Mechanistic insights into the inhibition of prostate specific antigen by beta-lactam class compounds

Prostate Specific Antigen (PSA) is a biomarker used in the diagnosis of prostate cancer and to monitor therapeutic response. However, its precise role in prostate carcinogenesis and metastasis remains largely unknown. A number of studies arguing in the favor of an active role of PSA in prostate cancer development and progression have implicated this serine protease in the release and activation of growth factors such as insulin-like growth factor 1 (IGF1) through cleavage of insulin like growth factor binding protein 3 and Transforming Growth Factor beta (TGF-beta) through cleavage of Latent TGF-beta. In contrast, other studies suggest that PSA activity might hinder tumor development and progression. In light of these contradictory findings, efficient inhibitors of PSA are needed for exploring its biological role in tumor development and metastasis. Towards the goal of developing potent inhibitors of PSA, we have explored the molecular mechanism of a series of beta-lactam based compounds on binding to PSA using activity assays, matrix assisted laser desorption ionization with a time-of-flight mass spectrometry, and GOLD docking methodology. The mass spectrometry experiments and the activity assays confirmed the time-dependent and covalent nature of beta-lactam binding. To gain insights on the reaction intermediates at the molecular level, we docked beta-lactam inhibitors to a homology modeled PSA using the GOLD docking program in noncovalent and covalent binding modes. The docking studies elucidated the molecular details of the early noncovalent Michaelis complex, the acyl-enzyme covalent complex, and the nature of conformational reorganization required for the long term stability of the covalent complex. Additionally, the molecular basis for the effect of stereochemistry of the lactam ring on the inhibitory potency was elucidated through docking of beta-lactam enantiomers. As a validation of our docking methodology, two novel enantiomers were synthesized and evaluated for their inhibitory potency using fluorogenic substrate based activity assays. Additionally, cis enantiomers of eight beta-lactam compounds reported in a previous study were docked and their GOLD scores and binding modes were analyzed in order to assess the general applicability of our docking results. The close agreement of our docking results with the experimental data validates the mechanistic insights revealed through the docking studies and paves the way for the design and development of potent and specific inhibitors of PSA.     

The growth-hormone inducible transmembrane protein (Ghitm) belongs to the Bax inhibitory protein-like family

The conserved protein domain UPF0005 is a protein family signature distributed among many species including fungi and bacteria. Although of unknown functionality this motif has been found in newly identified antiapoptotic proteins comprising the BI-1 family, namely Bax-inhibitory Protein-1 (BI-1), Lifeguard (LFG), and h-GAAP. In a search for vertebrate proteins presumably belonging to the BI-1 family, we found that Growth-hormone inducible transmembrane protein (Ghitm) is another prospective member of the BI-1 family. Here we characterise Ghitm in a first analysis regarding its phylogeny, expression in cancer cell lines, and proteomical properties.     

Design,synthesis, and evaluation of novel 4-amino-2-(4-benzylpiperazin-1-yl)methylbenzonitrile compounds as Zika inhibitors

The prevalence of Zika virus (ZIKV) has become widespread in recent years. ZIKV infection is associated with severe congenital CNS malformations in both newborns and adults. However, neither vaccines nor therapeutics are available to control ZIKV infection until now. We started by hit screening our in-house small molecule library, then designed, synthesized, and evaluated a new class of 1, 4-bibenzylsubstituted piperazine derivatives for their cytopathic effect (CPE) protection effect in a ZIKV-infected Vero E6 cellular assay. A preliminary structure–activity relationship study identified five novel 4-amino-2-(4-benzylpiperazin-1-yl)methylbenzonitrile analogs with obvious CPE reduction effects against ZIKV at micromolar concentrations. Moreover, compound 3p exerted a significant antiviral effect on both Zika RNA replication and virus protein expression in a dose-dependent manner at low micromolar concentrations. This study demonstrated the potential of a novel 4-amino-2-(4-benzylpiperazin-1-yl)methylbenzonitrile scaffold for the development of anti-ZIKV candidates.     

Syntheses and structure–activity relationships for some triazolyl p38α MAPK inhibitors

The design, synthesis and biological evaluation of novel triazolyl p38α MAPK inhibitors with improved water solubility for formulation in cationic liposomes (SAINT-O-Somes) targeted at diseased endothelial cells is described. Water-solubilizing groups were introduced via a ‘click’ reaction of functional azides with 2-alkynyl imidazoles and isosteric oxazoles to generate two small libraries of 1,4-disubstituted 1,2,3-triazolyl p38α MAPK inhibitors. Triazoles with low IC₅₀ values and desired physicochemical properties were screened for in vitro downregulation of proinflammatory gene expression and were formulated in SAINT-O-Somes. Triazolyl p38α MAPK inhibitor 88 (IC₅₀ = 0.096 μM) displayed the most promising in vitro activity.     

Studies on new,centrally active and reversible acetylcholinesterase inhibitors

We have synthesized the tertiary amines of pyridostigmine and neostigmine, 3-pyridinol dimethylcarbamate (norpyridostigmine) and 3-dimethylaminophenol dimethylcarbamate (norneostigmine) respectively, and we have tested their abilities to cross the blood-brain barrier and inhibit mouse brainAChE activity. The in vivo inhibition of AChE activity by norpyridostigmine reaches 72% at 10 minutes which is comparable to that seen with physostigmine (73% at 10 minutes). Inhibition by norneostigmine is less effective (50% at 10 minutes) and approaches that obtained with tetrahydroaminoacridine (57% at 10 minutes). These data show that both norpyridostigmine and norneostigmine cross the blood-brain barrier and that they are effective inhibitors of mouse brain AChE activity. These drugs could be useful in the treatment of memory, impairment associated with Alzheimer's disease, and other memory disorders.     

Arabinose 5-phosphate isomerase as a target for antibacterial design: Studies with substrate analogues and inhibitors

Structural requirements of d-arabinose 5-phosphate isomerase (KdsD, E.C. 5.3.1.13) from Pseudomonas aeruginosa were analysed in detail using advanced NMR techniques. We performed epitope mapping studies of the binding between the enzyme and the most potent KdsD inhibitors found to date, together with studies of a set of newly synthesised arabinose 5-phosphate (A5P) mimetics. We report here the first experimental evidence that KdsD may bind the furanose form of A5P, suggesting that catalysis of ring opening may be an important part of KdsD catalysis.     

苦荞胰蛋白酶抑制剂的纯化及特性研究

利用固相胰蛋白酶亲和色谱及离子交换色谱,从苦荞种子中分离纯化了三个具有抑制胰蛋白酶活力的组份(BTI-Ⅰ,Ⅱ,Ⅲ)。经聚丙稀酰胺凝胶等电聚焦测定,三种抑制剂的等电点为6.87,6.7,5.14。根据Sephadex G-75凝胶过滤,SDS-PAGE测定,以及由当量抑制比值和氨基酸组份的推算,它们的分子量范围分别为5200-5700(Ⅰ),5500—6000(Ⅱ),5000—5600(III)。三者对胰蛋白酶的抑制常数分别为2.12×10~(-8),4.78×10~(-9),1.22×10~(-8)。氨基酸分析结果表明,它们均含有很高的极性氨基酸。在酸性条件下,它们均具有很高的热稳定性。化学修饰的结果表明,它们活性中心的氨基酸残基为Lys。     

Primary cells derived from Tuberous Sclerosis Complex patients show autophagy alteration in the haploinsufficiency state

Tuberous sclerosis complex (TSC) is an autosomal dominant cancer predisposition disorder caused by heterozygous mutations in TSC1 or TSC2 genes and characterized by mTORC1 hyperactivation. TSC-associated tumors develop after loss of heterozygosity mutations and their treatment involves the use of mTORC1 inhibitors. We aimed to evaluate cellular processes regulated by mTORC1 in TSC cells with different mutations before tumor development. Flow cytometry analyses were performed to evaluate cell viability, cell cycle and autophagy in non-tumor primary TSC cells with different heterozygous mutations and in control cells without TSC mutations, before and after treatment with rapamycin (mTORC1 inhibitor). We did not observe differences in cell viability and cell cycle between the cell groups. However, autophagy was reduced in mutated cells. After rapamycin treatment, mutated cells showed a significant increase in the autophagy process (p=0.039). We did not observe differences between cells with distinct TSC mutations. Our main finding is the alteration of autophagy in non-tumor TSC cells. Previous studies in literature found autophagy alterations in tumor TSC cells or knock-out animal models. We showed that autophagy could be an important mechanism that leads to TSC tumor formation in the haploinsufficiency state. This result could guide future studies in this field.     

N-terminal sequence analysis of atrial granule serine proteinase purified by affinity chromatography

Atrial granule serine proteinase is considered the leading candidate endoproteolytic processing enzyme of pro-atrial natriuretic factor. Its cleavage specificity is directed toward a monobasic amino acid processing site, and as such, the atrial enzyme is distinguished from the family of prohormone convertases which act at dibasic amino acid processing sites. To delineate the molecular mechanisms which distinguish monobasic from dibasic amino acid-directed processing enzymes, pure atrial enzyme is needed for sequence determination leading to molecular cloning, and for preparation of antisera. An affinity chromatography purification scheme seemed a logical modification of our established procedures to yield suitable amounts of enzyme for further studies. Surprisingly, pseudo-peptide bond inhibitors of the atrial enzyme [Damodaran and Harris (1995),J. Protein Chem., this issue] formed ineffective affinity ligands, even though these compounds contain essential residues on either side of what would be the scissile bond in a peptide substrate. On the other hand, tripeptide aldehydes (based on the substrate recognition sequence of the atrial enzyme) linked to Sepharose formed effective affinity matrices, permitting purification of the enzyme in a single step from a subcellular fraction enriched for atrial granules and lysosomes. Hence, the enzyme was purified 2000-fold in 90% overall yield, and subjected to N-terminal sequence analysis through 26 residues. The sequence determined, XXPEAAGLPG[R, L]GNPVP[F, G]R[Q, I]XY[G, E]XR(N, A]V, indicates that the atrial enzyme is unique, showing little sequence homology to other proteins in the database.Abbreviations AGSP atrial granule serine proteinase - ANF atrial natriuretic factor - BSA bovine serum albumin - Bz benzoyl - EACA 6()-aminocaproic acid - HEPES N-2-hydroxyethylpiperazine-N'-propanesulfonic acid - HPLC high-performance liquid chromatography - PEG polyethylene glycol-3350 - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Single-letter abbreviations are used to denote amino acids