RÉSUMÉ
The design and synthesis of potent, tripeptidic acylsulfonamide inhibitors of HCV NS3 protease that contain a difluoromethyl cyclopropyl amino acid at P1 are described. A cocrystal structure of 18 with a NS3/4A protease complex suggests the presence of a H-bond between the polarized C-H of the CHF2 moiety and the backbone carbonyl of Leu135 of the enzyme. Structure-activity relationship studies indicate that this H-bond enhances enzyme inhibitory potency by 13- and 17-fold compared to the CH3 and CF3 analogues, respectively, providing insight into the deployment of this unique amino acid.
RÉSUMÉ
Often similar structures need to be compared to reveal local differences throughout the entire model or between related copies within the model. Therefore, a program to compare multiple structures and enable correction any differences not supported by the density map was written within the Phenix framework (Adams et al., Acta Cryst 2010; D66:213-221). This program, called Structure Comparison, can also be used for structures with multiple copies of the same protein chain in the asymmetric unit, that is, as a result of non-crystallographic symmetry (NCS). Structure Comparison was designed to interface with Coot(Emsley et al., Acta Cryst 2010; D66:486-501) and PyMOL(DeLano, PyMOL 0.99; 2002) to facilitate comparison of large numbers of related structures. Structure Comparison analyzes collections of protein structures using several metrics, such as the rotamer conformation of equivalent residues, displays the results in tabular form and allows superimposed protein chains and density maps to be quickly inspected and edited (via the tools in Coot) for consistency, completeness and correctness.
Sujet(s)
Modèles moléculaires , Protéines/composition chimique , Interface utilisateur , Protéines/génétiqueRÉSUMÉ
The design and synthesis of a series of tripeptide acylsulfonamides as potent inhibitors of the HCV NS3/4A serine protease is described. These analogues house a C4 aryl, C4 hydroxy-proline at the S2 position of the tripeptide scaffold. Information relating to structure-activity relationships as well as the pharmacokinetic and cardiovascular profiles of these analogues is provided.
Sujet(s)
Antiviraux/composition chimique , Hepacivirus/enzymologie , Oligopeptides/composition chimique , Inhibiteurs de protéases/composition chimique , Protéines virales non structurales/antagonistes et inhibiteurs , Animaux , Antiviraux/pharmacocinétique , Antiviraux/pharmacologie , Période , Coeur/effets des médicaments et des substances chimiques , Hepacivirus/effets des médicaments et des substances chimiques , Humains , Techniques in vitro , Oligopeptides/pharmacocinétique , Oligopeptides/pharmacologie , Proline/composition chimique , Inhibiteurs de protéases/pharmacocinétique , Inhibiteurs de protéases/pharmacologie , Lapins , Rats , Relation structure-activité , Sulfonamides/composition chimique , Protéines virales non structurales/métabolismeRÉSUMÉ
The discovery of a back-up to the hepatitis C virus NS3 protease inhibitor asunaprevir (2) is described. The objective of this work was the identification of a drug with antiviral properties and toxicology parameters similar to 2, but with a preclinical pharmacokinetic (PK) profile that was predictive of once-daily dosing. Critical to this discovery process was the employment of an ex vivo cardiovascular (CV) model which served to identify compounds that, like 2, were free of the CV liabilities that resulted in the discontinuation of BMS-605339 (1) from clinical trials. Structure-activity relationships (SARs) at each of the structural subsites in 2 were explored with substantial improvement in PK through modifications at the P1 site, while potency gains were found with small, but rationally designed structural changes to P4. Additional modifications at P3 were required to optimize the CV profile, and these combined SARs led to the discovery of BMS-890068 (29).
Sujet(s)
Antiviraux/composition chimique , Hepacivirus/effets des médicaments et des substances chimiques , Isoquinoléines/usage thérapeutique , Oligopeptides/composition chimique , Sulfonamides/composition chimique , Protéines virales non structurales/antagonistes et inhibiteurs , Animaux , Antiviraux/administration et posologie , Antiviraux/pharmacocinétique , Antiviraux/pharmacologie , Chiens , Calendrier d'administration des médicaments , Résistance virale aux médicaments , Hepacivirus/génétique , Macaca fascicularis , Mâle , Modèles moléculaires , Oligopeptides/administration et posologie , Oligopeptides/pharmacocinétique , Oligopeptides/pharmacologie , Lapins , Rat Sprague-Dawley , Réplicon , Stéréoisomérie , Relation structure-activité , Sulfonamides/administration et posologie , Sulfonamides/pharmacocinétique , Sulfonamides/pharmacologie , Sulfonamides/usage thérapeutiqueRÉSUMÉ
The discovery of BMS-605339 (35), a tripeptidic inhibitor of the NS3/4A enzyme, is described. This compound incorporates a cyclopropylacylsulfonamide moiety that was designed to improve the potency of carboxylic acid prototypes through the introduction of favorable nonbonding interactions within the S1' site of the protease. The identification of 35 was enabled through the optimization and balance of critical properties including potency and pharmacokinetics (PK). This was achieved through modulation of the P2* subsite of the inhibitor which identified the isoquinoline ring system as a key template for improving PK properties with further optimization achieved through functionalization. A methoxy moiety at the C6 position of this isoquinoline ring system proved to be optimal with respect to potency and PK, thus providing the clinical compound 35 which demonstrated antiviral activity in HCV-infected patients.
Sujet(s)
Antiviraux/usage thérapeutique , Découverte de médicament , Hépatite C/traitement médicamenteux , Isoquinoléines/usage thérapeutique , Inhibiteurs de protéases/usage thérapeutique , Sulfonamides/usage thérapeutique , Protéines virales non structurales/antagonistes et inhibiteurs , Animaux , Cristallographie aux rayons X , Chiens , Évaluation préclinique de médicament , Humains , Isoquinoléines/composition chimique , Modèles moléculaires , Inhibiteurs de protéases/composition chimique , Sulfonamides/composition chimiqueRÉSUMÉ
The discovery of asunaprevir (BMS-650032, 24) is described. This tripeptidic acylsulfonamide inhibitor of the NS3/4A enzyme is currently in phase III clinical trials for the treatment of hepatitis C virus infection. The discovery of 24 was enabled by employing an isolated rabbit heart model to screen for the cardiovascular (CV) liabilities (changes to HR and SNRT) that were responsible for the discontinuation of an earlier lead from this chemical series, BMS-605339 (1), from clinical trials. The structure-activity relationships (SARs) developed with respect to CV effects established that small structural changes to the P2* subsite of the molecule had a significant impact on the CV profile of a given compound. The antiviral activity, preclincial PK profile, and toxicology studies in rat and dog supported clinical development of BMS-650032 (24).
Sujet(s)
Antiviraux/usage thérapeutique , Hépatite C/traitement médicamenteux , Isoquinoléines/usage thérapeutique , Inhibiteurs de protéases/usage thérapeutique , Sulfonamides/usage thérapeutique , Protéines virales non structurales/antagonistes et inhibiteurs , Animaux , Antiviraux/sang , Antiviraux/composition chimique , Chiens , Humains , Isoquinoléines/sang , Isoquinoléines/composition chimique , Modèles moléculaires , Inhibiteurs de protéases/sang , Inhibiteurs de protéases/composition chimique , Lapins , Rats , Sulfonamides/sang , Sulfonamides/composition chimiqueRÉSUMÉ
The process of iterative structure-based drug design involves the X-ray crystal structure determination of upwards of 100 ligands with the same general scaffold (i.e. chemotype) complexed with very similar, if not identical, protein targets. In conjunction with insights from computational models and assays, this collection of crystal structures is analyzed to improve potency, to achieve better selectivity and to reduce liabilities such as absorption, distribution, metabolism, excretion and toxicology. Current methods for modeling ligands into electron-density maps typically do not utilize information on how similar ligands bound in related structures. Even if the electron density is of sufficient quality and resolution to allow de novo placement, the process can take considerable time as the size, complexity and torsional degrees of freedom of the ligands increase. A new module, Guided Ligand Replacement (GLR), was developed in Phenix to increase the ease and success rate of ligand placement when prior protein-ligand complexes are available. At the heart of GLR is an algorithm based on graph theory that associates atoms in the target ligand with analogous atoms in the reference ligand. Based on this correspondence, a set of coordinates is generated for the target ligand. GLR is especially useful in two situations: (i) modeling a series of large, flexible, complicated or macrocyclic ligands in successive structures and (ii) modeling ligands as part of a refinement pipeline that can automatically select a reference structure. Even in those cases for which no reference structure is available, if there are multiple copies of the bound ligand per asymmetric unit GLR offers an efficient way to complete the model after the first ligand has been placed. In all of these applications, GLR leverages prior knowledge from earlier structures to facilitate ligand placement in the current structure.
Sujet(s)
Cristallographie aux rayons X/méthodes , Conception de médicament , Protéase du VIH/composition chimique , Protéase du VIH/métabolisme , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/enzymologie , Humains , Ligands , Modèles moléculaires , Liaison aux protéines , p38 Mitogen-Activated Protein Kinases/composition chimique , p38 Mitogen-Activated Protein Kinases/métabolismeRÉSUMÉ
High-throughput drug-discovery and mechanistic studies often require the determination of multiple related crystal structures that only differ in the bound ligands, point mutations in the protein sequence and minor conformational changes. If performed manually, solution and refinement requires extensive repetition of the same tasks for each structure. To accelerate this process and minimize manual effort, a pipeline encompassing all stages of ligand building and refinement, starting from integrated and scaled diffraction intensities, has been implemented in Phenix. The resulting system is able to successfully solve and refine large collections of structures in parallel without extensive user intervention prior to the final stages of model completion and validation.
Sujet(s)
Cristallographie aux rayons X/méthodes , Protéines/composition chimique , Animaux , Conception de médicament , Facteur Xa/composition chimique , Facteur Xa/métabolisme , Protéase du VIH/composition chimique , Protéase du VIH/métabolisme , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/enzymologie , Humains , Ligands , Modèles moléculaires , Liaison aux protéines , Protéines/métabolisme , Thrombine/composition chimique , Thrombine/métabolismeRÉSUMÉ
Human ß-glucuronidase (GUS) cleaves ß-D-glucuronic acid residues from the non-reducing termini of glycosaminoglycan and its deficiency leads to mucopolysaccharidosis type VII (MPSVII). Here we report a high resolution crystal structure of human GUS at 1.7 Å resolution and present an extensive analysis of the structural features, unifying recent findings in the field of lysosome targeting and glycosyl hydrolases. The structure revealed several new details including a new glycan chain at Asn272, in addition to that previously observed at Asn173, and coordination of the glycan chain at Asn173 with Lys197 of the lysosomal targeting motif which is essential for phosphotransferase recognition. Analysis of the high resolution structure not only provided new insights into the structural basis for lysosomal targeting but showed significant differences between human GUS, which is medically important in its own right, and E. coli GUS, which can be selectively inhibited in the human gut to prevent prodrug activation and is also widely used as a reporter gene by plant biologists. Despite these differences, both human and E. coli GUS share a high structure homology in all three domains with most of the glycosyl hydrolases, suggesting that they all evolved from a common ancestral gene.
Sujet(s)
Glucuronidase/composition chimique , Glucuronidase/métabolisme , Lysosomes/métabolisme , Séquence d'acides aminés , Cristallographie aux rayons X , Glycosaminoglycanes/métabolisme , Humains , Données de séquences moléculairesRÉSUMÉ
Optimization of a 5-oxopyrrolopyridine series based upon structure-activity relationships (SARs) developed from our previous efforts on a number of related bicyclic series yielded compound 2s (BMS-767778) with an overall activity, selectivity, efficacy, PK, and developability profile suitable for progression into the clinic. SAR in the series and characterization of 2s are described.
Sujet(s)
Acétamides/composition chimique , Acétamides/pharmacologie , Composés hétérocycliques bicycliques/composition chimique , Composés hétérocycliques bicycliques/pharmacologie , Dipeptidyl peptidase 4/métabolisme , Inhibiteurs de la dipeptidyl-peptidase IV/composition chimique , Inhibiteurs de la dipeptidyl-peptidase IV/pharmacologie , Conception de médicament , Pyrroles/composition chimique , Pyrroles/pharmacologie , Acétamides/synthèse chimique , Animaux , Composés hétérocycliques bicycliques/synthèse chimique , Domaine catalytique , Dipeptidyl peptidase 4/composition chimique , Inhibiteurs de la dipeptidyl-peptidase IV/synthèse chimique , Hyperglycémie provoquée , Humains , Mâle , Souris , Modèles moléculaires , Pyrroles/synthèse chimique , Spécificité du substratRÉSUMÉ
The design, synthesis and characterization of a phosphonate inhibitor of N-acetylneuraminate-9-phosphate phosphatase (HDHD4) is described. Compound 3, where the substrate C-9 oxygen was replaced with a nonlabile CH2 group, inhibits HDHD4 with a binding affinity (IC50 11µM) in the range of the native substrate Neu5Ac-9-P (compound 1, Km 47µM). Combined SAR, modeling and NMR studies are consistent with the phosphonate group in inhibitor 3 forming a stable complex with native Mg(2+). In addition to this key interaction, the C-1 carboxylate of the sugar interacts with a cluster of basic residues, K141, R104 and R72. Comparative NMR studies of compounds 3 and 1 with Ca(2+) and Mg(2+) are indicative of a highly dynamic process in the active site for the HDHD4/Mg(2+)/3 complex. Possible explanations for this observation are discussed.
Sujet(s)
Conception de médicament , Antienzymes/synthèse chimique , Phosphoric monoester hydrolases/antagonistes et inhibiteurs , Acides sialiques/synthèse chimique , Oses phosphates/synthèse chimique , Animaux , Sites de fixation , Cristallographie aux rayons X , Antienzymes/composition chimique , Antienzymes/métabolisme , Humains , Cinétique , Spectroscopie par résonance magnétique , Simulation de docking moléculaire , Phosphoric monoester hydrolases/métabolisme , Structure tertiaire des protéines , Rats , Acides sialiques/composition chimique , Acides sialiques/métabolisme , Oses phosphates/composition chimique , Oses phosphates/métabolismeRÉSUMÉ
The design, synthesis and SAR of a novel class of valerolactam-based arylsulfonamides as potent and selective FXa inhibitors is reported. The arylsulfonamide-valerolactam scaffold was derived based on the proposed bioisosterism to the arylcyanoguanidine-caprolactam core in known FXa inhibitors. The SAR study led to compound 46 as the most potent FXa inhibitor in this series, with an IC(50) of 7 nM and EC(2×PT) of 1.7 µM. The X-ray structure of compound 40 bound to FXa shows that the sulfonamide-valerolactam scaffold anchors the aryl group in the S1 and the novel acylcytisine pharmacophore in the S4 pockets.
Sujet(s)
Anticoagulants/composition chimique , Inhibiteurs du facteur Xa , Pipéridones/composition chimique , Inhibiteurs de la sérine protéinase/composition chimique , Anticoagulants/synthèse chimique , Anticoagulants/pharmacologie , Sites de fixation , Cristallographie aux rayons X , Activation enzymatique/effets des médicaments et des substances chimiques , Facteur Xa/métabolisme , Humains , Lactames/composition chimique , Conformation moléculaire , Pipéridones/synthèse chimique , Pipéridones/pharmacologie , Structure tertiaire des protéines , Inhibiteurs de la sérine protéinase/synthèse chimique , Inhibiteurs de la sérine protéinase/pharmacologie , Relation structure-activitéRÉSUMÉ
Design, synthesis, and SAR of 7-oxopyrrolopyridine-derived DPP4 inhibitors are described. The preferred stereochemistry of these atropisomeric biaryl analogs has been identified as Sa. Compound (+)-3t, with a K(i) against DPP4, DPP8, and DPP9 of 0.37 nM, 2.2, and 5.7 µM, respectively, showed a significant improvement in insulin response after single doses of 3 and 10 µmol/kg in ob/ob mice.
Sujet(s)
Cytochrome P-450 CYP3A/métabolisme , Dipeptidyl peptidase 4/métabolisme , Inhibiteurs de la dipeptidyl-peptidase IV/composition chimique , Inhibiteurs de la dipeptidyl-peptidase IV/pharmacologie , Canaux potassiques éther-à-go-go/métabolisme , Pyridines/composition chimique , Pyridines/pharmacologie , Animaux , Domaine catalytique , Diabète/traitement médicamenteux , Inhibiteurs de la dipeptidyl-peptidase IV/pharmacocinétique , Humains , Insuline/sang , Insuline/métabolisme , Souris , Souris de lignée C57BL , Modèles moléculaires , Pyridines/pharmacocinétique , Pyrroles/composition chimique , Pyrroles/pharmacocinétique , Pyrroles/pharmacologie , Rats , StéréoisomérieRÉSUMÉ
CARM1 (co-activator-associated arginine methyltransferase 1) is a PRMT (protein arginine N-methyltransferase) family member that catalyses the transfer of methyl groups from SAM (S-adenosylmethionine) to the side chain of specific arginine residues of substrate proteins. This post-translational modification of proteins regulates a variety of transcriptional events and other cellular processes. Moreover, CARM1 is a potential oncological target due to its multiple roles in transcription activation by nuclear hormone receptors and other transcription factors such as p53. Here, we present crystal structures of the CARM1 catalytic domain in complex with cofactors [SAH (S-adenosyl-L-homocysteine) or SNF (sinefungin)] and indole or pyazole inhibitors. Analysis of the structures reveals that the inhibitors bind in the arginine-binding cavity and the surrounding pocket that exists at the interface between the N- and C-terminal domains. In addition, we show using ITC (isothermal titration calorimetry) that the inhibitors bind to the CARM1 catalytic domain only in the presence of the cofactor SAH. Furthermore, sequence differences for select residues that interact with the inhibitors may be responsible for the CARM1 selectivity against PRMT1 and PRMT3. Together, the structural and biophysical information should aid in the design of both potent and specific inhibitors of CARM1.
Sujet(s)
Indoles/antagonistes et inhibiteurs , Indoles/composition chimique , Protein-arginine N-methyltransferases/antagonistes et inhibiteurs , Protein-arginine N-methyltransferases/composition chimique , Pyrazoles/antagonistes et inhibiteurs , Pyrazoles/composition chimique , Séquence d'acides aminés , Domaine catalytique/effets des médicaments et des substances chimiques , Cristallographie aux rayons X , Antienzymes/composition chimique , Antienzymes/métabolisme , Antienzymes/pharmacologie , Humains , Indoles/métabolisme , Données de séquences moléculaires , Liaison aux protéines/effets des médicaments et des substances chimiques , Protein-arginine N-methyltransferases/métabolisme , Pyrazoles/métabolismeRÉSUMÉ
Continued structure-activity relationship (SAR) exploration within our previously disclosed azolopyrimidine containing dipeptidyl peptidase-4 (DPP4) inhibitors led us to focus on an imidazolopyrimidine series in particular. Further study revealed that by replacing the aryl substitution on the imidazole ring with a more polar carboxylic ester or amide, these compounds displayed not only increased DPP4 binding activity but also significantly reduced human ether-a-go-go related gene (hERG) and sodium channel inhibitory activities. Additional incremental adjustment of polarity led to permeable molecules which exhibited favorable pharmacokinetic (PK) profiles in preclinical animal species. The active site binding mode of these compounds was determined by X-ray crystallography as exemplified by amide 24c. A subsequent lead molecule from this series, (+)-6-(aminomethyl)-5-(2,4-dichlorophenyl)-N-(1-ethyl-1H-pyrazol-5-yl)-7-methylimidazo[1,2-a]pyrimidine-2-carboxamide (24s), emerged as a potent, selective DPP4 inhibitor that displayed excellent PK profiles and in vivo efficacy in ob/ob mice.
Sujet(s)
Inhibiteurs de la dipeptidyl-peptidase IV , Hypoglycémiants/synthèse chimique , Imidazoles/synthèse chimique , Pyrimidines/synthèse chimique , Animaux , Domaine catalytique , Cristallographie aux rayons X , Diabète de type 2/traitement médicamenteux , Dipeptidyl peptidase 4/composition chimique , Chiens , Canal potassique ERG1 , Canaux potassiques éther-à-go-go/antagonistes et inhibiteurs , Humains , Hypoglycémiants/pharmacocinétique , Hypoglycémiants/pharmacologie , Imidazoles/pharmacocinétique , Imidazoles/pharmacologie , Mâle , Souris , Souris obèse , Modèles moléculaires , Pyrimidines/pharmacocinétique , Pyrimidines/pharmacologie , Rats , Bloqueurs de canaux sodiques/pharmacologie , Stéréoisomérie , Relation structure-activitéRÉSUMÉ
We report the design and synthesis of a novel class of N,N'-disubstituted aroylguanidine-based lactam derivatives as potent and orally active FXa inhibitors. The structure-activity relationships (SAR) investigation led to the discovery of the nicotinoyl guanidine 22 as a potent FXa inhibitor (FXa IC(50)=4 nM, EC(2xPT)=7 microM). However, the potent CYP3A4 inhibition activity (IC(50)=0.3 microM) of 22 precluded its further development. Detailed analysis of the X-ray crystal structure of compound 22 bound to FXa indicated that the substituent at the 6-position of the nicotinoyl group of 22 would be solvent-exposed, suggesting that efforts to attenuate the unwanted CYP activity could focus at this position without affecting FXa potency significantly. Further SAR studies on the 6-substituted nicotinoyl guanidines resulted in the discovery of 6-(dimethylcarbamoyl) nicotinoyl guanidine 36 (BMS-344577, IC(50)=9 nM, EC(2xPT)=2.5 microM), which was found to be a selective, orally efficacious FXa inhibitor with an excellent in vitro liability profile, favorable pharmacokinetics and pharmacodynamics in animal models.
Sujet(s)
Anticoagulants/composition chimique , Inhibiteurs du facteur Xa , Guanidines/composition chimique , Inhibiteurs de la sérine protéinase/composition chimique , Anticoagulants/pharmacologie , Cytochrome P-450 CYP3A , Inhibiteurs du cytochrome P-450 CYP3A , Découverte de médicament , Guanidines/pharmacologie , Humains , Concentration inhibitrice 50 , Inhibiteurs de la sérine protéinase/pharmacologie , Relation structure-activitéRÉSUMÉ
The N,N'-disubstituted cyanoguanidine is an excellent bioisostere of the thiourea and ketene aminal functional groups. We report the design and synthesis of a novel class of cyanoguanidine-based lactam derivatives as potent and orally active FXa inhibitors. The SAR studies led to the discovery of compound 4 (BMS-269223, K(i)=6.5nM, EC(2xPT)=32muM) as a selective, orally bioavailable FXa inhibitor with an excellent in vitro liability profile, favorable pharmacokinetics and pharmacodynamics in animal models. The X-ray crystal structure of 4 bound in FXa is presented and key ligand-protein interactions are discussed.
Sujet(s)
Antithrombine-III/pharmacologie , Benzofuranes/pharmacologie , Guanidines/composition chimique , Lactames/composition chimique , Administration par voie orale , Animaux , Antithrombine-III/composition chimique , Benzofuranes/composition chimique , Chimie pharmaceutique/méthodes , Cristallographie aux rayons X/méthodes , Chiens , Haplorhini , Humains , Concentration inhibitrice 50 , Cinétique , Lactames/pharmacologie , Ligands , Modèles chimiques , Rats , Relation structure-activité , Thiourée/composition chimiqueRÉSUMÉ
An indole-based P1 moiety was incorporated into a previously established factor Xa inhibitor series. The indole group was designed to hydrogen-bond with the carbonyl of Gly218, while its 3-methyl or 3-chloro substituent was intended to interact with Tyr228. These interactions were subsequently observed in the X-ray crystal structure of compound 18. SAR studies led to the identification of compound 20 as the most potent FXa inhibitor in this series (IC(50) = 2.4 nM, EC(2xPT) = 1.2 microM). An in-depth energetic analysis suggests that the increased binding energy of 3-chloroindole-versus 3-methylindole-containing compounds in this series is due primarily to (a) the more hydrophobic nature of chloro- versus methyl-containing compounds and (b) an increased interaction of 3-chloroindole versus 3-methylindole with Gly218 backbone. The stronger hydrophobicity of chloro- versus methyl-substituted aromatics may partly explain the general preference for chloro- versus methyl-substituted P1 groups in FXa, which extends beyond the current series.
Sujet(s)
Conception de médicament , Antienzymes , Inhibiteurs du facteur Xa , Indoles , Théorie quantique , Animaux , Sites de fixation/effets des médicaments et des substances chimiques , Simulation numérique , Cristallographie aux rayons X , Relation dose-effet des médicaments , Antienzymes/synthèse chimique , Antienzymes/composition chimique , Antienzymes/pharmacologie , Facteur Xa/effets des médicaments et des substances chimiques , Humains , Liaison hydrogène , Indoles/synthèse chimique , Indoles/composition chimique , Indoles/pharmacologie , Souris , Modèles chimiques , Modèles moléculaires , Relation structure-activité , Analyse de survie , Venins/pharmacologie , Thrombose veineuse/traitement médicamenteux , Thrombose veineuse/enzymologieRÉSUMÉ
The inhibition of DPP-IV by saxagliptin has been proposed to occur through formation of a covalent but reversible complex. To evaluate further the mechanism of inhibition, we determined the X-ray crystal structure of the DPP-IV:saxagliptin complex. This structure reveals covalent attachment between S630 and the inhibitor nitrile carbon (C-O distance <1.3 A). To investigate whether this serine addition is assisted by the catalytic His-Asp dyad, we generated two mutants of DPP-IV, S630A and H740Q, and assayed them for ability to bind inhibitor. DPP-IV H740Q bound saxagliptin with an approximately 1000-fold reduction in affinity relative to DPP-IV WT, while DPP-IV S630A showed no evidence for binding inhibitor. An analog of saxagliptin lacking the nitrile group showed unchanged binding properties to the both mutant proteins, highlighting the essential role S630 and H740 play in covalent bond formation between S630 and saxagliptin. Further supporting mechanism-based inhibition by saxagliptin, NMR spectra of enzyme-saxagliptin complexes revealed the presence of three downfield resonances with low fractionation factors characteristic of short and strong hydrogen bonds (SSHB). Comparison of the NMR spectra of various wild-type and mutant DPP-IV:ligand complexes enabled assignment of a resonance at approximately 14 ppm to H740. Two additional DPP-IV mutants, Y547F and Y547Q, generated to probe potential stabilization of the enzyme-inhibitor complex by this residue, did not show any differences in inhibitor binding either by ITC or NMR. Together with the previously published enzymatic data, the structural and binding data presented here strongly support a histidine-assisted covalent bond formation between S630 hydroxyl oxygen and the nitrile group of saxagliptin.