A docking screen identified reversible, non-covalent inhibitors (e.g. 1) of the parasite cysteine protease cruzain. Chemical optimization of 1 led to a series of oxadiazoles possessing interpretable SAR and potencies as much as 500-fold greater than 1. Detailed investigation of the SAR series subsequently revealed that many members of the oxadiazole class (and surprisingly also 1) act via divergent modes of inhibition – competitive or via colloidal aggregation – depending on the assay conditions employed.
Pharmacological inhibitors of cyclin-dependent kinases (CDKs) have a wide therapeutic potential. Among the CDK inhibitors currently under clinical trials, the 2,6,9-trisubstituted purine (R)-roscovitine displays rather high selectivity, low toxicity, and promising antitumor activity. In an effort to improve this structure, we synthesized several bioisosteres of roscovitine. Surprisingly, one of them, pyrazolo[1,5-a]-1,3,5-triazine 7a (N-&-N1, GP0210), displayed significantly higher potency, compared to (R)-roscovitine and imidazo[2,1-f]-1,2,4-triazine 13 (N-&-N2, GP0212), at inhibiting various CDKs and at inducing cell death in a wide variety of human tumor cell lines. This approach may thus provide second generation analogues with enhanced biomedical potential.
Ring size reduction of the central piperidine ring of lobelane yielded pyrrolidine analogs that showed marked inconsistencies in their ability to bind to the dihydrotetrabenazine (DTBZ) binding site on the vesicular monoamine transporter-2 (VMAT2) and their ability to inhibit VMAT2 function. The structure activity relationships indicate that structural modification within the pyrrolidine series resulted in analogs that interact with two different sites, i.e., the DTBZ binding site and an alternative site on VMAT2 to inhibit transporter function.
VMAT2; 2,5-disubstituted pyrrolidines; dihydrotetrabenazine; methamphetamine abuse
With the aim to develop a positron emission tomography (PET) tracer to assess the distribution of P-glycoprotein (P-gp) at the blood-brain barrier (BBB) in vivo, the potent third-generation P-gp inhibitor elacridar (1) was labeled with 11C by reaction of O-desmethyl 1 with [11C]-methyl triflate. In vitro autoradiography and small-animal PET imaging of [11C]-1 was performed in rats (n=3), before and after administration of unlabeled 1, as well as in wild-type, Mdr1a/b(−/−) and Bcrp1(−/−) mice (n=3). In PET experiments in rats, administration of unlabeled 1 increased brain activity uptake 5.4-fold, whereas blood activity levels remained unchanged. In Mdr1a/b(−/−) mice, brain activity uptake was 2.5-fold higher compared to wild-type animals, whereas in Bcrp1(−/−) mice brain activity uptake was only 1.3-fold higher. In vitro autoradiography showed that 63% of [11C]-1 binding was displaceable by an excess of unlabeled 1. As the signal obtained with [11C]-1 appeared to be specific for P-gp at the BBB, its utility for the visualization of cerebral P-gp merits further investigation.
P-gp; elacridar; 11C; PET; blood-brain barrier
In order to obtain a metabolically more stable analgesic peptide derivative, O-β-glycosylated serine (Ser(Glc)) was introduced into TY027 (Tyr-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH-3’,5’-Bzl(CF3)2) which was a previously reported bifunctional compound with delta/mu opioid agonist and neurokinin-1 receptor antagonist activities, and with a half life of 4.8 h in rat plasma. Incorporation of Ser(Glc) into various positions of TY027 gave analogues with variable bioactivities. Analogue 6 (Tyr-D-Ala-Gly-Phe-Nle-Pro-Leu-Ser(Glc)-Trp-NH-3’,5’-Bzl(CF3)2) was found to have effective bifunctional activities with a well-defined conformation with two β-turns based on the NMR conformational analysis in the presence of DPC micelles. In addition, 6 showed significant improvement in its metabolic stability (70 ± 9 % of 6 was intact after 24 h incubation in rat plasma). This improved metabolic stability, along with its effective and delta selective bifunctional activities, suggests that 6 could be an interesting research tool and possibly a promising candidate as a novel analgesic drug.
bifunctional peptide derivatives; glycopeptides; analgesics; opioid induced tolerance; opioid receptor agonist; neurokinin-1 receptor antagonist; conformation-activity relationships; NMR structure; DPC micelles
We measured the “druggability” of the ATP-dependent luciferase derived from the firefly Photuris pennsylvanica that was optimized using directed evolution (Ultra-Glo™, Promega). Quantitative high throughput screening (qHTS) was used to determine IC50’s of 198,899 samples against a formulation of Ultra-Glo luciferase (Kinase-Glo™). We found that only 0.1% of the Kinase-Glo inhibitors showed an IC50 < 10 μM compared to 0.9% found from a previous qHTS against the firefly luciferase from Photinus pyralis (lucPpy). Further, the maximum affinity identified in the lucPpy qHTS was 50 nM while for Kinase-Glo this value increased to 600 nM. Compounds with interactions stretching outside the luciferin binding pocket were largely lost with Ultra-Glo luciferase. Therefore, Ultra-Glo luciferase will show less compound interference when used as an ATP sensor compared to lucPpy. This study demonstrates the power of large-scale quantitative analysis of structure-activity relationships (>100K compounds) in addressing important questions such as a target's druggability.
chemical profiling; enzyme assay; PubChem; luciferase; quantitative high-throughput screening
Structural modifications affecting the efficacy of analogs of the endogenous opioid peptide dynorphin (Dyn) A have focused on the N-terminal “message” sequence, based on the “message-address” concept. To test the hypothesis that changes in the C-terminal “address” domain could affect efficacy, modified amino acids and cyclic constraints were incorporated into this region of the partial agonist [N-benzylTyr1]Dyn A-(1-11). Modifications in the C-terminal domain of [N-benzylTyr1]Dyn A-(1-11)NH2 resulted in increased kappa opioid receptor (KOR) affinity for all of the linear analogs, but did not affect the efficacy of these peptides at KOR. Cyclization between positions 5 and 8 yielded [N-benzylTyr1,cyclo(D-Asp5,Dap8)]Dyn A-(1-11)NH2 (13) (Patkar et al. J. Med. Chem. 2005, 48, 4500-4503) with high selectivity for KOR. In contrast to the linear peptides, this peptide exhibits negligible efficacy in the AC assay and is a KOR antagonist. These data are consistent with our hypothesis that appropriate modifications in the “address” domain of Dyn A analogs may affect efficacy.
We have examined 46 tetramethylrosamine/rhodamine derivatives with structural diversity in the heteroatom of the xanthylium core, the amino substituents of the 3- and 6-positions, and the alkyl, aryl, or heteroaryl group at the 9-substituent. These compounds were examined for affinity and ATPase stimulation in isolated MDR3 CL P-gp and human P-gp-His10, for their ability to promote uptake of calcein AM and vinblastine in multidrug-resistant MDCKII-MDR1 cells, and for transport in monolayers of MDCKII-MDR1 cells. Thioamide 31-S gave KM of 0.087 μM in human P-gp. Small changes in structure among this set of compounds affected affinity as well as transport rate (or flux) even though all derivatives examined were substrates for P-gp. With isolated protein, tertiary amide groups dictate high affinity and high stimulation while tertiary thioamide groups give high affinity and inhibition of ATPase activity. In MDCKII-MDR1 cells, the tertiary thioamide-containing derivatives promote uptake of calcein AM and have very slow passive, absorptive, and secretory rates of transport relative to transport rates for tertiary amide-containing derivatives. Thioamide 31-S promoted uptake of calcein AM and inhibited efflux of vinblastine with IC50’s of ~2 μM in MDCKII-MDR1 cells.
Multidrug resistance; P-glycoprotein; rhodamines; rosamines; inhibition
Inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel are predicted to slow cyst enlargement in polycystic kidney disease and reduce intestinal fluid loss in secretory diarrheas. Screening of ~110000 small synthetic and natural compounds for inhibition of halide influx in CFTR-expressing epithelial cells yielded a new class of pyrimido-pyrrolo-quinoxalinedione (PPQ) CFTR inhibitors. Testing of 347 analogues established structure–activity relationships. The most potent compound, 7,9-dimethyl-11-phenyl-6-(5-methylfuran-2-yl)-5,6-dihydro-pyrimido-[4′,5′-3,4]pyrrolo[1,2-a]quinoxaline-8,10-(7H,9H)-dione, PPQ-102, completely inhibited CFTR chloride current with IC50 ~90 nM. The PPQs, unlike prior CFTR inhibitors, are uncharged at physiological pH, and therefore not subject to membrane potential-dependent cellular partitioning or block efficiency. Patch-clamp analysis confirmed voltage-independent CFTR inhibition by PPQ-102 and showed stabilization of the channel closed state. PPQ-102 prevented cyst expansion and reduced the size of preformed cysts in a neonatal kidney organ culture model of polycystic kidney disease. PPQ-102 is the most potent CFTR inhibitor identified to date.
The Aryl Hydrocarbon Receptor (AhR) is a ligand-activated transcription factor; the AhR Per-AhR/Arnt-Sim (PAS) domain binds ligands. We developed homology models of the AhR PAS domain to characterize previously observed intra- and inter-species differences in ligand binding using Molecular Docking. In silico structure-based virtual ligand screening using our model resulted in the identification of pinocembrin and 5-hydroxy-7-methoxyflavone, which promoted nuclear translocation and transcriptional activation of AhR and AhR-dependent induction of endogenous target genes.
Twenty-two lycorine-related compounds were investigated for in vitro anti-tumor activity using four cancer cell lines displaying different levels of resistance to pro-apoptotic stimuli and two cancer cell lines sensitive to pro-apoptotic stimuli. Lycorine and six of its congeners exhibited potency in the single-digit micromolar range, while no compound appeared more active than lycorine. Lycorine also displayed the highest potential (in vitro) therapeutic ratio, being at least 15 times more active against cancer than normal cells. Our studies also showed that lycorine exerts its in vitro anti-tumor activity through cytostatic rather than cytotoxic effects. Furthermore, lycorine provided significant therapeutic benefit in mice bearing brain grafts of the B16F10 melanoma model at non-toxic doses. Thus, the results of the current study make lycorine an excellent lead for the generation of compounds able to combat cancers, which are naturally resistant to pro-apoptotic stimuli, such as glioblastoma, melanoma, non-small-cell-lung cancers, metastatic cancers, among others.
This Letter describes the synthesis and SAR of a series of analogs of the mGlu5 partial antagonist 5-(phenylethynyl)pyrimidine. New molecular switches are identified that modulate the pharmacological activity of the lead compound. Slight structural modifications around the proximal pyrimidine ring change activity of the partial antagonist lead to that of potent and selective full negative allosteric modulators and positive allosteric modulators, that demonstrate in vivo efficacy in rodent models for anxiolytic activity and antipsychotic, respectively.
The incidence of obesity and other diseases associated with an increased triacylglycerol mass is growing rapidly, particularly in the United States. Glycerol 3-phosphate acyltransferase (GPAT) catalyzes the rate-limiting step of glycerolipid biosynthesis, the acylation of glycerol 3-phosphate with saturated long chain acyl-CoAs. In an effort to produce small molecule inhibitors of this enzyme, a series of benzoic and phosphonic acids was designed and synthesized. In vitro testing of this series has led to the identification of several compounds, in particular 2-(nonylsulfonamido)benzoic acid (15g), possessing moderate GPAT inhibitory activity in an intact mitochondrial assay.
Clustering of 99 available X-ray crystal structures of HIV-1 reverse transcriptase (RT) at the flexible non-nucleoside inhibitor binding pocket (NNIBP) provides information about features of the conformational landscape for binding non-nucleoside inhibitors (NNRTIs), including effects of mutation and crystal forms. The ensemble of NNIBP conformations is separated into eight discrete clusters based primarily on the position of the functionally important primer grip, the displacement of which is believed to be one of the mechanisms of inhibition of RT. Two of these clusters are populated by structures in which the primer grip exhibits novel conformations that differ from the predominant cluster by over 4 Å and are induced by the unique inhibitors capravirine and rilpivirine/TMC278. This work identifies a new conformation of the NNIBP that may be used to design NNRTIs. It can also be used to guide more complete exploration of the NNIBP free energy landscape using advanced sampling techniques.
A series of cationic gold(I) complexes containing 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea (1), [AuL(1)]n+ (where L is Cl−, Br-, SCN−, PEt3, PPh3, or 1), derived from a class of analogous platinum(II) antitumor agents, has been synthesized. Unlike platinum, gold does not form permanent adducts with DNA, and its complexes are two orders of magnitude less cytotoxic in non-small-cell lung cancer cells than the most active platinum-based agent. Instead, several gold analogues show submicromolar and selective antimicrobial activity against Mycobacterium tuberculosis.
Current cancer chemotherapeutic agents clinically deployed today are designed to be indiscriminately cytotoxic, however achieving selective targeting of cancer malignancies would allow for improved diagnostic and chemotherapeutic tools. Integrin α4β1, a heterodimeric cell surface receptor, is believed to have a relaxed conformation in normal cells and an active conformation in cancerous cells, specifically T- and B-cell lymphomas. This highly attractive yet poorly understood receptor has been selectively targeted with the bisaryl urea peptidomimetic antagonist 1. However, concerns regarding its preliminary pharmacokinetic (PK) profile provided an impetus to change the pharmacophore from a bisaryl urea to a 2-arylaminobenzothiazole moiety, resulting in an analog with improved physicochemical properties, solubility and kidney:tumor ratio while maintaining potency (6; IC50 = 53 pM). The results presented herein utilized heterocyclic and solid-phase chemistry, cell adhesion assay, and in vivo optical imaging using the cyanine dye Cy5.5 conjugate.
Dynorphin A (Dyn A) is an endogenous ligand for kappa (κ) opioid receptors. To restrict the conformational mobility, we synthesized several cyclic Dyn A-(1-11)NH2 analogs on solid phase utilizing ring-closing metathesis (RCM) between the side chains of allylglycine (AllGly) residues incorporated in positions 2, 5 and/or 8. Cyclizations between the side chains of AllGly gave reasonable yields (56–74%) of all of the desired cyclic peptides. Both the cis and trans isomers were obtained for all of the cyclic peptides, with the ratio of cis to trans isomers depending on the position and stereochemistry of the AllGly. Most of the cyclic Dyn A-(1-11)NH2 analogs examined exhibit low nanomolar binding affinity for κ opioid receptors (Ki = 0.84–11 nM). In two of the three cases the configuration of the double bond has a significant influence on the opioid receptor affinity and agonist potency. All of the peptides inhibited adenylyl cyclase (AC) activity in a concentration-dependent manner with full or close to full agonist activity. These potent Dyn A analogs are the first ones cyclized by RCM.
We describe here the design, synthesis, molecular modeling, and biological evaluation of a series of small molecule, nonpeptide inhibitors of SARS-CoV PLpro. Our initial lead compound was identified via high-throughput screening of a diverse chemical library. We subsequently carried out structure-activity relationship studies, and optimized the lead structure to potent inhibitors that have shown antiviral activity against SARS-CoV infected Vero E6 cells. Based upon the X-ray crystal structure of one of the potent inhibitors 24-bound to SARS-CoV PLpro, a drug-design template was created. Our structure-based modification led to the design of a more potent inhibitor, 2 (enzyme IC50 = 0.46 μM; antiviral EC50 = 12.5 μM). Interestingly, its methylamine derivative 49 displayed good enzyme inhibitory potency (IC50 = 1.3 μM) and most potent SARS antiviral activity (EC50 = 2.5 μM) in the series. We have carried out computational docking studies and generated a predictive 3D-QSAR model for SARS-CoV PLpro inhibitors.
We developed an in-cell NMR assay for screening small molecule interactor libraries (SMILI-NMR) for compounds capable of disrupting or enhancing specific interactions between two or more components of a biomolecular complex. The method relies on the formation of a well-defined biocomplex and utilizes in-cell NMR spectroscopy to identify the molecular surfaces involved in the interaction at atomic scale resolution. Changes in the interaction surface caused by a small molecule interfering with complex formation are used as a read-out of the assay. The in-cell nature of the experimental protocol insures that the small molecule is capable of penetrating the cell membrane and specifically engaging the target molecule(s). Utility of the method was demonstrated by screening a small dipeptide library against the FKBP–FRB protein complex involved in cell cycle arrest. The dipeptide identified by SMILI-NMR showed biological activity in a functional assay in yeast.
(N)-Methanocarba nucleosides containing bicyclo[3.1.0]hexane replacement of the ribose ring previously demonstrated selectivity as A3 adenosine receptor (AR) agonists (5′-uronamides) or antagonists (5′-truncated). Here, these two series were modified in parallel at the adenine C2 position. N6-3-Chlorobenzyl-5′-N-methyluronamides derivatives with functionalized 2-alkynyl chains of varying length terminating in a reactive carboxylate, ester, or amine group were full, potent human A3AR agonists. Flexibility of chain substitution allowed the conjugation with a fluorescent cyanine dye (Cy5) and biotin, resulting in binding Ki values of 17 and 36 nM, respectively. The distal end of the chain was predicted by homology modeling to bind at the A3AR extracellular regions. Corresponding l-nucleosides were nearly inactive in AR binding. In the 5′-truncated nucleoside series, 2-Cl analogues were more potent at A3AR than 2-H and 2-F, functional efficacy in adenylate cyclase inhibition varied, and introduction of a 2-alkynyl chain greatly reduced affinity. SAR parallels between the two series lost stringency at distal positions. The most potent and selective novel compounds were amine congener 15 (Ki = 2.1 nM) and truncated partial agonist 22 (Ki = 4.9 nM).
New nitric oxide synthase (NOS) inhibitors were designed de novo with knowledge gathered from the studies on the nNOS-selective dipeptide inhibitors. Each of the new inhibitors consists of three fragments: an aminopyridine ring, a pyrrolidine, and a tail of various length and polarity. The in vitro inhibitory assays indicate good potency and isoform selectivity for some of the compounds. Crystal structures of these inhibitors bound to either wild type or mutant nNOS and eNOS have confirmed design expectations. The aminopyridine ring mimics the guanidinium group of L-arginine and functions as an anchor to place the compound in the NOS active site where it hydrogen bonds to a conserved Glu. The rigidity of the pyrrolidine ring places the pyrrolidine ring nitrogen between the same conserved Glu and the selective residue nNOS Asp597/eNOS Asn368 which results in similar interactions observed with the α-amino group of dipeptide inhibitors bound to nNOS. These structures provide additional information to help in the design of inhibitors with greater potency, physico-chemical properties, and isoform selectivity.
Ligands from the naltrexamine series have consistently demonstrated agonist activity at kappa opioid receptors (KOR), with varying activity at the mu opioid receptor (MOR). Various 6β-cinnamoylamino derivatives were made with the aim of generating ligands with a KOR agonist/MOR partial agonist profile, as ligands with this activity may be of interest as treatment agents for cocaine abuse. The ligands all displayed the desired high affinity, non-selective binding in vitro and in the functional assays were high efficacy KOR agonists with some partial agonist activity at MOR. Two of the new ligands (12a, 12b) have been evaluated in vivo, with 12a acting as a KOR agonist, and therefore somewhat similar to the previously evaluated analogues 3–6, while 12b displayed predominant MOR agonist activity.
14-O-cinnamoyl esters of naltrexone (6) were synthesised and evaluated in isolated tissue assays in vitro and in vivo in mouse antinociceptive assays. Their predominant opioid receptor activity was mu receptor (MOR) antagonism but the unsubstituted cinnamoyl derivative (6a) and the p-methylcinnamoyl derivative (6c) had partial MOR agonist activity in vitro and in vivo. When compared to the equivalent 14-cinnamoylaminomorphinones (5) the cinnamoyloxy morphinones (6) as MOR antagonists had shorter duration of action and were less effective as pseudoirreversible antagonists. The antinociceptive activity of the cinnamoyloxycodeinones (7) was not significantly greater than that of the morphinones (6) but they showed no evidence of any pseudoirreversible MOR antagonism. In both respects these profiles differed from those of the equivalent 14-cinnamoylaminocodeinones (4).
We report herein the design of potent and orally active small-molecule inhibitors of the MDM2-p53 interaction. Compound 5 binds to MDM2 with a Ki value of 0.6 nM, activates p53 at concentrations as low as 40 nM, and potently and selectively inhibits cell growth in tumor cells with wild-type p53 over tumor cells with mutated/deleted p53. Compound 5 has a good oral bioavailability and effectively inhibits tumor growth in the SJSA-1 xenograft model.
Described is the SAR of 18 Di-Sansalvamide A derivatives and the mechanism of action of the most potent compound. We show that this scaffold is a promising lead in the development of novel cancer therapeutics because it is: cytotoxic at nanomolar potency, inhibits a well-established oncogenic target (Hsp90), and does not share structural motifs with current drugs on the market.