Continued exploration of the triazolopyridine scaffold as a platform for p38 MAP kinase inhibition

The structure based drug design, synthesis and structure–activity relationship of a series of C6 sulfur linked triazolopyridine based p38 inhibitors are described. The metabolic deficiencies of this series were overcome through changes in the C6 linker from sulfur to methylene, which was predicted by molecular modeling to be bioisosteric. X-ray of the ethylene linked compound 61 confirmed the predicted binding orientation of the scaffold in the p38 enzyme.     

Potent, selective, and orally bioavailable matrix metalloproteinase-13 inhibitors for the treatment of osteoarthritis

Modification of -biphenylsulfonamidocarboxylic acids led to potent and selective MMP-13 inhibitors. Compound 16 showed 100% oral bioavailability in rats and demonstrated >50% inhibition of bovine cartilage degradation at 10 ng/mL.     

A systematic computational analysis of human matrix metalloproteinase 13 (MMP-13) crystal structures and structure-based identification of prospective drug candidates as MMP-13 inhibitors repurposable for osteoarthritis

Abstract

Osteoarthritis (OA) is the most common form of arthritis with no available disease-modifying treatments, and is a major cause of disability. Matrix metalloproteinase 13 (MMP-13) is vital for OA progression and thus, inhibition of MMP-13 is an effective strategy to treat OA. Since the past few decades, drug repurposing has gained substantial popularity worldwide as a time- and cost-effective approach to find new indications for the existing drugs. Therefore, more than 40 X-ray co-crystal structures of the human MMP-13 with bound inhibitors are investigated to gain the structural insights such as conserved direct interactions with binding site residues, namely Ala-238, Thr-245 and Thr-247. Afterwards, enrichment study using active and decoy set of ligands revealed three MMP-13 structures (PDB-IDs: 1XUC, 3WV1 and 5BPA) with optimal enrichment performance. Docking-based screening of existing drugs against the three crystal structures followed by binding free-energy calculation suggested drugs namely eltrombopag, cilostazol and domperidone as potential MMP-13 inhibitors that need further experimental validation. These insights may serve as a potential starting point of further experimental validation and structure-based drug design/repurposing of MMP-13 inhibitors for the treatment of OA. Abbreviations 2D two-dimensional

3D three-dimensional

FDA Food and Drug Administration

MM-GBSA Molecular Mechanics Generalized Born Surface Area

MMPs matrix metalloproteinases

MMP-13 matrix metalloproteinase 13

NMR nuclear magnetic resonance

OA osteoarthritis

PDB Protein Data Bank

PDB-ID Protein Data Bank ID

PLIP protein–ligand interaction profiler

ROC receiver operating characteristic,

RMSD root mean square deviation

Communicated by Ramaswamy H. Sarma     

Orally active achiral N-hydroxyformamide inhibitors of ADAM-TS4 (aggrecanase-1) and ADAM-TS5 (aggrecanase-2) for the treatment of osteoarthritis

A new achiral class of N-hydroxyformamide inhibitor of both ADAM-TS4 and ADAM-TS5, 2 has been discovered through modification of the complex P1 group present in historical inhibitors 1. This structural change improved the DMPK properties and greatly simplified the synthesis whilst maintaining excellent cross-MMP selectivity profiles. Investigation of structure-activity and structure-property relationships in the P1 group resulted in both ADAM-TS4 selective and mixed ADAM-TS4/5 inhibitors. This led to the identification of a pre-clinical candidate with excellent bioavailability across three species and predicting once daily dosing kinetics.     

慢性睡眠限制状态下大鼠髁突软骨MMP-1,MMP-13表达的变化

目的:探讨MMP-1,MMP-13在慢性睡眠限制引起大鼠髁突软骨结构变化中的表达变化及作用。方法:180只雄性Wistar大鼠随机分为3组(n=60):慢性睡眠限制组(CSR)、大平台组(LC)、笼养组(CON)。每组根据试验时间不同分别分为3个亚组(n=20):7天(7D)、14天(14D)、21天(21D)组。参考改良多平台法(MMPM)建立大鼠的慢性睡眠限制模型。通过HE染色观察大鼠髁突软骨的结构变化。通过免疫组化和实时定量PCR分别检测MMP-1和MMP-13的蛋白水平及m RNA水平的表达变化。结果:HE染色和扫描电镜结果显示,CSR组的大鼠髁突软骨出现了病理性的改变。与CON和LC组比较,CSR组MMP-1和MMP-13的m RNA转录和蛋白表达水平明显升高(P0.05)。结论:慢性睡眠限制能够引起大鼠颞下颌关节髁突软骨的病理性变化。MMP-1和MMP-13的表达水平的变化可能在大鼠髁突软骨病理性改变中起关键作用。     

The design and synthesis of novel N-hydroxyformamide inhibitors of ADAM-TS4 for the treatment of osteoarthritis

Two series of N-hydroxyformamide inhibitors of ADAM-TS4 were identified from screening compounds previously synthesised as inhibitors of matrix metalloproteinase-13 (collagenase-3). Understanding of the binding mode of this class of compound using ADAM-TS1 as a structural surrogate has led to the discovery of potent and very selective inhibitors with favourable DMPK properties. Synthesis, structure-activity relationships, and strategies to improve selectivity and lower in vivo metabolic clearance are described.     

Crystal structures of the catalytic domain of human stromelysin-1 (MMP-3) and collagenase-3 (MMP-13) with a hydroxamic acid inhibitor SM-25453

Crystal structures of the catalytic domain of human stromelysin-1 (MMP-3) and collagenase-3 (MMP-13) with a hydroxamic acid inhibitor SM-25453 have been solved at 2.01 and 2.37A resolutions, respectively. The results revealed that the binding modes for this inhibitor to MMP-3 and -13 were quite similar. However, subtle comparative differences were observed at the bottom of S1' pockets, which were occupied with the guanidinomethyl moiety of the inhibitor. A remarkable feature of the inhibitor was the deep penetration of its long aliphatic chain into the S1' pocket and exposure of the guanidinomethyl moiety to the solvent.     

Molecular docking,synthesis, and biological evaluation of naphthoquinone as potential novel scaffold for H5N1 neuraminidase inhibition

A series of dimeric naphthoquinones containing natural 2-hydroxy-1-4-naphthoquinone moiety was designed, synthesized, and evaluated against neuraminidase of H5N1 virus. p-hydroxy derivatives showed higher inhibition when compared to p-halogenated compounds. Molecular docking studies conducted with H5N1 neuraminidase clearly demonstrated different binding modes of the most active compound onto the open and closed conformations of loop 150. The results thus provide not only evidences of a novel scaffold evaluated as inhibitor, but also a rational explanation involving molecular modeling and the role of loop 150 in the binding.     

Indolyl-pyrrolone as a new scaffold for Pim1 inhibitors

Pim1 belongs to a family of serine/threonine kinases, which is involved in the control of cell growth, differentiation, and apoptosis. Pim1 plays a pivotal role in cytokine signaling and is implicated in the development of a large number of tumors, representing a very attractive target for anticancer therapy. In this work, we applied a virtual screening protocol aimed at identifying small molecules able to inhibit Pim1 activity. The search of novel inhibitors was performed through a structure-based molecular modeling approach, taking advantage of the availability of the three-dimensional crystal structure of inhibitors bound to Pim1. Starting from the knowledge of protein–ligand complexes, the software LigandScout was used to generate pharmacophoric models, in turn used as queries to perform a virtual screening of databases, followed by docking experiments. As a result, a restricted set of candidates for biological testing was identified. Finally, among the six compounds selected as potential inhibitors of Pim1, two candidates endowed with a significant activity against Pim1 emerged. Interestingly, one of these compounds has a chemical scaffold different from inhibitors previously identified.     

Calcium pentosan polysulfate directly inhibits enzymatic activity of ADAMTS4 (aggrecanase-1) in osteoarthritic chondrocytes

Aggrecanases that include ADAMTS1, 4, 5, 8, 9 and 15 are considered to play key roles in aggrecan degradation in osteoarthritic cartilage. Here we demonstrate that calcium pentosan polysulfate (CaPPS) directly inhibits the aggrecanase activity of ADAMTS4 without affecting the mRNA expression of the ADAMTS species in interleukin-1alpha-stimulated osteoarthritic chondrocytes. Synthetic peptides corresponding to specific regions of the thrombospondin type 1 repeat, cysteine-rich or spacer domain of ADAMTS4 inhibit the binding to immobilized CaPPS. These data suggest that CaPPS could function as chondroprotective agent for the treatment of osteoarthritis by inhibition of ADAMTS4 through interaction with the C-terminal ancillary domain.     

GRASP-1 is a neuronal scaffold protein for the JNK signaling pathway

GRASP-1 is a neuronally enriched protein that interacts with the AMPA-type glutamate receptor/GRIP complex. GRASP-1 can be cleaved by Caspase-3 in both normal and ischemic brains although the functional significance of this cleavage remains elusive. We investigated signal transduction pathways that might lie downstream of GRASP-1 and found that GRASP-1 potently activates JNK pathway signaling, with no effect on ERK signaling. Such JNK pathway activating activity requires binding of GRASP-1 to both JNK and the upstream JNK pathway activator MEKK-1. Furthermore, mutations that prevent Caspase 3-cleavage of GRASP-1 dramatically inhibit the JNK pathway activating activity of GRASP-1, suggesting a novel link between Caspase-3 activation and JNK pathway signaling. These results suggest that GRASP-1 serves as a scaffold protein to facilitate MEKK-1 activation of JNK signaling in neurons.     

TEM-1 beta-lactamase as a scaffold for protein recognition and assay

A large number of different proteins or protein domains have been investigated as possible scaffolds to engineer antibody-like molecules. We have previously shown that the TEM-1 beta-lactamase can accommodate insertions of random sequences in two loops surrounding its active site without compromising its activity. From the libraries that were generated, active enzymes binding with high affinities to monoclonal antibodies raised against prostate-specific antigen, a protein unrelated to beta-lactamase, could be isolated. Antibody binding was shown to affect markedly the enzyme activity. As a consequence, these enzymes have the potential to be used as signaling molecules in direct or competitive homogeneous immunoassay. Preliminary results showed that beta-lactamase clones binding to streptavidin could also be isolated, indicating that some enzymes in the libraries have the ability to recognize proteins other than antibodies. In this paper, we show that, in addition to beta-lactamases binding to streptavidin, beta-lactamase clones binding to horse spleen ferritin and beta-galactosidase could be isolated. Affinity maturation of a clone binding to ferritin allowed obtaining beta-lactamases with affinities comprised between 10 and 20 nM (Kd) for the protein. Contrary to what was observed for beta-lactamases issued from selections on antibodies, enzyme complexation induced only a modest effect on enzyme activity, in the three cases studied. This kind of enzyme could prove useful in replacement of enzyme-conjugated antibodies in enzyme-linked immunosorbant assays (ELISA) or in other applications that use antibodies conjugated to an enzyme.     

A novel function of CEP135 as a platform protein of C-NAP1 for its centriolar localization

A proteomic study predicted that about one hundred kinds of proteins constitute a basic structure of the centrosome. Most of the core centrosomal proteins contain extensive coiled-coil domains, suggesting that the protein-protein interaction is a critical force for the core centrosome configuration. In the present study, we investigated a novel interaction between CEP135 and C-NAP1, two core centriolar proteins. Depletion of CEP135 caused a premature centrosome splitting. Reduction of the centrosomal C-NAP1 level was accompanied in a specific manner. Ectopic expression of the CEP135 mutant proteins also caused centrosome splitting in association with the reduction of the centrosomal C-NAP1 levels. Based on these results, we propose that CEP135 acts as a platform protein for C-NAP1 at the centriole.     

兔后交叉韧带断裂后外侧胫骨平台退变的组织学研究

目的：探讨兔后交叉韧带（Posterior cruciate ligament,PCL）断裂对外侧胫骨平台组织学的影响。方法：48只家兔膝关节随机配对为实验侧和对照侧造模,造模后第4、8、16、24周各随机处死12只,行外侧胫骨平台大体观察、HE染色、免疫组化检测基质金属蛋白酶13（matrix metalloproteinase-13,MMP-13）、基质金属蛋白酶抑制剂1（Tisse inhibitor-1 of matrix metalloproteinasel,TIMP-1）表达。结果：①大体观察,随时间延长,实验组外侧胫骨平台软骨出现磨损,呈灰黄色,弹性差,骨赘形成。②组织学观察,随时间延长,胫骨平台软骨纤维化,细胞排列紊乱,簇聚细胞出现频率增加。⑧实验组MMP-13、TIMP—1表达均高于对照组,有显著性差异。P〈0．05。④实验组MMP-13、TIMP-1表达阳性率第4、8、16周逐渐升高,24周下降,各组比较有显著性差异,P〈0．05。结论：①兔膝关节PCL断裂会引起外侧胫骨平台软骨退行性变,且该退变随着时间的推移逐渐加重。②MMP-13与TIMP-1在PCL断裂膝关节外侧胫骨平台中的表达呈现先高后低的变化规律,造成MMP-13与T1MP-1的失衡,加速软骨退变。⑨MMP-13与T／MP-1表达增高提示MMP-13与TIMP—1可能参与了PCL断裂后外侧胫骨平台软骨的退变过程。