RÉSUMÉ
The UDP glucuronosyltransferases (UGT) deactivate many therapeutics via glucuronidation while being required for clearance of normal metabolites and xenobiotics. There are 19 UGT enzymes categorized into UGT1A and UGT2B families based on sequence conservation. This presents a challenge in terms of targeting specific UGTs to overcome drug resistance without eliciting overt toxicity. Here, we identified for the first time that UGT1A4 is highly elevated in acute myeloid leukemia (AML) patients and its reduction corresponded to objective clinical responses. To develop inhibitors to UGT1A4, we leveraged previous NMR-based fragment screening data against the C-terminal domain of UGT1A (UGT1A-C). NMR and medicinal chemistry strategies identified novel chemical matter based on fragment compounds with the capacity to bind â¼20 fold more tightly to UGT1A-C (Kd â¼ 600 µM vs â¼30 µM). Some compounds differentially inhibited UGT1A4 versus UGT1A1 enzyme activity and restored drug sensitivity in resistant human cancer cells. NMR-based NOE experiments revealed these novel compounds recognised a region distal to the catalytic site suggestive of allosteric regulation. This binding region is poorly conserved between UGT1A and UGT2B C-terminal sequences, which otherwise exhibit high similarity. Consistently, these compounds did not bind to the C-terminal domain of UGT2B7 nor a triple mutant of UGT1A-C replaced with UGT2B7 residues in this region. Overall, we discovered a site on UGTs that can be leveraged to differentially target UGT1As and UGT2Bs, identified UGT1A4 as a therapeutic target, and found new chemical matter that binds the UGT1A C-terminus, inhibits glucuronidation and restores drug sensitivity.
Sujet(s)
Découverte de médicament , Résistance aux médicaments antinéoplasiques , Antienzymes , Glucuronosyltransferase , Humains , Domaine catalytique , Chimie pharmaceutique , Glucuronosyltransferase/antagonistes et inhibiteurs , Uridine diphosphate , Antienzymes/composition chimique , Antienzymes/pharmacologie , Spectroscopie par résonance magnétique/méthodesRÉSUMÉ
Protease-activated receptor 4 (PAR4) is a G-protein coupled receptor that is expressed on human platelets and activated by the coagulation enzyme thrombin. PAR4 plays a key role in blood coagulation, and its importance in pathological thrombosis has been increasingly recognized in recent years. Herein, we describe the optimization of a series of imidazothiadiazole PAR4 antagonists to a first-in-class clinical candidate, BMS-986120 (43), and a backup clinical candidate, BMS-986141 (49). Both compounds demonstrated excellent antithrombotic efficacy and minimal bleeding time prolongation in monkey models relative to the clinically important antiplatelet agent clopidogrel and provide a potential opportunity to improve the standard of care in the treatment of arterial thrombosis.
Sujet(s)
Agrégation plaquettaire , Thrombose , Benzofuranes , Plaquettes , Humains , Imidazoles , Morpholines , Récepteur de type PAR-1 , Récepteurs à la thrombine , Thiazoles , Thrombine , Thrombose/traitement médicamenteuxRÉSUMÉ
Trimethoprim (TMP) is widely used to treat infections in humans and in livestock, accelerating the incidence of TMP resistance. The emergent and largely untracked type II dihydrofolate reductases (DfrBs) are intrinsically TMP-resistant plasmid-borne Dfrs that are structurally and evolutionarily unrelated to chromosomal Dfrs. We report kinetic characterization of the known DfrB family members. Their kinetic constants are conserved and all are poorly inhibited by TMP, consistent with TMP resistance. We investigate their inhibition with known and novel bisubstrate inhibitors of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK). Importantly, all are inhibited by the HPPK inhibitors, making these molecules dual-target inhibitors of two folate pathway enzymes that are strictly microbial.
RÉSUMÉ
The worldwide use of the broad-spectrum antimicrobial trimethoprim (TMP) has induced the rise of TMP-resistant microorganisms. In addition to resistance-causing mutations of the microbial chromosomal dihydrofolate reductase (Dfr), the evolutionarily and structurally unrelated type II Dfrs (DfrBs) have been identified in TMP-resistant microorganisms. DfrBs are intrinsically TMP-resistant and allow bacterial proliferation when the microbial chromosomal Dfr is TMP-inhibited, making these enzymes important targets for inhibitor development. Furthermore, DfrBs occur in multiresistance plasmids, potentially accelerating their dissemination. We previously reported symmetrical bisbenzimidazoles that are the first selective inhibitors of the only well-characterized DfrB, DfrB1. Here, their diversification provides a new series of inhibitors (K i = 1.7-12.0 µM). Our results reveal two prominent features: terminal carboxylates and inhibitor length allow the establishment of essential interactions with DfrB1. Two crystal structures demonstrate the simultaneous binding of two inhibitor molecules in the symmetrical active site. Observations of those dimeric inhibitors inspired the design of monomeric analogues, binding in a single copy yet offering similar inhibition potency (K i = 1.1 and 7.4 µM). Inhibition of a second member of the DfrB family, DfrB4, suggests the generality of these inhibitors. These results provide key insights into inhibition of the highly TMP-resistant DfrBs, opening avenues to downstream development of antibiotics for combatting this emergent source of resistance.
RÉSUMÉ
In an effort to identify novel antithrombotics, we have investigated protease-activated receptor 4 (PAR4) antagonism by developing and evaluating a tool compound, UDM-001651, in a monkey thrombosis model. Beginning with a high-throughput screening hit, we identified an imidazothiadiazole-based PAR4 antagonist chemotype. Detailed structure-activity relationship studies enabled optimization to a potent, selective, and orally bioavailable PAR4 antagonist, UDM-001651. UDM-001651 was evaluated in a monkey thrombosis model and shown to have robust antithrombotic efficacy and no prolongation of kidney bleeding time. This combination of excellent efficacy and safety margin strongly validates PAR4 antagonism as a promising antithrombotic mechanism.
Sujet(s)
Benzofuranes/pharmacologie , Fibrinolytiques/pharmacologie , Hémorragie/prévention et contrôle , Récepteurs à la thrombine/antagonistes et inhibiteurs , Thrombose/prévention et contrôle , Animaux , Benzofuranes/composition chimique , Benzofuranes/pharmacocinétique , Biodisponibilité , Modèles animaux de maladie humaine , Fibrinolytiques/composition chimique , Fibrinolytiques/pharmacocinétique , Cellules HEK293 , Hémorragie/métabolisme , Humains , Macaca fascicularis , Modèles chimiques , Structure moléculaire , Agrégation plaquettaire/effets des médicaments et des substances chimiques , Récepteurs à la thrombine/génétique , Récepteurs à la thrombine/métabolisme , Relation structure-activité , Thrombose/métabolismeRÉSUMÉ
Secretion systems are protein complexes essential for bacterial virulence and potential targets for antivirulence drugs. In the intracellular pathogen Brucella suis, a type IV secretion system mediates the translocation of virulence factors into host cells and it is essential for pathogenicity. VirB8 is a core component of the secretion system and dimerization is important for functionality of the protein complex. We set out to study dimerization and possible conformational changes of VirB8 from B. suis (VirB8s) using nuclear magnetic resonance, X-ray crystallography, and differential scanning fluorimetry. We identified changes of the protein induced by a concentration-dependent monomer-to-dimer transition of the periplasmic domain (VirB8sp). We also show that the presence of the detergent CHAPS alters several signals in the heteronuclear single quantum coherence (HSQC) spectra and some of these chemical shift changes correspond to those observed during monomer-dimer transition. X-ray analysis of a monomeric variant (VirB8spM102R ) demonstrates that significant structural changes occur in the protein's α-helical regions (α2 and α4). We localized chemical shift changes of residues at the dimer interface as well as to the α1 helix that links this interface to a surface groove that binds dimerization inhibitors. Fragment-based screening identified small molecules that bind to VirB8sp and two of them have differential binding affinity for wild-type and the VirB8spM102R variant underlining their different conformations. The observed chemical shift changes suggest conformational changes of VirB8s during monomer-dimer transition that may play a role during secretion system assembly or function and they provide insights into the mechanism of inhibitor action. DATABASE: BMRB accession no. 26852 and PDB 5JBS.
Sujet(s)
Protéines bactériennes/composition chimique , Brucella suis/métabolisme , Systèmes de sécrétion de type IV/composition chimique , Cristallographie aux rayons X , Dimérisation , Fluorimétrie , Micelles , Simulation de docking moléculaire , Résonance magnétique nucléaire biomoléculaire , Périplasme/composition chimique , Conformation des protéinesRÉSUMÉ
The biphenyl derivatives 2 and 3 are prototypes of a novel class of NS5A replication complex inhibitors that demonstrate high inhibitory potency toward a panel of clinically relevant HCV strains encompassing genotypes 1-6. However, these compounds exhibit poor systemic exposure in rat pharmacokinetic studies after oral dosing. The structure-activity relationship investigations that improved the exposure properties of the parent bis-phenylimidazole chemotype, culminating in the identification of the highly potent NS5A replication complex inhibitor daclatasvir (33) are described. An element critical to success was the realization that the arylglycine cap of 2 could be replaced with an alkylglycine derivative and still maintain the high inhibitory potency of the series if accompanied with a stereoinversion, a finding that enabled a rapid optimization of exposure properties. Compound 33 had EC50 values of 50 and 9 pM toward genotype-1a and -1b replicons, respectively, and oral bioavailabilities of 38-108% in preclinical species. Compound 33 provided clinical proof-of-concept for the NS5A replication complex inhibitor class, and regulatory approval to market it with the NS3/4A protease inhibitor asunaprevir for the treatment of HCV genotype-1b infection has recently been sought in Japan.
Sujet(s)
Antiviraux/pharmacologie , Antienzymes/pharmacologie , Hepacivirus/effets des médicaments et des substances chimiques , Imidazoles/pharmacologie , Protéines virales non structurales/antagonistes et inhibiteurs , Réplication virale/effets des médicaments et des substances chimiques , Animaux , Antiviraux/composition chimique , Antiviraux/pharmacocinétique , Aire sous la courbe , Carbamates , Chiens , Découverte de médicament , Antienzymes/composition chimique , Antienzymes/pharmacocinétique , Hepacivirus/enzymologie , Hepacivirus/physiologie , Imidazoles/composition chimique , Imidazoles/pharmacocinétique , Spectroscopie par résonance magnétique , Pyrrolidines , Rats , Spectrométrie de masse ESI , Relation structure-activité , Valine/analogues et dérivésRÉSUMÉ
Hepatitis C virus (HCV) non-structural protein 5A (NS5A) is a multi-functional protein that is expressed in basally phosphorylated (p56) and in hyperphosphorylated (p58) forms. NS5A phosphorylation has been implicated in regulating multiple aspects of HCV replication. We recently reported the identification of a class of compounds that potently inhibit HCV RNA replication by targeting NS5A. Although the precise mechanism of inhibition of these compounds is not well understood, one activity that has been described is their ability to block expression of the hyperphosphorylated form of NS5A. Here, we report that an NS5A inhibitor impaired hyperphosphorylation without affecting basal phosphorylation at the C-terminal region of NS5A. This inhibitor activity did not require NS5A domains II and III and was distinct from that of a cellular kinase inhibitor that also blocked NS5A hyperphosphorylation, results that are consistent with an inhibitor-binding site within the N-terminal region of NS5A. In addition, we observed that an NS5A inhibitor promoted the accumulation of an HCV polyprotein intermediate, suggesting that inhibitor binding to NS5A may occur prior to the completion of polyprotein processing. Finally, we observed that NS5A p56 and p58 separated into different membrane fractions during discontinuous sucrose gradient centrifugation, consistent with these NS5A phosphoforms performing distinct replication functions. The p58 localization pattern was disrupted by an NS5A inhibitor. Collectively, our results suggest that NS5A inhibitors probably impact several aspects of HCV expression and regulation. These findings may help to explain the exceptional potency of this class of HCV replication complex inhibitors.
Sujet(s)
Antiviraux/pharmacologie , Antienzymes/pharmacologie , Hepacivirus/effets des médicaments et des substances chimiques , Hepacivirus/enzymologie , Protéines virales non structurales/antagonistes et inhibiteurs , Protéines virales non structurales/métabolisme , Phosphorylation , RNA replicase/antagonistes et inhibiteurs , RNA replicase/métabolismeRÉSUMÉ
Novel C-5 substituted pyrrolotriazines were optimized for dual EGFR and HER2 protein tyrosine kinase inhibition. The lead compound exhibited promising oral efficacy in both EGFR and HER2 driven human tumor xenograft models. It is hypothesized that its C-5 morpholine side chain binds in the ribose phosphate portion of the ATP binding pocket.
Sujet(s)
Chimie pharmaceutique/méthodes , Récepteurs ErbB/antagonistes et inhibiteurs , Pyrroles/synthèse chimique , Récepteur ErbB-2/antagonistes et inhibiteurs , Triazines/synthèse chimique , Adénosine triphosphate/composition chimique , Animaux , Cellules Caco-2 , Conception de médicament , Antienzymes/pharmacologie , Humains , Concentration inhibitrice 50 , Souris , Transplantation tumorale , Phosphates/composition chimique , Pyrroles/pharmacologie , Ribose/composition chimique , Triazines/pharmacologieRÉSUMÉ
We have recently identified BMS-345541 (1) as a highly selective and potent inhibitor of IKK-2 (IC50 = 0.30 microM), which however was considerably less potent against IKK-1 (IC50 = 4.0 microM). In order to further explore the SAR around the imidazoquinoxaline tricyclic structure of 1, we prepared a series of tetracyclic analogues (7, 13, and 18). The synthesis and biological activities of these potent IKK inhibitors are described.
Sujet(s)
Composés hétérocycliques avec 4 noyaux ou plus/synthèse chimique , Composés hétérocycliques avec 4 noyaux ou plus/pharmacologie , I-kappa B Kinase/antagonistes et inhibiteurs , Benzimidazoles/synthèse chimique , Benzimidazoles/pharmacologie , Lignée cellulaire , Humains , Concentration inhibitrice 50 , Quinazolines/synthèse chimique , Quinazolines/pharmacologie , Quinoléines/synthèse chimique , Quinoléines/pharmacologie , Quinoxalines/synthèse chimique , Quinoxalines/pharmacologie , Relation structure-activité , Spécificité du substratRÉSUMÉ
A series of fluoroglycosylated fluoroindolocarbazoles was examined with respect to their topoisomerase I activity, cytotoxicity, and selectivity. The lead clinical candidate from this series, BMS-250749, displays broad spectrum antitumor activity superior to CPT-11 against some preclinical xenograft models, including curative antitumor activity against Lewis lung carcinoma.
Sujet(s)
Antinéoplasiques/synthèse chimique , Camptothécine/analogues et dérivés , Camptothécine/pharmacologie , Carbazoles/synthèse chimique , Glucosides/synthèse chimique , Indoles/synthèse chimique , Animaux , Antinéoplasiques/composition chimique , Antinéoplasiques/pharmacologie , Carbazoles/composition chimique , Carbazoles/pharmacologie , Lignée cellulaire tumorale , Évaluation préclinique de médicament , Résistance aux médicaments antinéoplasiques , Tests de criblage d'agents antitumoraux , Glucosides/composition chimique , Glucosides/pharmacologie , Humains , Techniques in vitro , Indoles/composition chimique , Indoles/pharmacologie , Irinotécan , Souris , Microsomes du foie/métabolisme , Inhibiteurs de la topoisomérase-I , Transplantation hétérologueRÉSUMÉ
[reaction: see text] Both 6'- and 4'-fluoro-glycosylated indolo[2,3-a]carbazoles are substrates for base-induced loss of fluorine as a leaving group from sp3 carbon. In the case of alpha-N-glycosylated substrate 3, loss of fluorine from the 6'-position leads to 3,6-anhydroglucose analogue 1. A novel N12,N13-bridged sugar analogue 2 results from loss of 4'-fluorine from beta-N-glycosylated analogue 4. Both analogues 1 and 2 display topo I inhibitory potencies similar to camptothecin.
Sujet(s)
Carbazoles/synthèse chimique , Carbone/composition chimique , Antienzymes/synthèse chimique , Fluor/composition chimique , Hétérosides/synthèse chimique , Composés hétérocycliques, cycle ponté/synthèse chimique , Hydrocarbures fluorés/synthèse chimique , Indoles/synthèse chimique , Inhibiteurs de la topoisomérase-I , Animaux , Carbazoles/pharmacologie , Antienzymes/pharmacologie , Hétérosides/pharmacologie , Composés hétérocycliques, cycle ponté/pharmacologie , Hydrocarbures fluorés/composition chimique , Indoles/composition chimique , Leucémie P388 , Conformation moléculaire , Structure moléculaire , Relation structure-activitéRÉSUMÉ
A series of fluoroindolocarbazoles were studied with respect to their topoisomerase I activity, cytotoxicity, selectivity, and in vivo antitumor activity. Emerging from this series was BMS-251873, a potential clinical candidate possessing a robust pharmacological profile including curative antitumor activity against prostate carcinoma.
Sujet(s)
Antinéoplasiques/synthèse chimique , Carbazoles/synthèse chimique , Glucosides/synthèse chimique , Tumeurs de la prostate/traitement médicamenteux , Inhibiteurs de la topoisomérase-I , Animaux , Antinéoplasiques/composition chimique , Antinéoplasiques/pharmacologie , Carbazoles/composition chimique , Carbazoles/pharmacologie , Glucosides/composition chimique , Glucosides/pharmacologie , Mâle , Souris , Transplantation tumorale , Solubilité , Relation structure-activité , Eau , Tests d'activité antitumorale sur modèle de xénogreffeRÉSUMÉ
beta-Alanine derivative 2 (IC(50)=16 nM) and related compounds were found to be potent MC4R agonists.
Sujet(s)
Dipeptides/synthèse chimique , Dipeptides/pharmacologie , Récepteur de la mélanocortine de type 4/agonistes , bêta-Alanine/pharmacologie , Animaux , Sites de fixation , Dipeptides/composition chimique , Dipeptides/pharmacocinétique , Conception de médicament , Comportement alimentaire/effets des médicaments et des substances chimiques , Humains , Cinétique , Modèles animaux , Modèles moléculaires , Relation structure-activité , bêta-Alanine/pharmacocinétiqueRÉSUMÉ
2-arylamino-4-trifluoromethyl-5-aminomethylthiazoles represent a novel series of high-affinity corticotropin releasing factor-1 receptor (CRF(1)R) antagonists that are prepared in three steps in good overall yields. Herein, we report binding SAR as well as anxiolytic activity of an exemplary compound (7a, K(i)=8.6 nM) in a mouse canopy model.