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AMP-activated protein kinase (AMPK) is a eukaryotic energy sensor and protector from mitochondrial/energetic stress that is also a therapeutic target for cancer and metabolic disease. Metformin is an AMPK inducer that has been used in cancer therapeutic trials. Through screening we isolated cetylpyridinium chloride (CPC), a drug known to dose-dependently inhibit mitochondrial complex 1, as a potent and dose-dependent AMPK stimulator. Mitochondrial biogenesis and bioenergetics changes have also been implicated in glioblastoma, which is the most aggressive form of brain tumors. Cetylpyridinium chloride has been administered in humans as a safe drug-disinfectant for several decades, and we report here that under in vitro conditions, cetylpyridinium chloride kills glioblastoma cells in a dose dependent manner at a higher efficacy compared to current standard of care drug, temozolomide.

The development of efficient and sustainable methods for the synthesis of nitrogen heterocycles is an important goal for the chemical industry. In particular, substituted chiral piperidines are prominent targets due to their prevalence in medicinally relevant compounds and their precursors. A potential biocatalytic approach to the synthesis of this privileged scaffold would be the asymmetric dearomatization of readily assembled activated pyridines. However, nature has yet to yield a suitable biocatalyst specifically for this reaction. Here, by combining chemical synthesis and biocatalysis, we present a general chemo-enzymatic approach for the asymmetric dearomatization of activated pyridines for the preparation of substituted piperidines with precise stereochemistry. The key step involves a stereoselective one-pot amine oxidase/ene imine reductase cascade to convert N-substituted tetrahydropyridines to stereo-defined 3- and 3,4-substituted piperidines. This chemo-enzymatic approach has proved useful for key transformations in the syntheses of the antipsychotic drugs Preclamol and OSU-6162, as well as for the preparation of two important intermediates in synthetic routes of the ovarian cancer monotherapeutic Niraparib.

The selective laser melting process is a growing technology for the manufacture of parts with very complex geometry. However, not all materials are suitable for this process, involving rapid localized melting and solidification. Tungsten has difficulties due to the high melting temperature. This study focuses on the possibility of processing a WC/Co/Cr composite powder using selective laser melting. Samples were fabricated and characterized in terms of density, defects, microstructure and hardness. Tests were conducted with hatch spacing of 120 &mu, m and process speed of 40 mm/s. A constant laser power of 100 W and a powder layer thickness of 30 &mu, m were used. A relative density of 97.53%, and therefore a low porosity, was obtained at an energy density of 12.5 J/mm2. Microscopic examination revealed the presence of small cracks and a very heterogeneous distribution of the grain size.

One of the many applications of direct laser metal deposition (DLMD) is the realization of multilayer thick coatings having particular mechanical characteristics, such as high hardness. The objective of this work was to obtain a thick, very hard and wear resistant coating, containing a high percentage of tungsten carbide (WC), on an AISI 304 stainless steel substrate. In order to achieve this result, a tungsten carbide?cobalt?chrome (WC/Co/Cr) powder was processed by the DLMD method. WC/Co/Cr is a composite widely used as a wear-resistant material for cutting tools, molds, coatings and other severe applications. Because of its high hardness, poor ductility and low thermal expansion coefficient, depositing this material directly on the stainless steel substrate is very difficult. In order to overcome this problem, the strategy of functionally graded materials (FGM) was used. Colmonoy 227-F nickel alloy was chosen for this purpose in order to generate a mixture with the WC/Co/Cr powder. Four different materials were deposited, layer by layer, by mixing Colmonoy 227-F with an increasing amount of WC/Co/Cr powders, until obtaining a thick surface coating with a maximum amount of WC of 77.4 wt%. For each powder mixture, a mathematical model was applied to calculate optimal values of translation speed and overlap percentages. A metallographic examination was performed in order to detect macro- and micro-structures of the different materials. Finally, Vickers micro-hardness was measured at various locations along the transverse section to appreciate the gradual increase of the FGM hardness, starting from the substrate and culminating at the top surface of the last deposited material.

Glioblastoma (GBM) is an incurable form of brain cancer with a median survival time (MdST) of ~15 months after treatment with chemoradiotherapy. Identification of a CpG Island Methylator Phenotype (CIMP) subtype of GBM (G-CIMP), represents a significant clinical discovery, as these patients have an enhanced survival, with a MdST of 3 years. G-CIMP is characterized by a mutation in isocitrate dehydrogenase 1 or 2 (IDH1/2), which results in production of the oncometabolite 2-hydroxglutarate, an inhibitor of α-ketoglutarate-dependent demethylases. This results in aberrant DNA methylation, transcriptional repression, and may drive tumorigenesis. Here, we present data showing that G-CIMP is significantly more sensitive in vitro to inhibitors of the two arms of epigenetic regulation of transcription: the transcriptional activation arm and the transcriptional repression arm. Drug targets include the Bromodomain and Extraterminal (BET) and Histone Deacetylase (HDAC) proteins, involved in epigenetic transcriptional activation of oncogenes and repression of tumor suppressors, respectively. G-CIMP cells show a profound loss of cell viability at submicromolar concentrations using the small molecule inhibitors JQ1 and Panobinostat, which target BET and HDAC proteins, respectively. Western blot analysis indicates that BET inhibition elicits preferential loss of histone phosphorylation and acetylation in G-CIMP, notably serine 10 of Histone 3 and lysine 27 of Histone 3 (H3K27Ac), respectively. Both epigenetic marks are associated with positively regulating transcription. H3K27Ac is a superenhancer with known functions in activating oncogene transcription. These data ultimately suggest that G-CIMP tumor cells are reliant on established epigenetic transcriptional regulation to maintain cell viability. The global methylation status of these tumors may make them particularly sensitive to pharmacologic disruption of these epigenetic pathways. These studies provide a strong foundation for future preclinical work evaluating the prospective treatment of G-CIMP with combinatorial therapy using BET and/or HDAC inhibitors. Citation Format: Thomas K. Sears, Sandipan Datta, Gino Cortopassi, James Angelastro, Kevin Woolard. Targeting epigenetic dysregulation in isocitrate dehydrogenase mutant glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3846.

Peripherin is a type III intermediate filament present in peripheral and certain CNS neurons. We report here that peripherin contains a phosphotyrosine residue and, as such, is the only identified intermediate filament protein known to be modified in this manner. Antiserum specific for phosphotyrosine recognizes peripherin present in PC12 cells (with or without nerve growth factor treatment) and in rat sciatic nerve as well as that expressed in Sf-9 cells and SW-13 cl. 2 vim− cells. The identity of peripherin as a tyrosine-phosphorylated protein in PC12 cells was confirmed by immunoprecipitation, two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, and phosphoamino acid analysis. Unlike serine/threonine phosphorylation, tyrosine phosphorylation of peripherin is not regulated by depolarization or nerve growth factor treatment. To identify the site of tyrosine phosphorylation, rat peripherin was mutated at several tyrosine residues and expressed in SW-13 cl. 2 vim− cells. Tyrosine phosphorylation was selectively lost only for peripherin mutants in which the carboxy-terminal tyrosine (Y474) was mutated. Indirect immunofluorescence staining indicated that both wild-type peripherin and peripherin Y474F form a filamentous network in SW-13 cl. 2 vim− cells. This indicates that tyrosine phosphorylation of the peripherin C-terminal residue is not required for assembly and leaves open the possibility that this modification serves other functions.

20-Fluoro-17(20)-pregnenolone derivatives were designed as enol mimics of pregnenolone. All of the targeted, novel fluoroolefins were potent inhibitors of C 17(20) lyase.

Androstenes bearing a cyclopropyl group attached to the C-17β position with a heteroatom linker, designed as mechanism-based inhibitors of steroid C 17(20) lyase, were found to be potent, time-dependent inhibitors of this enzyme.

Valylprolylvalyl pentafluoroethyl ketones with different N-protecting groups were evaluated in vitro and in vivo as inhibitors of human neutrophil elastase (HNE). Several of these compounds were found to be orally active in HNE-induced rat and hamster lung hemorrhage models. The compound with 4-(4-morpholinylcarbonyl)benzoyl as the protecting group, 71 (MDL 101,146), was studied in greater detail. Hydration and epimerization studies were performed on 71 and related compounds in various media, including human blood serum. High-performance liquid chromatography studies on a reversed-phase system as a measure of the lipophilicity of 71 and related compounds revealed a small range of relative retention times wherein the orally active compounds fell. The K i value determined for 71 vs HNE was 25 nM

Treatment of the amido esters (9) and (17) with PPSE (polyphosphoric acid trimethylsilyl ester) followed by NaBH 4 in ethanol gave the qui- nolizidinones (11-14) and (19) via a vinylogous Bischler-Napieralski nitrilium ion cyclization-reductive lactamization two-step process. Subsequent ozonolysis and reduction afforded chiral hydroxylated quino- lizidines in moderate to good yield. In contrast to five-membered-ring formation, six-membered-ring formation via nitrilium-ion cyclization requires a p-methoxy-substituted styryl terminator. The effect para-su- bstituted styryl terminators have on the energy of activation and ΔH for the cyclization process has been calculated by semiemprical and ab initio methods

Direct Laser Metal Deposition (DLMD) is actually one of the most attractive techniques in the group of Material Accretion Manufacturing (MAM) processes.In fact, the DLMD technology is able to realize, to repair and to restore, directly from the 3D CAD model, in a rapid and economic way, objects, moulds and tools, also with a very complex geometry, using a great variety of metals, including those very difficult to work with the conventional techniques. Moreover, this technique is well suited to produce very hard coatings. Metallic parts, with good mechanical properties, can be directly obtained through melting coaxially fed powders with a laser.The primary goal to pursue in the DLMD process is to produce samples with minimum porosity and with a good adhesion to the substrate.The objective of this work was to deposit layers of tungsten carbide-cobalt-chrome (WC/Co/Cr) powder on a substrate of AISI 316 stainless steel. WC/Co/Cr is a composite widely used as a wear-resistant material for cutting tools, moulds, coatings and other applications.In order to achieve good levels of adhesion, the metallic material used for the substrate and the deposited one should have a certain degree of ductility and a similar thermal linear expansion coefficient.Previous experimental studies performed on direct laser deposition of tungsten carbide showed considerable difficulties in processing this type of powder; in fact, the deposited material has a considerable hardness, but a low ductility. In addition, there is a large difference between values of the thermal expansion coefficients of the substrate compared to that of the deposited layers. This leads, generally, to the partial or total lack of adhesion of the deposit on the substrate, affecting, in a definitive way, the applicability of the process parameters used, as they have been optimized to achieve high values of hardness and relative density.In order to overcome this problem, it was decided to use the strategy of Functionally Graded Materials (FGM), which consists in interposing layers of materials with variable composition between the substrate and the coating of tungsten carbide. The nickel alloy Colmonoy 227, to mix with the powder of WC/Co/Cr, was chosen for this purpose.Results of experimentation showed that it is possible to obtain coatings of WC/Co/Cr with a good adhesion, between the first layer and the substrate and between layers, and with good mechanical properties.Direct Laser Metal Deposition (DLMD) is actually one of the most attractive techniques in the group of Material Accretion Manufacturing (MAM) processes.In fact, the DLMD technology is able to realize, to repair and to restore, directly from the 3D CAD model, in a rapid and economic way, objects, moulds and tools, also with a very complex geometry, using a great variety of metals, including those very difficult to work with the conventional techniques. Moreover, this technique is well suited to produce very hard coatings. Metallic parts, with good mechanical properties, can be directly obtained through melting coaxially fed powders with a laser.The primary goal to pursue in the DLMD process is to produce samples with minimum porosity and with a good adhesion to the substrate.The objective of this work was to deposit layers of tungsten carbide-cobalt-chrome (WC/Co/Cr) powder on a substrate of AISI 316 stainless steel. WC/Co/Cr is a composite widely used as a wear-resistant material for cutting tools, moulds...

Extracellular signal-regulated protein kinases (ERKs) constitute a family of protein serine-threonine kinases implicated in a variety of cell-signaling pathways. In cultured rat pheochromocytoma PC12 cells, ERK1 and ERK2 are activated by nerve growth factor (NGF), which also induces rapid association between ERK1 and the high affinity gp140prototrk tyrosine kinase NGF receptor. In the present work, we investigated the possible association between ERKs and the low affinity NGF receptor, p75. Extracts of PC12 cells (before and after NGF treatment) were subjected to immunoprecipitation with anti-p75 antibodies or antiserum; the immune complexes were then assessed for the presence of ERK proteins and tyrosine phosphorylation or for ERK activity using a specific substrate peptide. ERK1 and, to a lesser extent, ERK2 were found to be constitutively associated with p75. NGF did not modulate the total amount of ERK proteins coimmunoprecipitated with p75 but did markedly stimulate the level of p75-associated ERK catalytic activity. NGF treatment also enhanced the tyrosine phosphorylation of a p75-associated species that co-migrates with ERK1 in Western blots. Finally, K-252a, a compound that specifically inhibits activation by NGF of gp140prototrk, abolished the latter effect. These findings indicate that NGF, via activation of gp140prototrk, leads to association of enzymatically active ERKs with p75 and raise the possibility that this interaction may play a role in the NGF mechanism of action.

Treatment of the amido esters (9) and (17) with PPSE (polyphosphoric acid trimethylsilyl ester) followed by NaBH 4 in ethanol gave the qui- nolizidinones (11-14) and (19) via a vinylogous Bischler-Napieralski nitrilium ion cyclization-reductive lactamization two-step process. Subsequent ozonolysis and reduction afforded chiral hydroxylated quino- lizidines in moderate to good yield. In contrast to five-membered-ring formation, six-membered-ring formation via nitrilium-ion cyclization requires a p-methoxy-substituted styryl terminator. The effect para-su- bstituted styryl terminators have on the energy of activation and ΔH for the cyclization process has been calculated by semiemprical and ab initio methods

Androstenes bearing a cyclopropyl group attached to the C-17β position with a heteroatom linker, designed as mechanism-based inhibitors of steroid C 17(20) lyase, were found to be potent, time-dependent inhibitors of this enzyme.

The ketomethylene phenylalanal and alanal analogues of Cbz-Val-Phe-H and Cbz-Val-Ala-H have been prepared and the K i values versus chicken gizzard smooth muscle calpain were determined. The ketomethylene isosteres were significantly less potent than the corresponding dipeptide aldehydes, and this loss in activity is attributed to the absence of a critical interaction between the P1–P2 amide bond and the peptide binding region of calpain.

Tubulin, widely recognized as a GTP/GDP-binding protein, has been isolated in its polymerized state from rat PC12 cells and embryonic chick dorsal root ganglion neurons by Triton X-100 detergent extraction of the cytoskeletal fraction. Perchloric acid extraction and deproteinization of this fraction permitted subsequent analysis of nucleotide identity and content by high performance liquid chromatography. PC12 cells grown in the absence of nerve growth factor (NGF) contained ADP, ATP, GDP, and GTP at levels consistent with the actin and tubulin content of the cytoskeletal fraction. Microtubules from PC12 cells cultured in the presence of NGF contain an additional nucleotide that we have identified as dGTP. Analysis of whole cell nucleotide extracts from PC12 cells grown in the absence or presence of NGF revealed no evidence for the presence of dGTP at 4 and 14 days, respectively. We have determined that embryonic chick dorsal root ganglion neurons also contain this deoxyribonucleotide, and we found virtually no ADP or ATP in the extracted dorsal root ganglion cytoskeletal fraction. On the basis of metabolic labeling studies with [14C] guanine, we have inferred that the presence of dGTP in NGF-treated PC12 cells probably arises either from binding to the nonexchangeable nucleotide site of tubulin undergoing dynamic assembly/disassembly or from binding to the exchangeable site of tubulin subsequently incorporated into highly stabilized microtubules.

Ethyl 3-(benzylamino)-2,2-difluoro-4-methylpentanoate (4) was prepared via a Reformatsky reaction of ethyl bromodifluoroacetate with the imine from isovaleraldehyde and benzylamine. N-Debenzylation of 4 gave amino ester 6, which was coupled to Boc·Ala·Ala·Pro·OH to provide a potential proteinase inhibitor.

Comparison of MeO-Suc-Val-Pro-Phe-CO2Me (29) and MeO-Suc-Ala-Ala-Pro-Phe- CO2Me (25) with their corresponding trifluoromethyl ketones 9a and 9b, respectively, in rat and human neutrophil cathepsin G assays showed the alpha-keto esters to be more potent inhibitors. Likewise, Ac-Pro-Ala-Pro-Ala-CO2Me (21) was more potent than its corresponding trifluoromethyl ketone (9c) in both porcine pancreatic elastase and human neutrophil elastase assays. Within a set of Ala-Ala-Pro-Val-CF3 elastase inhibitors, the carbobenzyloxy (Cbz) N-protecting group conferred greater potency as a P5 site recognition unit for elastase than did dansyl, methoxysuccinyl, or tert-butyloxycarbonyl. Initial inhibition of elastase was greater when trifluoromethyl ketone 9f was added from a stock solution of dimethyl sulfoxide than when it had been buffer-equilibrated prior to assay, which suggests that the nonhydrated ketone is the more effective form of the inhibitor. The most potent elastase inhibitor we report is Na-(Ad-SO2)-N epsilon-(MeO-Suc)Lys-Pro-Val-CF3 (16) which has a Ki of 0.58 nM.

Background The purpose of this study was to validate the anti-neoplastic activity of a novel ATF5-targeted therapeutic approach against various recalcitrant human malignancies. Activating transcription factor 5 (ATF5) is a member of the cAMP response element binding (CREB)/ATF subfamily of basic leucine zipper transcription factors and has been shown to be overexpressed in many human cancers. CP-d/n-ATF5 is a novel peptide specifically designed to contain a cell-penetrating domain and a domain inhibiting the function of ATF5 in a unique and novel targeted therapeutic approach. This therapeutic strategy was tested among a large panel of different therapy refractory human cancers including glioblastoma, melanoma, breast, prostate-, lung- and pancreatic cancer. Methods Cellular viability was assessed by MTT-assays. The induction of apoptosis was examined by staining for annexin V/propidium iodide or JC-1 (loss of mitochondrial membrane potential) prior to flow cytometric analysis. Western blotting was performed for further molecular analysis detecting caspase cleavage and expression of Bcl-2 family members. Subcutaneous xenograft models were used to examine the anti-neoplastic effects of CP-d/n-ATF5 in vivo. Results Our data show that CP-d/n-ATF5 yields a significant increase in apoptosis across a panel of 10 different cancer cell lines when compared to treatment with a mock-mutated control peptide or penetratin. On the molecular level, treatment with CP-d/n-ATF5 resulted in a marked down-regulation of the anti-apoptotic Bcl-2 family members Mcl-1 and Bcl-2. This effect was amplified in a synergistic manner by adding either ABT-263 or TRAIL to a combined targeted therapeutic approach. In vivo, treatment with CP-d/n-ATF5 resulted in a significant reduction of tumor growth in a heterotopic glioblastoma- and an orthotopic melanoma model. Conclusions The novel ATF5-targeting compound CP-d/n-ATF5 provides a broad and pronounced anti-neoplastic activity against human cancers in vitro and in vivo. On the molecular level, this effect is at least in part due to a shift of the cellular homeostasis towards a pro-apoptotic cellular phenotype by inhibiting anti-apoptotic Bcl-2 family proteins. Overall, CP-d/n-ATF5 may represent a promising novel anti-cancer agent. Citation Format: Georg Karpel-Massler, Chang Shu, Lily Chau, James M. Angelastro, Lloyd A. Greene, Markus D. Siegelin. A novel cell-penetrating peptide targeting ATF5 (CP-d/n-ATF5) exerts anti-cancer activity in vitro and in vivo against a broad spectrum of human cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2923. doi:10.1158/1538-7445.AM2015-2923

6-Hydroxydopamine, 1-methyl-4-phenyl-pyridinium (MPP(+)), and rotenone cause the death of dopaminergic neurons in vitro and in vivo and are widely used to model Parkinson's disease. To identify regulated genes in such models, we performed serial analysis of gene expression on neuronal PC12 cells exposed to 6-hydroxydopamine. This revealed a striking increase in transcripts associated with the unfolded protein response. Immunoblotting confirmed phosphorylation of the key endoplasmic reticulum stress kinases IRE1α and PERK (PKR-like ER kinase) and induction of their downstream targets. There was a similar response to MPP(+) and rotenone, but not to other apoptotic initiators. As evidence that endoplasmic reticulum stress contributes to neuronal death, sympathetic neurons from PERK null mice in which the capacity to respond to endoplasmic reticulum stress is compromised were more sensitive to 6-hydroxydopamine. Our findings, coupled with evidence from familial forms of Parkinson's disease, raise the possibility of widespread involvement of endoplasmic reticulum stress and the unfolded protein response in the pathophysiology of this disease.

The synthesis of [1-[(5-hydroxy-4-(phenylmethyl)-3-oxazolidinyl)carbonyl]-2-ethylpropyl]carbamic acid phenylmethyl ester (2; MDL 104,903), a potent inhibitor of calpain, is described. Synthesis of related compounds, which offer insights into the mechanism of action for 2, are also described, as is an O-acetyl prodrug derivative of 2.

The ketomethylene phenylalanal and alanal analogues of Cbz-Val-Phe-H and Cbz-Val-Ala-H have been prepared and the K i values versus chicken gizzard smooth muscle calpain were determined. The ketomethylene isosteres were significantly less potent than the corresponding dipeptide aldehydes, and this loss in activity is attributed to the absence of a critical interaction between the P1–P2 amide bond and the peptide binding region of calpain.

Les composes du titre sont synthetises a partir de la «N»-benzyloxocarbonyl phenylalanine, ou de la N-benzyloxycarbonylvalyl-phenylalanine

Extracellular signal-regulated protein kinases (ERKs) are members of the mitogen-activated protein kinase family that are rapidly phosphorylated and activated in response to various extracellular stimuli, including growth factors. Of these, the ERK1 and ERK2 forms are by far the most abundant and the most studied. Much less is known about other ERK forms, including one previously designated ERK4 on the basis of its cross-reactivity with ERK1 and ERK2. We report here that ERK4 in rat PC12 pheochromocytoma cells can be immunoprecipitated by anti-ERK antiserum R2 and have used this reagent to characterize this species further. We find that ERK4 rapidly becomes tyrosine-phosphorylated in response to nerve growth factor (NGF) and epidermal growth factor (EGF) and, to a lesser degree, in response to insulin and a permeant cyclic AMP analogue. As in the case of ERK1 and ERK2, tyrosine phosphorylation of ERK4 occurs by a ras-dependent pathway in response to NGF and EGF and shows prolonged kinetics for NGF but not EGF treatment. Recognition by multiple antisera directed against various domains of ERK1 supports classification of ERK4 within the ERK family; however, two-dimensional gel analysis clearly distinguishes ERK4 from isoforms of ERK1. These findings thus reveal an additional member of the ERK family that is responsive to growth factors and that could play a distinct role in intracellular signaling.

We have recently shown that alpha-MAPI, a peptidic aldehyde of microbial origin, inhibits the HIV protease with a potency comparable to pepstatin, having, differently from pepstatin, no activity on other aspartic proteases. In this study different peptide derivatives containing a C-terminal aldehyde have been tested to assess the potential of this function for the inhibition of HIV protease. The results of our analysis correspond with the recently published subsite preferences of the viral enzyme, indicating that aldehydes bind to the active site of the HIV protease. Our data suggest that peptide aldehydes can act in their hydrated forms as transition state analogues with the most potent inhibitor having an IC50 of 0.9 microM.

Determination of the adenine and guanine nucleotides in Triton X-100-extracted cytoskeletal fractions was utilized to estimate the actin and tubulin content of the assembled cytoskeletons in nonmuscle cells. Results with stable cell lines (i.e., rat pheochromocytoma PC12 and neuroblastoma NB41A3) and with primary cultures (i.e., human foreskin fibroblasts and chick embryonic dorsal root ganglion neurons) exhibited levels of cytoskeletal fraction ADP and GDP consistent with their assembly-induced nucleoside-5′-triphosphatase activities only previously analyzed in vitro . Likewise, estimates of actin and tubulin content fall in the range of values obtained by other experimental approaches. In contrast, analysis of whole cell nucleotides showed high [ATP]/[ADP] and [GTP]/[GDP] ratios, suggesting there is little, if any, contamination of the cytoskeletal nucleotide pool by other cellular nucleotides.

Two squalene derivatives, trisnorsqualene cyclopropylamine and trisnorsqualene N-methylcyclopropylamine, were synthesized and tested for inhibition of lanosterol and squalene epoxide formation from squalene in rat hepatic microsomes, and for the inhibition of cholesterol synthesis in human cultured hepatoblastoma (HepG2) cells. Trisnorsqualene cyclopropylamine inhibited [3H]-squalene conversion to [3H]squalene epoxide in microsomes (IC50 = 5.0 microM), indicating that this derivative inhibited squalene mono-oxygenase. Trisnorsqualene N-methylcyclopropylamine inhibited [3H]squalene conversion to [3H]lanosterol (IC50 = 12.0 microM) and caused [3H]-squalene epoxide to accumulate in microsomes, indicating that this derivative inhibited 2,3-oxidosqualene cyclase. Cholesterol biosynthesis from [14C]acetate in HepG2 cells was inhibited by both derivatives (IC50 = 1.0 microM for trisnorsqualene cyclopropylamine; IC50 = 0.5 microM for trisnorsqualene N-methylcyclopropylamine). Cells incubated with trisnorsqualene cyclopropylamine accumulated [14C]squalene, while cells incubated with trisnorsqualene N-methylcyclopropylamine accumulated [14C]squalene epoxide and [14C]squalene diepoxide. The concentration range of inhibitor which caused these intermediates to accumulate coincided with that which inhibited cholesterol synthesis. The results indicate that cyclopropylamine derivatives of squalene are effective inhibitors of cholesterol synthesis, and that substitutions at the nitrogen affect enzyme selectivity and thus the mechanism of action of the compounds.

Neutrophil elastase and cathepsin G are serine proteases that can damage connective tissue and trigger other pathological reactions. Compounds containing a peptide sequence to impart specificity and bearing an alpha-dicarbonyl unit (alpha-diketone or alpha-keto ester) at the carboxy terminus are potent inhibitors of the neutrophil serine proteases (human neutrophil elastase: R-Val-COCH3, Ki = 0.017 microM; R-Val-COOCH3, Ki = 0.002 microM; human neutrophil cathepsin G: R-Phe-COCH3, Ki = 0.8 microM; R-Phe-COOCH3, Ki = 0.44 microM; R = N-(4-[(4-chlorophenyl)sulfonylaminocarbonyl]phenylcarbonyl)+ ++ValylProlyl).

Enol acetates of a-keto esters with E configuration were prepared as potential prodrugs for human neutrophil elastase (HNE) inhibitors.

Substituted hydroxyquinolizidines have been synthesized as conformationally restricted analogs of the type II high energy intermediate formed during the cyclization of 2,3-oxidosqualene to lanosterol. Compounds 4a and 4b were found to be potent inhibitors of rat liver oxidosqualene cyclase (OSC) with Kn values of 0.51 μM and 0.11 μM, respectively.

A synthetic inhibitor of calpain protects rat erythrocyte membrane-associated cytoskeletal proteins from proteolytic degradation (IC50 = 1 microM) which occurs when the cells are rendered permeable to Ca++. Leupeptin, a naturally occurring inhibitor of the enzyme, does not afford any protection at concentrations up to 100 microM.