the synthesis of peptidomimetic compounds that are based on a benzodiazepine scaffold flanked with positively charged and hydrophobic amino acids. These compounds mimic the essential properties of cationic AMPs. The new design possesses the benzodiazepine scaffold that is comprised of two glycine amino acids and which confers flexibility and aromatic hydrophobic 'back', and two arms used for further synthesis on solid phase for incorporation of charged and hydrophobic amino acids. This approach allowed us a better understanding of the influence of these features on the antimicrobial activity and selectivity. A novel compound was discovered which has MICs of 12.5 µg/ml against Staphylococcus aureus and 25 µg/ml against Escherichia coli, similar to the well-known antimicrobial peptide MSI-78. In contrast to MSI-78, the above mentioned compound has lower lytic effect against mammalian red blood cells. These peptidomimetic compounds will pave the way for future design of potent synthetic mimics of AMPs for therapeutic and biomedical applications.

Synthesis and evaluation of a photoresponsive quencher for fluorescent hybridization probes.

Nowadays, most nucleic acid detections using fluorescent probes rely on quenching of fluorescence by energy transfer from one fluorophore to another or to a non-fluorescent molecule (quencher). The most widely used quencher in fluorescent probes is 4-((4-(dimethylamino)phenyl)azo)benzoic acid (DABCYL). We targeted a nucleoside-DABCYL analogue which could be incorporated anywhere in an oligonucleotide sequence and in any number, and used as a quencher in different hybridization sensitive probes. Specifically, we introduced a 5-(4-((dimethylamino)phenyl)azo)benzene)-2'-deoxy-uridine (dU(DAB)) quencher. The photoisomerization and dU(DAB)'s ability to quench fluorescein emission have been investigated. We incorporated dU(DAB) into a series of oligonucleotide (ON) probes including strand displacement probes, labeled with both fluorescein (FAM) and dU(DAB), and TaqMan probes bearing one or two dU(DAB) and a FAM fluorophore. We used these probes for the detection of a DNA target in real-time PCR (RT-PCR). All probes showed amplification of targeted DNA. A dU(DAB) modified TaqMan RT-PCR probe was more efficient as compared to a DABCYL bearing probe (93% vs. 87%, respectively). Furthermore, dU(DAB) had a stabilizing effect on the duplex, causing an increase in Tm up to 11 °C. In addition we showed the photoisomerisation of the azobenzene moiety of dU(DAB) and the dU(DAB) triply-labeled oligonucleotide upon irradiation. These findings suggest that dU(DAB) modified probes are promising probes for gene quantification in real-time PCR detection and as photoswitchable devices.

Phenyl-imidazolo-cytidine analogues: structure-photophysical activity relationship and ability to detect single DNA mismatch.

To expand the arsenal of fluorescent cytidine analogues for the detection of genetic material, we synthesized para-substituted phenyl-imidazolo-cytidine ((Ph)ImC) analogues 5a-g and established a relationship between their structure and fluorescence properties. These analogues were more emissive than cytidine (λem 398-420 nm, Φ 0.009-0.687), and excellent correlation was found between Φ of 5a-g and σp(-) of the substituent on the phenyl-imidazolo moiety (R(2) = 0.94). Calculations suggested that the dominant tautomer of (Ph)ImC in methanol solution is identical to that of cytidine. DFT calculations of the stable tautomer of selected (Ph)ImC analogues suggested a relationship between the HOMO-LUMO gap and Φ and explained the loss of fluorescence in the nitro analogue. Incorporation of the CF3-(Ph)ImdC analogue into a DNA probe resulted in 6-fold fluorescence quenching of the former. A 17-fold reduction of fluorescence was observed for the G-matched duplex vs ODN(CF3-(Ph)ImdC), while for A-mismatched duplex, only a 2-fold decrease was observed. Furthermore, since the quantum yield of ODN(CF3-(Ph)ImdC):ODN(G) was reduced 17-fold vs that of a single strand, whereas that of ODN(CF3-(Ph)ImdC):ORN(G) was reduced only 3.8-fold, ODN(CF3-(Ph)ImdC) appears to be a DNA-selective probe. We conclude that the ODN(CF3-(Ph)ImdC) probe, exhibiting emission sensitivity upon single nucleotide replacement, may be potentially useful for DNA single nucleotide polymorphism (SNP) typing.

Detection of cyclin D1 mRNA by hybridization sensitive NIC-oligonucleotide probe.

A large group of fluorescent hybridization probes, includes intercalating dyes for example thiazole orange (TO). Usually TO is coupled to nucleic acids post-synthetically which severely limits its use. Here, we have developed a phosphoramidite monomer, 10, and prepared a 2'-OMe-RNA probe, labeled with 5-(trans-N-hexen-1-yl-)-TO-2'-deoxy-uridine nucleoside, dU(TO), (Nucleoside bearing an Inter-Calating moiety, NIC), for selective mRNA detection. We investigated a series of 15-mer 2'-OMe-RNA probes, targeting the cyclin D1 mRNA, containing one or several dU(TO) at various positions. dU(TO)-2'-OMe-RNA exhibited up to 7-fold enhancement of TO emission intensity upon hybridization with the complementary RNA versus that of the oligomer alone. This NIC-probe was applied for the specific detection of a very small amount of a breast cancer marker, cyclin D1 mRNA, in total RNA extract from cancerous cells (250 ng/µl). Furthermore, this NIC-probe was found to be superior to our related NIF (Nucleoside with Intrinsic Fluorescence)-probe which could detect cyclin D1 mRNA target only at high concentrations (1840 ng/µl). Additionally, dU(T) can be used as a monomer in solid-phase oligonucleotide synthesis, thus avoiding the need for post-synthetic modification of oligonucleotide probes. Hence, we propose dU(TO) oligonucleotides, as hybridization probes for the detection of specific RNA in homogeneous solutions and for the diagnosis of breast cancer.