Expression of activities of two 20 alpha-hydroxysteroid dehydrogenase isozymes in rat corpora lutea.

The rat ovary contains two isozymes of 20 alpha-hydroxysteroid dehydrogenase (HSD-1 and HSD-2). In this study, the expression of activity of each isozyme was investigated in ovaries that contained a single generation of corpora lutea during pseudopregnancy. This condition was induced by cervical stimulation in rats that had been rendered anovulatory by housing them in a continuously lit environment. The total activity of cytosolic 20 alpha-HSD was lower in the ovaries of these pseudopregnant rats than in ovaries containing multiple generations of corpora lutea. In normal pseudopregnancy, HSD-1 activity was low on days 5 and 9 and increased markedly on day 15, whereas HSD-2 was lower than HSD-1 and did not vary throughout pseudopregnancy. However, on days 5 and 9 of continuous-light pseudopregnancy, low activity of HSD-1 only was detected; by day 15, HSD-1 activity had increased sixfold and HSD-2 activity could be detected. Immunohistochemical methods using a specific antibody recognizing both HSD-1 and HSD-2 revealed that the number of 20 alpha-HSD-positive luteal cells increased by day 15. Thus, the increase in total enzyme activity and appearance of HSD-2 activity observed at late pseudopregnancy was accompanied by an increase in the number of 20 alpha-HSD-positive luteal cells.     

人精子膜蛋白片段在沙门氏菌中的表达——一种制备抗生育疫苗的途径

合成编码一种人精子膜蛋白ＹＷＫ－Ⅱ胞外区的一段多肽片段的双链寡核苷酸链,ＨＳＤ－２ａ。用平端连接的方法将其插入到沙门氏菌鞭毛基因ｆｌｉＣ（ｄ）的抗原表位ＩＶ高变区ＥｃｏＲＶ位点,构建了重组质粒ｐＬＳ４０８－Ｈ１。重组基因在鞭毛负性ａｒｏＡ基因缺失的无致病性沙门氏菌Ｓ．ｄｕｂｌｉｎ ＳＬ５９２８疫苗菌株中表达。经ＥＬＩＳＡ、电镜免疫胶体金法检测,表明ＨＳＤ－２ａ编码的多肽片段成功地在沙门氏菌鞭毛表面     

Genetic identification of HSD-1, a conserved steroidogenic enzyme that directs larval development in Caenorhabditis elegans

In C. elegans, steroid hormones function in conjunction with insulin/IGF-1-like signaling in promoting reproductive development over entry into the diapausal dauer stage. The NCR-1 and -2 (NPC1-related) intracellular cholesterol transporters function redundantly in preventing dauer arrest, presumably by regulating the availability of substrates for steroid hormone synthesis. We have identified hsd-1 as a new component of this cholesterol trafficking/processing pathway, using an ncr-1 enhancer screen. HSD-1 is orthologous to 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4) isomerases (3beta-HSDs), which are key steroidogenic enzymes in vertebrates, and is exclusively expressed in two neuron-like XXX cells that are crucial in preventing dauer arrest, suggesting that it is involved in biosynthesis of dauer-preventing steroid hormones. The hsd-1 null mutant displays defects in inhibiting dauer arrest: it forms dauers in the deletion mutant backgrounds of ncr-1 or daf-28/insulin; as a single mutant, it is hypersensitive to dauer pheromone. We found that hsd-1 defects can be rescued by feeding mutant animals with several steroid intermediates that are either downstream of or in parallel to the 3beta-HSD function in the dafachronic acid biosynthetic pathway, suggesting that HSD-1 functions as a 3beta-HSD. Interestingly, sterols that rescued hsd-1 defects also bypassed the need for the NCR-1 and/or -2 functions, suggesting that HSD-1-mediated steroid hormone production is an important functional output of the NCR transporters. Finally, we found that the HSD-1-mediated signal activates insulin/IGF-I signaling in a cell non-autonomous fashion, suggesting a novel mechanism for how these two endocrine pathways intersect in directing development.     

Assignment of chromosomal locus and evidence for alternatively spliced mRNAs of a human sperm membrane protein (hSMP-1).

The gene (HSD-1) coding a human sperm membrane protein (hSMP-1) was isolated from a human testis cDNA expression library using antibodies found in the serum of an infertile woman. HSD-1 was localized to a single locus on chromosome 9 and assigned to band 9p12-p13 by fluorescent in situ hybridization (FISH) mapping and DAPI (4,6-diamidino-2-phenylindole) banding, using rat/human somatic cell hybrids and metaphase chromosomes of human lymphocytes. In rescreening a testis lambdagt10 cDNA expression library, the full-length cDNA (HSD-1) and several truncated cDNAs with heterologous regions were isolated from positive clones. The heterology consisted of deletion, insertion and alteration of the 5'-end. These heterologous truncated fragments may be produced by alternative splicing of mRNAs. Two recombinant prokaryotic expression vectors were constructed with one of the heterologous fragment (clone #26) with and without the alternative 5'-end. Escherichia coli transfected with the construct containing the alternative 5'-end failed to produce the recombinant product, whereas those transfected with the vector lacking the 5'-end produced hSMP-1. DNASIS analysis of the structure of #26 mRNA suggests that the 5'-end has a stable secondary configuration that may maintain the mRNA in an inactivated state, whereby hindering its translation and preventing the expression of the gene.     

Discovery of potent and selective PARP-1 and PARP-2 inhibitors: SBDD analysis via a combination of X-ray structural study and homology modeling

We disclose herein our efforts aimed at discovery of selective PARP-1 and PARP-2 inhibitors. We have recently discovered several novel classes of quinazolinones, quinazolidinones, and quinoxalines as potent PARP-1 inhibitors, which may represent attractive therapeutic candidates. In PARP enzyme assays using recombinant PARP-1 and PARP-2, the quinazolinone derivatives displayed relatively high selectivity for PARP-1 and quinoxaline derivatives showed superior selectivity for PARP-2, and the quinazolidinone derivatives did not have selectivity for PARP-1/2. Structure-based drug design analysis via a combination of X-ray structural study utilizing the complexes of inhibitors and human PARP-1 catalytic domain, and homology modeling using murine PARP-2 suggested distinct interactions of inhibitors with PARP-1 and PARP-2. These findings provide a new structural framework for the design of selective inhibitors for PARP-1 and PARP-2.     

Sequence selectivity of the cleavage sites induced by topoisomerase I inhibitors: a molecular dynamics study

Topoisomerase IB (Top1) inhibitors, such as camptothecin (CPT), stabilize the Top1-DNA cleavage complex in a DNA sequence-dependent manner. The sequence selectivity of Top1 inhibitors is important for targeting specific genomic sequences of therapeutic value. However, the molecular mechanisms underlying this selectivity remain largely unknown. We performed molecular dynamics simulations to delineate structural, dynamic and energetic features that contribute to the differential sequence selectivity of the Top1 inhibitors. We found the sequence selectivity of CPT to be highly correlated with the drug binding energies, dynamic and structural properties of the linker domain. Chemical insights, gained by per-residue binding energy analysis revealed that the non-polar interaction between CPT and nucleotide at the +1 position of the cleavage site was the major (favorable) contributor to the total binding energy. Mechanistic insights gained by a potential of mean force analysis implicated that the drug dissociation step was associated with the sequence selectivity. Pharmaceutical insights gained by our molecular dynamics analyses explained why LMP-776, an indenoisoquinoline derivative under clinical development at the National Institutes of Health, displays different sequence selectivity when compared with camptothecin and its clinical derivatives.     

Structure-activity relationships for the selectivity of hepatitis C virus NS3 protease inhibitors

The selectivity of hepatitis C virus (HCV) non-structural protein 3 (NS3) protease inhibitors was determined by evaluating their inhibitory effect on other serine proteases (human leukocyte elastase (HLE), porcine pancreatic elastase (PPE), bovine pancreatic chymotrypsin (BPC)) and a cysteine protease (cathepsin B). For these peptide inhibitors, the P1-side chain and the C-terminal group were the major determinants of selectivity. Inhibitors with electrophilic C-terminal residues were generally non-selective while compounds with non-electrophilic C-terminal residues were more selective. Furthermore, compounds with P1 aminobutyric acid residues were non-selective, while 1-aminocyclopropane-1-carboxylic acid (ACPC) and norvaline-based inhibitors were generally selective. The most potent and selective inhibitors of NS3 protease tested contained a non-electrophilic phenyl acyl sulfonamide C-terminal residue. HLE was most likely to be inhibited by the HCV protease inhibitors, in agreement with similar substrate specificities for these enzymes. The identified structure-activity relationships for selectivity are of significance for design of selective HCV NS3 protease inhibitors.     

Crystal structures of MMP-1 and -13 reveal the structural basis for selectivity of collagenase inhibitors

The X-ray crystal structures of the catalytic domain of human collagenase-3 (MMP-13) and collagenase-1 (MMP-1) with bound inhibitors provides a basis for understanding the selectivity profile of a novel series of matrix metalloprotease (MMP) inhibitors. Differences in the relative size and shape of the MMP S1' pockets suggest that this pocket is a critical determinant of MMP inhibitor selectivity. The collagenase-3 S1' pocket is long and open, easily accommodating large P1' groups, such as diphenylether. In contrast, the collagenase-1 S1' pocket must undergo a conformational change to accommodate comparable P1' groups. The selectivity of the diphenylether series of inhibitors for collagenase-3 is largely determined by their affinity for the preformed S1' pocket of collagenase-3, as compared to the induced fit in collagenase-1.     

Encounter with unexpected collagenase-1 selective inhibitor: switchover of inhibitor binding pocket induced by fluorine atom

Phosphonamide-based inhibitors having trifluoromethyl moiety showed highly selective inhibition against MMP-1. A possible mechanism of the selectivity of MMP-1 inhibitors through the switchover of the binding pocket was speculated by computational calculations. As a consequence of the unexpected selectivity, the specific interaction of CF3 group of the inhibitor and Arg214 in the S1' pocket of MMP-1 conducted a low binding energy.     

The design strategy of selective PTP1B inhibitors over TCPTP

Protein tyrosine phosphatase 1B (PTP1B) has already been well studied as a highly validated therapeutic target for diabetes and obesity. However, the lack of selectivity limited further studies and clinical applications of PTP1B inhibitors, especially over T-cell protein tyrosine phosphatase (TCPTP). In this review, we enumerate the published specific inhibitors of PTP1B, discuss the structure–activity relationships by analysis of their X-ray structures or docking results, and summarize the characteristic of selectivity related residues and groups. Furthermore, the design strategy of selective PTP1B inhibitors over TCPTP is also proposed. We hope our work could provide an effective way to gain specific PTP1B inhibitors.     

Rational design and synthesis of selective BACE-1 inhibitors

An effective approach for enhancing the selectivity of beta-site amyloid precursor protein cleaving enzyme (BACE 1) inhibitors is developed based on the unique features of the S1' pocket of the enzyme. A series of low molecular weight (<600) compounds were synthesized with different moieties at the P1' position. The selectivity of BACE 1 inhibitors versus cathepsin D and renin was enhanced 120-fold by replacing the hydrophobic propyl group with a hydrophilic propionic acid group.     

Thermodynamic rules for the design of high affinity HIV-1 protease inhibitors with adaptability to mutations and high selectivity towards unwanted targets

Protease inhibitors are key components in the chemotherapy of HIV-1 infection. However, the long term efficacy of antiretroviral therapies is hampered by issues of patient compliance often associated with the presence of severe side effects, and above all by the appearance of drug resistance. The development of new protease inhibitors with high potency, low susceptibility to mutations and minimal affinity for unwanted targets is an urgent goal. The engineering of these adaptive inhibitors requires identification of the critical determinants of affinity, adaptability, and selectivity. Analysis of the binding database for existing clinical and experimental inhibitors has allowed us to address the following questions in a quantitative fashion: (1) Is there an optimal binding affinity? Or, are the highest affinity inhibitors necessarily the best inhibitors? (2) What is the dependence of optimal affinity on adaptability and selectivity? (3) What are the determinants of adaptability to mutations associated with drug resistance? (4) How selectivity against unwanted targets can be improved? It is shown that the optimal affinity is a function of the effective target concentration and the desired adaptability and selectivity factors. Furthermore, knowledge of the enthalpic and entropic contributions to the binding affinity to the wild type provides a way of anticipating the response of an inhibitor to mutations associated with drug resistance, and therefore, a valuable guideline for optimization.     

Optimization of biaryl Selective HDAC1&2 Inhibitors (SHI-1:2)

A class of biaryl benzamides was identified and optimized as selective HDAC1&2 inhibitors (SHI-1:2). These agents exhibit selectivity over class II HDACs 4-7, as well as class I HDACs 3 and 8; providing examples of selective HDAC inhibitors for the HDAC isoforms most closely associated with cancer. The hypothesis for the increased selectivity is the binding of a pendant aromatic group in the internal cavity of the HDAC1&2 enzymes. SAR development based on an initial lead led to a series of potent and selective inhibitors with reduced off-target activity and tumor growth inhibition activity in a HCT-116 xenograft model.     

The structural basis for the selectivity of benzotriazole inhibitors of PTP1B

Protein tyrosine phosphatase 1B (PTP1B) has been implicated in the regulation of the insulin signaling pathway and represents an attractive target for the design of inhibitors in the treatment of type 2 diabetes and obesity. Inspection of the structure of PTP1B indicates that potent PTP1B inhibitors may be obtained by targeting a secondary aryl phosphate-binding site as well as the catalytic site. We report here the crystal structures of PTP1B in complex with first and second generation aryldifluoromethyl-phosphonic acid inhibitors. While all compounds bind in a previously unexploited binding pocket near the primary binding site, the second generation compounds also reach into the secondary binding site, and exhibit moderate selectivity for PTP1B over the closely related T-cell phosphatase. The molecular basis for the selectivity has been confirmed by single point mutation at position 52, where the two phosphatases differ by a phenylalanine-to-tyrosine switch. These compounds present a novel platform for the development of potent and selective PTP1B inhibitors.     

Benzoylbenzimidazole-based selective inhibitors targeting Cryptosporidium parvum and Toxoplasma gondii calcium-dependent protein kinase-1

Calcium-dependent protein kinase-1 (CDPK1) from Cryptosporidium parvum (CpCDPK1) and Toxoplasma gondii (TgCDPK1) have become attractive targets for discovering selective inhibitors to combat infections caused by these protozoa. We used structure-based design to improve a series of benzoylbenzimidazole-based compounds in terms of solubility, selectivity, and potency against CpCDPK1 and TgCDPK1. The best inhibitors show inhibitory potencies below 50nM and selectivity well above 200-fold over two human kinases with small gatekeeper residues.     

Discovery of quinazolinone and quinoxaline derivatives as potent and selective poly(ADP-ribose) polymerase-1/2 inhibitors

Two classes of quinazolinone derivatives and quinoxaline derivatives were identified as potent and selective poly(ADP-ribose) polymerase-1 and 2 (PARP-1) and (PARP-2) inhibitors, respectively. In PARP enzyme assays using recombinant PARP-1 and PARP-2, quinazolinone derivatives displayed relatively high selectivity for PARP-1 and quinoxaline derivatives showed superior selectivity for PARP-2. SBDD analysis via a combination of X-ray structural study and homology modeling suggested distinct interactions of inhibitors with PARP-1 and PARP-2. These findings provide a new structural framework for the design of selective inhibitors for PARP-1 and PARP-2.     

Synthesis and evaluation of trans 3,4-cyclopropyl L-arginine analogues as isoform selective inhibitors of nitric oxide synthase

Four optically pure conformationally restricted L-arginine analogues syn- 1 and anti- 2 trans-3,4-cyclopropyl L-arginine, and syn- 3 and anti-trans-3,4-cyclopropyl N-(1-iminoethyl) L-ornithine 4 were synthesized. These compounds were tested as potential inhibitors against the three isoforms of nitric oxide synthase (NOS). Compound 1 was determined to be a poor substrate of NOS, while compound 2 was determined to be a poor mixed type inhibitor and did not exhibit any isoform selectivity. Syn- 3 and anti-trans-3,4-cyclopropyl N-(1-iminoethyl) L-ornithine 4 were found to be competitive inhibitors of NOS. These compounds were time dependent inhibitors of inducible NOS (iNOS), but not of neuronal NOS (nNOS) or endothelial NOS (eNOS). Compound 3 was 10- to 100-fold more potent an inhibitor than 4, exhibited a 5-fold increase in nNOS/iNOS and eNOS/iNOS selectivity over 4, and displayed tight binding characteristics against iNOS. These results indicate that the relative configuration of the cyclopropyl ring in the L-arginine analogues significantly affects their inhibitory potential and NOS isoform selectivity.     

Differential protein acetylation induced by novel histone deacetylase inhibitors

Histone deacetylase (HDAC) inhibitors induce the hyperacetylation of nucleosomal histones in carcinoma cells resulting in the expression of repressed genes that cause growth arrest, terminal differentiation, and/or apoptosis. In vitro selectivity of several novel hydroxamate HDAC inhibitors including succinimide macrocyclic hydroxamates and the non-hydroxamate alpha-ketoamide inhibitors was investigated using isolated enzyme preparations and cellular assays. In vitro selectivity for the HDAC isozymes (HDAC1/2, 3, 4/3, and 6) was not observed for these HDAC inhibitors or the reference HDAC inhibitors, MS-275 and SAHA. In T24 and HCT116 cells these compounds caused the accumulation of acetylated histones H3 and H4; however, the succinimide macrocyclic hydroxamates and the alpha-ketoamides did not cause the accumulation of acetylated alpha-tubulin. These data suggest "selectivity" can be observed at the cellular level with HDAC inhibitors and that the nature of the zinc-chelating moiety is an important determinant of activity against tubulin deacetylase.