Neurotensin (NT) is an endogenous neuropeptide involved in a variety of central and peripheral neuromodulatory effects. Here we show effects of site-specific glycosylation on the in vitro and in vivo pharmacological properties of this neuropeptide. NT analogs containing O-linked disaccharides (β-melibiose and α-TF antigen) or β-lactose unit linked via a PEG3-spacer were designed and chemically synthesized using Fmoc chemistry. For the latter analog, Fmoc-Glu-(β-Lac-PEG3-amide) was prepared. Our results indicate that the addition of the disaccharides did not negatively affect the subnanomolar affinity nor the low nanomolar agonist potency for the neurotensin receptor subtype 1 (NTS1). Interestingly, three glycosylated analogs exhibited subpicomolar potency in the 6 Hz limbic seizure mouse model of pharmacoresistant epilepsy following intracerebroventricular administration. Our results suggest for the first time that chemically-modified NT analogs may lead to novel antiepileptic therapies.
neurotensin; anticonvulsant; glycosylation; binding; agonist
The endogenous neuropeptide galanin has anticonvulsant and analgesic properties mediated by galanin receptors expressed in the central and peripheral nervous systems. Our previous work showed that combination of truncation of the galanin peptide along with N-and C-terminal modifications afforded analogs that suppressed seizures or pain following intraperitoneal administration. To generate orally-active galanin analogs, the previously reported lead compound Gal-B2 (NAX 5055) was redesigned by (1) central truncation, (2) introduction of D-amino acids, (3) and addition of backbone spacers. Analog D-Gal(7-Ahp)-B2, containing 7-amino heptanoic acid as a backbone spacer and oligo-D-lysine motif at the C-terminus, exhibited anticonvulsant and analgesic activity post intraperitoneal administration. Oral administration of D-Gal(7-Ahp)-B2 demonstrated analgesic activity with reduction in both acute and inflammatory pain in the mouse formalin model of pain at doses as low as 8 mg/kg.
galanin; analgesic peptides; orally-active; backbone prosthesis; neuropeptide
Prodrugs are effective tools in overcoming drawbacks typically associated with drug formulation and delivery. Those employing esterase-triggered functional groups are frequently utilized to mask polar carboxylic acids and phenols, increasing drug-like properties such as lipophilicity. Herein we detail a comprehensive assessment for strategies that effectively release hydroxyl and phenolic moieties in the presence of an esterase. Matrix metalloproteinases (MMPs) serve as our proof-of-concept target. Three distinct ester-responsive protecting groups are incorporated into MMP proinhibitors containing hydroxyl moieties. Analytical evaluation of the proinhibitors demonstrates that the use of a benzyl ether group appended to the esterase trigger leads to considerably faster kinetics of conversion and enhanced aqueous stability when compared to more conventional approaches where the trigger is directly attached to the inhibitor. Biological assays confirm that all protecting groups effectively cleave in the presence of esterase to generate the active inhibitor.
esterase; matrix metalloproteinases; metalloenzymes; prodrugs
A novel lead compound for inhibition of the antibacterial drug target, glutamate racemase, is optimized for both ligand efficiency and lipophilic efficiency. A previously developed hybrid MD-docking and scoring scheme, FERM-SMD, is utilized to predict relative potencies of potential derivatives prior to chemical synthesis. This scheme was successful in distinguishing between high and low affinity binders with minimal experimental structural information, saving time and resources in the process. In vitro potency is increased approximately 4-fold against glutamate racemase from the model organism, B. subtilis. Lead derivatives show 2- to 4-fold increased antimicrobial potency over the parent scaffold. In addition, specificity toward B. subtilis, over E. coli and S. aureus, show dependency on the chemical substituent added to the parent scaffold. Finally, insight is gained into the capacity for these compounds to reach the target enzyme in vivo using a bacterial cell wall lysis assay. The result of this study is a novel small molecule inhibitor of GR with the following characteristics: Ki = 2.5 μM, LE = 0.45 kcal/mol/atom, LiPE = 6.0, MIC50 = 260 μg/mL against B. subtilis, EC50,lysis = 520 μg/mL against B. subtilis
Antibiotics; Fragment-Based Drug Discovery; Glutamate Racemase; Heterocyclic Aromatics; Inhibitors
allosteric; kinesin Eg5; STD-NMR; CORCEMA-ST; drug discovery
Earlier, we found estrogen receptor (ER) ligands having a novel three-dimensional oxabicyclo[2.2.1]heptene core scaffold and good ER binding affinity acted as partial agonists via small alkyl ester substitutions on the bicyclic core that indirectly modulate the critical switch helix in the ER ligand-binding domain, helix 12, by interactions with helix 11. This contrasts with the mechanism of action of tamoxifen, which directly pushes helix 12 out of the conformation required for gene activation. We now report that a much larger substitution can be tolerated at this position of the bicyclic core scaffold, namely a phenyl sulfonate group, which defines a novel binding epitope for the estrogen receptor. We prepared an array of 14 of these oxabicycloheptene sulfonates (OBHS), varying the phenyl sulfonate group. As with OBHS itself, these compounds showed preferential affinity for ERα, and the disposition and size of the phenyl substituents were important determinants of the binding affinity and selectivity of these compounds, with those having ortho substituents giving the highest, and para substituents the lowest affinities for ERα. A few analogs have ERα binding affinity that is comparable to or, in the case of the ortho chloro analog, higher than that of OBHS itself. In cell-based studies, we found several compounds with activity profiles comparable to tamoxifen, but acting entirely as indirect antagonists, allosterically interfering with recruitment of coactivator proteins to the receptor. Thus, the OBHS binding epitope represents a novel approach to the development of estrogen receptor antagonists via an indirect mechanism of antagonism.
Steroids; Cycloaddition; Estrogen; Estrogen Receptor; Hormones
A series of novel bis-tetrahydropyran 1,4-triazole analogues based on the acetogenin framework display low micromolar trypanocidal activities towards both bloodstream and insect forms of Trypanosoma brucei, the causative agent of African sleeping sickness. A divergent synthetic strategy was adopted for the synthesis of the key tetrahydropyran intermediates to enable rapid access to diastereochemical variation either side of the 1,4-triazole core. The resulting diastereomeric analogues displayed varying degrees of trypanocidal activity and selectivity in structure activity relationship studies.
Trypanosomiasis; acetogenins; neglected diseases; natural products
Water in the architecture of life: Potent and selective matrix metalloproteinase‐13 (MMP‐13) inhibitors were rationally designed by targeting multiple water‐mediated interactions between the target protein and small‐molecule inhibitors. This structure‐based design concept offers tremendous opportunities for the discovery of unique small molecules with tailored biological activity.imageWILEY-VCH
chemical biology; matrix metalloproteinase inhibitors; structure‐based drug design; structure–activity relationships; water‐mediated interactions
Freedom to merge: A combination of crystal structure examination and in silico predictions made it possible to overcome the conformational limitations of fragment merging and escape the internal strain in a series of weakly binding merged fragments that target M. tuberculosis CYP121. The insights attained provide a new perspective and guide for prioritizing synthetic efforts toward fragment merging in future and ongoing fragment‐based ligand discovery campaigns.imageWILEY-VCH
conformation analysis; cytochromes; drug discovery; fragment‐based; tuberculosis
A SAR translation strategy was used for the discovery of tetrahydroisoquinoline (THIQ)-based steroidomimetic and chimeric microtubule disruptors based upon a steroidal starting point. A steroid A,B-ring-mimicking THIQ core was connected to methoxy aryl D-ring ring mimics through methylene, carbonyl and sulfonyl linkers to afford a number of steroidomimetic hits (e.g. 20c GI50 2.1 μM). Optimisation and control experiments demonstrate the complementary SAR of this series and the steroid derivatives that inspired its design. Linkage of the THIQ-based A,B-mimic with the trimethoxy aryl motif prevalent in colchicine site binding microtubule disruptors delivered a series of chimeric molecules whose activity (to GI50 40 nM) surpasses that of the parent steroid derivatives. Validation of this strategy was obtained from the excellent oral activity of 20z relative to a benchmark steroidal bis-sulfamate in an in vivo model of multiple myeloma.
tetrahydroisoquinolines; microtubule disruptors; tubulin assembly; colchicine binding
Given the number of monogenic ocular diseases and the number of non-monogenic degenerative ocular diseases for which gene therapy has been considered as a treatment, the development of effective therapeutic delivery strategies for DNA is a critical research goal. Here we generate, characterize, and evaluate non-viral nanoparticles composed of glycol chitosan (GCS) and plasmid DNA (pDNA). We show that these particles are stable, do not aggregate in saline, are resistant to DNases, and have a hydrodynamic diameter of ∼250 nm. We further show that the plasmid in these NPs maintains its proper conformation and can be released and expressed inside the cell. To determine whether these NPs would be suitable for intraocular use, pDNA carrying the ubiquitously expressed CBA-eGFP expression cassette was compacted and subretinally injected into adult WT albino mice. At post-injection (PI) day 14, we observe substantial GFP expression exclusively in the retinal pigment epithelium (RPE) in eyes treated with GCS NPs but not in uncompacted pDNA or vehicle (saline) treated eyes. We observe no signs of gross retinal toxicity and at PI-30 days, there is no difference in electroretinogram function between GCS NP-, pDNA-, or vehicle-treated eyes. These results suggest that with further development GCS NPs may be a useful addition to our available repertoire of genetic therapies for the treatment of RPE-associated diseases.
Glycol Chitosan; Nanoparticles; Non-Viral Gene Delivery; Polymers; Retinal Pigment Epithelium
The urokinase receptor (uPAR) is a cell-surface protein that is part of an intricate web of transient and tight protein interactions that promote cancer cell invasion and metastasis. Here we evaluate the binding and biological activity of a new class of pyrrolidinone (3) and piperidinone (4) compounds, along with derivatives of previously-identified pyrazole (1) and propylamine (2) compounds. Competition assays revealed that the compounds displaced a fluorescently-labeled peptide (AE147-FAM) with inhibition constant Ki ranging from 6 to 63 μM. Structure-based computational pharmacophore analysis followed by extensive explicit-solvent molecular dynamics simulations and free energy calculations suggested pyrazole-based 1a and piperidinone-based 4 adopt different binding modes, despite their similar two-dimensional structures. In cells, compounds 1b and 1f showed significant inhibition of breast MDA-MB-231 and pancreatic ductal adenocarcinoma (PDAC) cell proliferation, but 4b exhibited no cytotoxicity even at concentrations of 100 μM. 1f impaired MDA-MB-231 invasion, adhesion, and migration in a concentration-dependent manner, while 4b inhibited only invasion. 1f inhibited gelatinase (MMP-9) activity in a concentration-dependent manner, while 4b showed no effect suggesting different mechanisms for inhibition of cell invasion. Signaling studies further highlighted these differences, showing that pyrazole compounds completely inhibited ERK phosphorylation and impaired HIF1α and NF-κB signaling, while pyrrolidinone and piperidinone (3 and 4b) had no effect. Annexin V staining suggested that the effect of pyrazole-based 1f on proliferation was due to cell killing through an apoptotic mechanism.
Large conductance, calcium- and voltage-gated potassium (BK) channels regulate various physiological processes and represent an attractive target for drug discovery. Numerous BK channel activators are available. However, these agents usually interact with the ubiquitously distributed channel-forming subunit and thus cannot selectively target a particular tissue. Here, we performed structure-activity relationship study of lithocholic acid (LCA), a cholane that activates BK channels via the accessory BK β1 subunit. The latter protein highly abundant in smooth muscle but scarce in most tissues. Modifications in the LCA lateral chain length and functional group yielded two novel smooth muscle BK channel activators, both having a small volume and a net negative charge in the substituent radical at C24. Our data provide detailed structural information that will be used to advance a pharmacophore in search of β1 subunit-selective BK channel activators. These compounds are expected to evoke smooth muscle relaxation, which would be beneficial in the pharmacotherapy of prevalent human disorders associated with increased smooth muscle degree of contraction, such as systemic hypertension, cerebral or coronary vasospasm, bronchial asthma, bladder hyperactivity, and erectile dysfunction.
MaxiK channel; structure-activity relationships; analogue series; BK β1 subunit; steroids
The transient nucleolus plays a central role in the upregulated synthesis of ribosomal RNA (rRNA) to sustain ribosome biogenesis, a hallmark of aberrant cell growth. This function, in conjunction with its unique pathohistological features in malignant cells and its ability to mediate apoptosis, renders this subnuclear structure a potential target for chemotherapeutic agents. In this Minireview, structurally and functionally diverse small molecules are discussed that have been reported to either interact with the nucleolus directly or perturb its function indirectly by acting on its dynamic components. These molecules include all major classes of nucleic acid-targeted agents, antimetabolites, kinase inhibitors, anti-inflammatory drugs, natural product antibiotics, oligopeptides, as well asnano-sized particles. Together, these molecules are invaluable probes of structure and function of the nucleolus. They also provide a unique opportunity to develop novel strategies for more selective and therefore better tolerated chemotherapeutic intervention. In this regard, inhibition of RNA polymerase I-mediated rRNA synthesis appears to be a promising mechanism of cancer cell kill. The recent development of molecules targeted at G-quadruplex forming rRNA gene sequences, which are currently undergoing clinical trials, seems to attest to the success of this approach.
antitumor agents; apoptosis; DNA-targeted; p53; ribosome biogenesis
We have screened a small library of thiuram disulfides for inhibition of lymphoid tyrosine phosphatase (LYP) activity. The parent thiuram disulfide, disulfiram, inhibited LYP activity in vitro and in Jurkat T cells whereas diethyldithiocarbamate failed to inihibit LYP at the concentrations tested. Compound 13, an N-(2-thioxothiazolidin-4-one) analog, was the most potent LYP inhibitor in this series, with an IC50 of 3 μM. Compound 13 was found to inhibit LYP pseudo-irreversibly, as evident by the time-dependence of inhibition with a Ki of 1.1 μM and a kinact of 0.004 s−1. The inhibition of LYP by compound 13 could not be reversed significantly by incubation with glutathione or by prolonged dialysis, but could be partially reversed by incubation with dithiothreitol. Compound 13 also inhibited LYP activity in Jurkat T cells.
Disulfiram; thiuram disulfides; PTP; screening; inhibitors
The development of responsive or “smart” magnetic resonance imaging (MRI) contrast agents that can report specific biomarker or biological events has been the focus of MRI contrast agent research over the past 20 years. Among various biological hallmarks of interest, tissue redox and hypoxia are particularly important owing to their roles in disease states and metabolic consequences. Herein we review the development of redox-/hypoxia-sensitive T1 shortening and paramagnetic chemical exchange saturation transfer (PARACEST) MRI contrast agents. Traditionally, the relaxivity of redox-sensitive Gd3+-based complexes is modulated through changes in the ligand structure or molecular rotation, while PARACEST sensors exploit the sensitivity of the metal-bound water exchange rate to electronic effects of the ligand-pendant arms and alterations in the coordination geometry. Newer designs involve complexes of redox-active metal ions in which the oxidation states have different magnetic properties. The challenges of translating redox- and hypoxia-sensitive agents in vivo are also addressed.
hypoxia; imaging agents; magnetic resonance imaging; PARACEST; redox chemistry
We have previously developed two potent chemical classes that inhibit the essential papain-like protease (PLpro) of severe acute respiratory syndrome coronavirus (SARS-CoV). In this study, we applied a novel approach to identify small fragments that act synergistically with these inhibitors. A fragment library was screened in combination with four previously developed lead inhibitors by fluorescence-based enzymatic assays. Several fragment compounds synergistically enhanced the inhibitory activity of the lead inhibitors by approximately an order of magnitude. Surface plasmon resonance (SPR) measurements showed that three fragments bind specifically to the PLpro enzyme. Mode of inhibition, computational solvent mapping, and molecular docking studies suggest that these fragments bind adjacent to the binding site of the lead inhibitors and further stabilize the inhibitor-bound state. We propose potential next generation compounds based upon a computational, fragment-merging approach. This approach provides an alternative strategy for lead optimization in cases where direct co-crystallization is difficult.
Papain-like protease; small molecule inhibitor; fragment-like inhibitor; Human SARS Coronavirus
Cyclic lipopeptides derived from the fusaricidin/LI-F family of naturally occurring antibiotics represent particularly attractive candidates for the development of new antibacterial agents. In comparison to natural products, these derivatives may offer better stability under physiologically relevant conditions and lower nonspecific toxicity, while preserving their antibacterial activity. In this study we have assessed the ability of cyclic lipodepsipeptide 1, and its amide 2, N-methyl amide 3 and linear peptide 4 analogues to interact with the cytoplasmic membranes of selected Gram-positive bacteria, as well as their bacteriostatic/bactericidal modes of action and in vivo potency using a Galleria mellonella model of MRSA infection. Cyclic lipopeptides 1 and 2 depolarize the cytoplasmic membranes of Gram-positive bacteria in a concentration-dependent manner. The degree of membrane depolarization was influenced by the structural and physical properties of 1 and 2, with more flexible and hydrophobic peptide 1 being most efficient. However, membrane depolarization does not correlate with bacterial cell lethality, suggesting that membrane-targeting activity is not the main mode of action for this class of antibacterial peptides. Conversely, substitution of the depsipeptide bond in 1 with an N-methyl amide bond 3, or its hydrolysis 4, lead to a complete loss of antibacterial activity, and indicate that the conformation of cyclic lipopeptides plays a role in their antibacterial activities. Cyclic lipopeptides 1 and 2 are also capable of improving the survival of G. mellonella larvae infected with MRSA with different efficiencies reflecting their in vitro activities. Gaining more insights into the structure-activity-relationship and mode of action of these cyclic lipopeptides may enable the development of new antibiotics of this class with improved antibacterial activity.
antibiotics; depsipeptide; biological activity; membranes; isosteric analogues
A SAR translation strategy adopted for the discovery of tetrahydroisoquinolinone (THIQ)‐based steroidomimetic microtubule disruptors has been extended to dihydroisoquinolinone (DHIQ)‐based compounds. A steroid A,B‐ring‐mimicking DHIQ core was connected to methoxyaryl D‐ring mimics through methylene, carbonyl, and sulfonyl linkers, and the resulting compounds were evaluated against two cancer cell lines. The carbonyl‐linked DHIQs in particular exhibit significant in vitro antiproliferative activities (e.g., 6‐hydroxy‐7‐methoxy‐2‐(3,4,5‐trimethoxybenzoyl)‐3,4‐dihydroisoquinolin‐1(2H)‐one (16 g): GI50 51 nm in DU‐145 cells). The broad anticancer activity of DHIQ 16 g was confirmed in the NCI 60‐cell line assay giving a mean activity of 33 nm. Furthermore, 6‐hydroxy‐2‐(3,5‐dimethoxybenzoyl)‐7‐methoxy‐3,4‐dihydroisoquinolin‐1(2H)‐one (16 f) and 16 g and their sulfamate derivatives 17 f and 17 g (2‐(3,5‐dimethoxybenzoyl)‐7‐methoxy‐6‐sulfamoyloxy‐3,4‐dihydroisoquinolin‐1(2H)‐one and 7‐methoxy‐2‐(3,4,5‐trimethoxybenzoyl)‐6‐sulfamoyloxy‐3,4‐dihydroisoquinolin‐1(2H)‐one, respectively) show excellent activity against the polymerization of tubulin, close to that of the clinical combretastatin A‐4, and bind competitively at the colchicine binding site of tubulin. Compounds 16 f and 17 f were also shown to demonstrate in vitro anti‐angiogenic activity. Additionally, X‐ray and computational analyses of 17 f reveal that electrostatic repulsion between the two adjacent carbonyl groups, through conformational biasing, dictates the adoption of a “steroid‐like” conformation that may partially explain the excellent in vitro activities.
colchicine; dihydroisoquinolinones; electrostatic repulsion; microtubules; tubulin
Through our focused effort to discover new and effective agents against toxoplasmosis, a structure-based drug design approach was utilized to develop a series of potent inhibitors of the enoyl-acyl carrier protein (ACP) reductase (ENR) enzyme in Toxoplasma gondii (TgENR). Modifications to positions 5 and 4′ of the well-known ENR inhibitor triclosan afforded a series of 29 new analogs. Among the resulting compounds, many showed high potency and improved physicochemical properties in comparison with the lead. The most potent compounds 16a and 16c have IC50 values of 250 nM against Toxoplasma gondii tachyzoites without apparent toxicity to the host cells. Their IC50 values against the recombinant TgENR were 43 and 26 nM, respectively. Additionally, 11 other analogs in this series had IC50 values ranging from 17 to 130 nM in the enzyme-based assay.
With respect to their excellent in vitro activity as well as improved drug-like properties, the lead compounds 16a and 16c are deemed to be an excellent starting point for the development of new medicines to effectively treat Toxoplasma gondii infections.
Enoyl Reductase; Inhibitors; Medicinal Chemistry; Toxoplasma gondii; triclosan
A series of analogs of the adamantyl arotinoid (AdAr) chalcone 5 (MX781) having halogenated benzyloxy substituents at C2′ and heterocyclic derivatives replacing the chalcone group were found to inhibit IκBα kinase α (IKKα) and IκBα kinase β (IKKβ) activities. The growth inhibitory activity of some analogs against Jurkat T cells as well as prostate carcinoma cells (PC-3) and chronic myelogenous leukemia cells (K562), which contain elevated basal IKK activity, correlates with induction of apoptosis and increased inhibition of recombinant IKKα and IKKβ in vitro, pointing towards inhibition of IKK/NFκB signaling as the most likely target of the anticancer activities of these AdArs. While the chalcone functional group present in many dietary compounds has been shown to mediate interactions with IKKβ via Michael addition with cysteine residues, AdArs containing five-membered heterocyclic ring (isoxazoles and pyrazoles) in place of the chalcone of the parent system are potent inhibitors of IKKs as well, which suggests that other mechanisms for inhibition exist that do not depend on the presence of a reactive α,β-unsaturated ketone.
Adamantyl arotinoids; chalcones; synthesis; kinase activities; IKK
Disulfide bridges, which stabilize the native conformation of conotoxins impose a challenge in the synthesis of smaller analogs. In this work, we describe the synthesis of a minimized analog of the analgesic μ-conotoxin KIIIA that blocks two sodium channel subtypes, the neuronal NaV1.2 and skeletal muscle NaV1.4. Three disulfide-deficient analogs of KIIIA were initially synthesized in which the native disulfide bridge formed between either C1-C9, C2-C15 or C4-C16 was removed. Deletion of the first bridge only slightly affected the peptide’s bioactivity. To further minimize this analog, the N-terminal residue was removed and two non-essential Ser residues were replaced by a single 5-amino-3-oxapentanoic acid residue. The resulting “polytide” analog retained the ability to block sodium channels and to produce analgesia. Until now, the peptidomimetic approach applied to conotoxins has progressed only modestly at best; thus, the disulfide-deficient analogs containing backbone spacers provide an alternative advance toward the development of conopeptide-based therapeutics.
conopeptide; conotoxin; sodium channels; backbone spacers; disulfide bridges
The unwanted psychoactive effects of cannabinoid receptor agonists have limited their development as medicines. These CB1 mediated side effects are due to the fact that CB1 receptors are largely expressed in the Central Nervous System (CNS). Since it is known that CB1 receptors are also located peripherally, there is a growing interest in targeting cannabinoid receptors located outside the brain. A library of chromenopyrazoles designed in analogy to the classical cannabinoid cannabinol were synthesized, characterized and tested for cannabinoid activity. Radiolabeled binding assays were used to determine their affinities at CB1 and CB2 receptors. Structural features required for CB1/CB2 affinity and selectivity were explored using molecular modeling. Within the chromenopyrazoles series, some of them showed to be selective CB1 ligands. These modeling studies suggest that CB1 full selectivity over CB2 can be accounted for the presence of a pyrazole ring in the structure. The functional activities of selected chromenopyrazoles were evaluated in isolated tissues. Behavioral tests, in vivo, were then carried on the most effective CB1 cannabinoid agonist (13a). Chromenopyrazole 13a did not induce modifications in any of the tested parameters on the mouse cannabinoid tetrad, discarding CNS-mediated effects. This lack of agonistic activity in the CNS suggests that it does not readily cross the blood-brain barrier. Moreover, compound 13a can induce antinociception in a peripheral model of orofacial pain in rat. Taking into account the negative results obtained in the hot plate test, it could be suggested that the antinociception induced by 13a in the orofacial test may be mediated through peripheral mechanisms.
agonist; cannabinoid; peripheral; protein model; receptor