Induction of fetal hemoglobin (HbF) is highly beneficial for patients carrying β-thalassemia, and novel HbF inducers are highly needed. Here, we describe a new class of promising HbF inducers characterized by an isoxazole chemical skeleton and obtained through modification of two natural molecules, geldanamycin and radicicol. After preliminary biological assays based on benzidine staining and RT-qPCR conducted on human erythroleukemic K562 cells, we employed erythroid precursors cells (ErPCs) isolated from β-thalassemic patients. ErPCs weretreated with appropriate concentrations of isoxazole derivatives. The accumulation of globin mRNAs was studied by RT-qPCR, and hemoglobin production by HPLC. We demonstrated the high efficacy of isozaxoles in inducing HbF. Most of these derivatives displayed an activity similar to that observed using known HbF inducers, such as hydroxyurea (HU) or rapamycin; some of the analyzed compounds were able to induce HbF with more efficiency than HU. All the compounds were active in reducing the excess of free α-globin in treated ErPCs. All the compounds displayed a lack of genotoxicity. These novel isoxazoles deserve further pre-clinical study aimed at verifying whether they are suitable for the development of therapeutic protocols for β-thalassemia.

In order to develop potential anticancer agents stimulating apoptosis, novel 3,4-isoxazolediamide and 4,5,6,7-tetrahydro-isoxazolo-[4,5-c]-pyridine derivatives have been synthetized. The original structures of geldanamycin and radicicol, which are known natural heat shock protein (HSP) inhibitors, were deeply modified because both of them exhibit several drawbacks, such as poor solubility, hepatotoxicity, intrinsic chemical instability or deprivation of the in vivo activity. This novel class of synthetic compounds containing the isoxazole nucleus exhibited potent and selective inhibition of HSP90 in previous studies. Biological assays (focusing on in vitro antiproliferative effects and pro-apoptotic activity) in human erythroleukemic K562 cells (as a model system referring to tumor cells grown in suspension), glioblastoma U251-MG and glioblastoma temozolomide (TMZ)-resistant T98G cell lines (two model systems referring to tumor cells grown attached to the flask), were performed. Almost all isoxazole derivatives demonstrated significant antiproliferative and pro-apoptotic activities, showing induction of both early and late apoptosis of K562 cells. Different effects were observed on the glioma U251-MG and T98G cells, depending on the structure of the analogues. Antiproliferative and pro-apoptotic activities in K562 cells were associated with the activation of the erythroid differentiation program. The present study demonstrated that 3,4-isoxazolediamide and 4,5,6,7-tetrahydro-isoxazolo-[4,5-c]-pyridine derivatives should be considered for in vivo studies focusing on the development of anticancer drugs acting, at least partially, via activation of apoptosis.

Fig. 2View Large Image Figure ViewerDownload Hi-res image Download (PPT) Enzymatic Synthesis of Deoxyribonucleotides. I. Formation of Deoxycytidine Diphosphate from Cytidine Diphosphate with Enzymes from Escherichia coli (Reichard, P. (1962) J. Biol. Chem. 237, 3513–3519) Electron Spin Resonance of the Iron-containing Protein B2 from Ribonucleotide Reductase (Ehrenberg, A., and Reichard, P. (1972) J. Biol. Chem. 247, 3485–3488) Activation of the Anaerobic Ribonucleotide Reductase from Escherichia coli by S-adenosylmethionine (Harder, J., Eliasson, R., Pontis, E., Ballinger, M. D., and Reichard, P. (1992) J. Biol. Chem. 267, 25548–25552) Peter Reichard was born in 1925 in Wiener Neustadt, a small town in Austria, about 40 km south of Vienna. With the annexation of Austria by Germany in 1938, Reichard and his family moved to Sweden where he attended a private boarding school near Stockholm. There, he developed an interest in science and upon graduating he decided to become a chemical engineer. However, in order to enter the school of engineering in Stockholm, Reichard needed practical experience so he spent a year working in a factory in the far north of Sweden. He recalls, “I enjoyed neither the long cold winter nor the dull work, which did not live up to my expectations. I decided no more chemistry for me (1Reichard P. To be there when the picture is painted.Annu. Rev. Biochem. 1995; 64: 1-28Crossref PubMed Google Scholar).” Instead, Reichard opted to go to medical school and was accepted by the Karolinska Institute in 1944. The school's introductory laboratory course involved qualitative analyses of mixtures of inorganic salts and it was then that Reichard realized how much he enjoyed “shaking test tubes and the outcome of simple experiments (1Reichard P. To be there when the picture is painted.Annu. Rev. Biochem. 1995; 64: 1-28Crossref PubMed Google Scholar).” Thinking that chemistry was not so bad after all, he began to consider a career in biological research and started to work with Einar Hammarsten. His first assignment with Hammarsten was to assist in the construction of a mass spectrometer. However, after just 1 week he dropped a screwdriver and broke a major glass part of the machine. Not surprisingly, he was discharged from his mass spec building duties. Hammarsten instead put him on a project crystallizing deoxyribonuclease. A subsequent visit by David Shemin steered Reichard's interests toward isolating pure 15N-labeled purines and pyrimidines from small amounts of nucleic acids, and using partition chromatography he was able to separate the four common ribonucleosides. In the fall of 1949 Reichard defended his thesis and received a doctorate in medicine. He spent the next 2 years at Karolinska, finishing medical school and doing research. During this time Reichard worked out a method to prepare 15N-nucleosides from biosynthetically labeled nucleic acids. He then injected the nucleosides into rats as precursors of nucleic acids. These experiments led him to conclude that ribonucleosides were incorporated into both RNA and DNA but deoxyribonucleosides were used exclusively for DNA synthesis. From these results, he proposed the existence of an enzyme that transformed ribose to deoxyribose. After finishing medical school in 1951, Reichard spent a year at Stanford University, doing a postdoctoral fellowship with Hubert Loring. He returned to the Karolinska Institute and was appointed Assistant Professor of Medical Chemistry in 1952. He continued his work on pyrimidine biosynthesis and was reasonably successful. His attempts to advance the ribonucleoside reduction story on the other hand were less fruitful. Incubation of labeled ribonucleotides with extracts from various cells never provided convincing evidence for the formation of deoxyribonucleotides. Suddenly, success arrived one day when Reichard added just the right amount of ATP and Mg2+ to the bacterial extract. He later learned that this delicate balance was due to the fact that the function of ATP is 2-fold: it acts as an allosteric effector for the reductase and it transforms cytidine 5′-phosphate (CMP) to cytidine 5′-diphosphate (CDP), the substrate reduced by the enzyme. Too much ATP causes all the CDP to be transformed into inactive cytidine 5′-triphosphate (CTP). In 1961, Reichard joined the faculty at the University of Uppsala as Professor of Medical Chemistry and continued to study ribonucleoside reduction. Using Escherichia coli, he managed to purify two enzyme fractions, A and B, that participated in the formation of deoxycytidine phosphates from CMP. This is the subject of the first Journal of Biological Chemistry (JBC) Classic reprinted here. Reichard found that Fraction A catalyzed the phosphorylation of cytidine 5′-phosphate (CMP) to cytidine 5′-diphosphate (CDP) whereas Fraction B carried out the reduction of the cytidine diphosphate to deoxycytidine diphosphate (dCDP). The reaction catalyzed by Fraction B required ATP, Mg2+ ions, and reduced lipoic acid. Later, Reichard determined that thioredoxin, rather than lipoic acid, was the hydrogen donor in the reaction. Reichard left Uppsala after 2 years to return to the Karolinska Institute as Professor of Medical Chemistry. Enzyme purification became his main focus, and he eventually purified E. coli ribonucleotide reductase using the reduction of CDP as an assay. He discovered that the enzyme, purified from Fraction B above, consists of two proteins, B1 and B2, which were later renamed R1 and R2. R1 contains two sets of allosteric sites that bind nucleoside triphosphates and is thus involved in regulation of the enzyme. R2 is made of two identical subunits and contains two atoms of iron. Although it was difficult to obtain large amounts of the E. coli reductase, Reichard ultimately arrived at a reproducible method that, after 2 weeks of hard work, gave him several milligrams of R1 and R2. This made structural work on the enzyme possible. He found that R2 gave a characteristic spectrum that suggested that the presence of iron in the enzyme was linked to its activity. Reichard then initiated a collaboration with his next-door neighbor, Anders Ehrenberg, who was a pioneer in the application of electron spin resonance (ESR) spectroscopy to biological problems. This work is the subject of the second JBC Classic reprinted here. Much to their surprise, Ehrenberg and Reichard discovered that R2 contains a signal characteristic of a free radical. The presence of this signal was also linked to the enzymatic activity of R2, suggesting that ribonucleotide reduction proceeds by radical chemistry. Eventually Britt-Marie Sjöberg localized the organic radical to tyrosine 122. In 1971 Reichard became Professor of Biochemistry at Karolinska. In the latter part of his career he began to look at how E. coli synthesizes dNTPs in the absence of oxygen. During anaerobic growth, the bacteria must acquire a different mode of ribonucleotide reduction because oxygen is needed for the generation of the tyrosyl radical. Reichard eventually purified two proteins, dA3 and dA1, involved in the anaerobic reduction of CTP to dCTP. dA3 was the actual anaerobic reductase, and dA1 was later determined to be ferredoxin NADP+ reductase. He found that the enzymes required the presence of S-adenosylmethionine (AdoMet), which was thought to be the precursor of an active radical on the enzyme that fulfilled the function of the tyrosyl radical in the aerobic enzyme. In addition to AdoMet, the reaction required NADPH, dithiothreitol, Mg2+, K+ ions, and a Chelex-treated boiled extract from E. coli, designated RT. Reichard eventually showed that the overall reduction of CTP occurred in two steps, which is the subject of the final JBC Classic reprinted here. His model can be seen in Fig. 2. The first step is the activation of the reductase (dA3) by dA1 and RT. During this step AdoMet is reductively cleaved into methionine and 5′-deoxyadenosine. The second step involves the actual reduction of CTP with dithiothreitol as the hydrogen donor. It was eventually determined that the anaerobic ribonucleotide reductase contains a glycyl radical. More information on the anaerobic ribonucleotide reductase from E. coli can be found in Reichard's JBC Minireview (2Reichard P. The anaerobic ribonucleotide reductase from Escherichia coli.J. Biol. Chem. 1993; 268: 8383-8386Abstract Full Text PDF PubMed Google Scholar). Reichard retired in 1991 and remains at the Karolinska Institute as Professor Emeritus. During his time at Karolinska he was a member of the Nobel Faculty and was heavily involved in the Nobel Committee, as he explains in his JBC Reflections (3Reichard P. Oswald T. Avery and the Nobel Prize in Medicine.J. Biol. Chem. 2002; 277: 13355-13362Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). Reichard has received many awards and honors including election to the National Academy of Sciences in 1980. He is also a Foreign Honorary Member of the American Academy of Arts and Sciences. 1Biographical information on Peter Reichard was taken from Ref. 1Reichard P. To be there when the picture is painted.Annu. Rev. Biochem. 1995; 64: 1-28Crossref PubMed Google Scholar. 1Biographical information on Peter Reichard was taken from Ref. 1Reichard P. To be there when the picture is painted.Annu. Rev. Biochem. 1995; 64: 1-28Crossref PubMed Google Scholar.

Introduction: HSP90 molecular chaperones (i.e., HSP90α, HSP90β, GRP94 and TRAP1) are potential therapeutic targets to design novel anticancer agents. However, despite numerous designed HSP90 inhibitors, most of them have failed due to unfavorable toxicity profiles and lack of specificity toward different HSP90 paralogs. Indeed, a major limitation in this field is the high structural homology between different HSP90 chaperones, which significantly limits our capacity to design paralog-specific inhibitors.Area covered: This review examines the relevance of TRAP1 in tumor development and progression, with an emphasis on its oncogenic/oncosuppressive role in specific human malignancies and its multifaceted and context-dependent functions in cancer cells. Herein, we discuss the rationale for considering TRAP1 as a potential molecular target and the strategies used to date, to achieve its compartmentalized inhibition directly in mitochondria.Expert opinion: TRAP1 targeting may represent a promising strategy for cancer therapy, based on the increasing and compelling evidence supporting TRAP1 involvement in human carcinogenesis. However, considering the complexity of TRAP1 biology, future strategies of drug discovery need to improve selectivity and specificity toward TRAP1 respect to other HSP90 paralogs. The characterization of specific human malignancies suitable for TRAP1 targeting is also mandatory.

The search for antimetastatic agents for cancer therapy may involve the ability of new compounds to maintain the tissue extracellular matrix integrity. Among known factors, heparanase, an endoglucuronidase responsible for heparan sulfate cleavage, is a promising target whose inhibition could represent a strong obstacle for metastatic cancerous mechanisms. The antimetastatic activity of some suramin derivatives reported in literature suggests a possible involvement of the heparanase enzyme. To confirm such hypothesis, we have investigated FCE27266, a molecule known for its antiangiogenic and antimetastatic properties. Other new derivatives were also synthesized and investigated. Our findings revealed that FCE27266 as well as some derivatives have a strong heparanase inhibition activity, together with no cytotoxic power. Moreover, a FCE27266 analogue (SST0546NA1; 17a) resulted also positive to lower gene expression of some proangiogenic factors.

STAT5 is a transcription factor, a member of the STAT family of signaling proteins. STAT5 is involved in many types of cancer, including chronic myelogenous leukemia (CML), in which this protein is found constitutively activated as a consequence of BCR-ABL expression. The neuroleptic drug pimozide was recently reported to act as an inhibitor of STAT5 phosphorylation and is capable of inducing apoptosis in CML cells invitro. Our research group has synthesized simple derivatives of pimozide with cytotoxic activity and that are able to decrease the levels of phosphorylated STAT5. In this work we continued the search for novel STAT5 inhibitors, synthesizing compounds in which the benzoimidazolinone ring of pimozide is either maintained or modified, in order to obtain further structure-activity relationship information for this class of STAT5 inhibitors. Two compounds of the series showed potent cytotoxic activity against BCR-ABL-positive and pSTAT5-overexpressing K562 cells and were able to markedly decrease the levels of phosphorylated STAT5.

We reported previously that a hemiasterlin derivative BF65 is a potent anticancer agent that can inhibit microtubule assembly. Here we show that a more potent stereospecific diastereomer (R)(S)(S)-BF65 can synergize with an allosteric Akt inhibitor MK-2206 to suppress the growth of SKOV3 ovarian cancer cells with constitutively active Akt. (R)(S)(S)-BF65 induced mitotic arrest and MK-2206 caused G0/G1 arrest, while the combination of both induced simultaneous G0/G1 and G2/M cell cycle arrest. (R)(S)(S)-BF65 induced phosphorylation and inactivation of Bcl-2, and downregulated Mcl-1, consequently may lead to apoptosis. (R)(S)(S)-BF65 inhibited mitogen-activated protein kinases (MAPKs), which may stimulate cell proliferation upon activation. (R)(S)(S)-BF65 also induced DNA damage after long-term treatment. MK-2206 is known to inhibit phosphorylation and activation of Akt and suppress cancer cell growth. The combination of (R)(S)(S)-BF65 and MK-2206 also inhibited the Akt pathway. Interestingly, MK-2206 upregulated Bcl-2 and induced activation of MAPKs in SKOV3 cells; however, when combined with (R)(S)(S)-BF65, these prosurvival effects were reversed. The combination also more significantly decreased Mcl-1 protein, increased PARP cleavage, and induced γ-H2AX, a DNA damage marker. Remarkably, MK-2206 enhanced the microtubule depolymerization effect of (R)(S)(S)-BF65. The combination of (R)(S)(S)-BF65 and MK-2206 also markedly inhibited cell migration. Thus, MK-2206 synergizes with (R)(S)(S)-BF65 to inhibit SKOV3 cell growth via downregulating the Akt signaling pathway, and enhancing the microtubule disruption effect of (R)(S)(S)-BF65. (R)(S)(S)-BF65 in turn suppresses Bcl-2 and MAPKs induced by MK-2206. (R)(S)(S)-BF65 and MK-2206 compensate each other leading to increased apoptosis and enhanced cytotoxicity, and may also suppress cancer cell invasion.

We report the design, synthesis and biological characterisation of a novel hybrid drug by conjugation of two tubulin inhibitors, a hemiasterlin derivative A (H-Mpa-Tle-Aha-OH), obtained by condensation of three non-natural amino acids, and cis-3,4',5-trimethoxy-3'aminostilbene (B). As we have previously demonstrated synergy between A and B, we used a monocarbonyl derivative of triethylene glycol as linker (L) to synthesise compounds A-L and A-L-B; via HPLC we analysed the release of its potential hydrolysis products A, A-L, B and B-L in physiological fluids: the hybrid A-L-B undergo hydrolysis in rat whole blood of the ester bond between A and L (half-life=118.2±9.5min) but not the carbamate bond between B and L; the hydrolysis product B-L was further hydrolyzed, but with a slower rate (half-life=288±12min). The compound A-L was the faster hydrolyzed conjugate (half-life=25.4±1.1min). The inhibitory activity of the compounds against SKOV3 ovarian cancer cell growth was analysed. The IC50 values were 7.48±1.27nM for A, 40.3±6.28nM for B, 738±38.5nM for A-L and 37.9±2.11nM for A-L-B. The anticancer effect of A-L-B was evidenced to be obtained via microtubule dynamics suppression. Finally, we stated the expression of the active efflux transporters P-gp (ABCB1) and MRP1 (ABCC1) in the human normal colon epithelial NCM460 cell line by reverse-transcription PCR. Via permeation studies across NCM460 monolayers we demonstrate the poor aptitude of A to interact with active efflux transporters (AET): indeed, the ratio between its permeability coefficients for the basolateral (B)→apical (A) and B→A transport was 1.5±0.1, near to the ratio of taltobulin (1.12±0.06), an hemiasterlin derivative able to elude AETs, and significantly different form the ratio of celiprolol (3.4±0.2), an AET substrate.

Signal Transducer and Activator of Transcription 5 (STAT5) protein, a component of the STAT family of signaling proteins, is considered to be an attractive therapeutic target because of its involvement in the progression of acute myeloid leukemia. In an effort to discover potent molecules able to inhibit the phosphorylation-activation of STAT5, twenty-two compounds were synthesized and evaluated on the basis of our knowledge of the activity of 2-(3′,4′,5′-trimethoxybenzoyl)-3-iodoacetamido-6-methoxy benzo[b]furan derivative 1 as a potent STAT5 inhibitor. Most of these molecules, structurally related to compound 1, were characterized by the presence of a common 3′,4′,5′-trimethoxybenzoyl moiety at the 2-position of different benzoheterocycles such as benzo[b]furan, benzo[b]thiophene, indole and N-methylindole. Effects on biological activity of the iodoacetamido group and of different moieties (methyl and methoxy) at the C-3 to C-7 positions were examined. In the series of benzo[b]furan derivatives, moving the iodoacetylamino group from the C-4 to the C-5 or C-6 positions did not significantly affect antiproliferative activity. Compounds 4, 15, 20 and 23 blocked STAT5 signals and induced apoptosis of K562 BCR–ABL positive cells. For compound 23, the trimethoxybenzoyl moiety at the 2-position of the benzo[b]furan core was not essential for potent inhibition of STAT5 activation.

Hsp90 is considered an interesting therapeutic target for anticancer drug development. Here we describe a new class of 4,5,6,7-tetrahydro-isoxazolo-[4,5-c]-pyridine compounds. A small library of derivatives has been synthesized and investigated. Some reported compounds show interesting properties combining both notable binding to Hsp90 and potent cell growth inhibitory activity. N-5 substitution with a 2,4 resorcinol carboxamide appears crucial for activity. Moreover, a derivative bearing a hydroxamic acid residue bound to C-3 amide portion was found to inhibit both Hsp90 and HDAC6.

STATs are transcription factors acting as intracellular signaling after stimulation with cytokines, growth factors and hormones. STAT5 is also constitutively active in many forms of cancers, including chronic myelogenous leukemia, acute lymphoblastic leukemia and Hodgkin's lymphoma. Recently, literature reported that the neuroleptic drug pimozide inhibits STAT5 phosphorylation inducing apoptosis in CML cells. We undertook an investigation from pimozide structure, obtaining simple derivatives with cytotoxic and STAT5-inhibitory activity, two of them markedly more potent than pimozide.

The identification of novel compounds modulating the expression/activity of molecular targets downstream to BCR-ABL could be a new approach in the treatment of chronic myeloid leukemias (CMLs) resistant to imatinib or other BCR-ABL-targeted molecules. Recently, we synthesized a new class of substituted 2-(3,4,5-trimethoxybenzoyl)-2-N, N-dimethylaminobenzo[b] furans, and among these 3-iodoacetylamino-6methoxybenzofuran-2-yl(3,5-trimethoxyphenyl) methanone (TR120) showed marked cytotoxic activity in BCR-ABL-expressing cells. Interestingly, TR120 was more potent than imatinib in cell growth inhibition and apoptosis induction in both BCR-ABL-expressing K562 and KCL22 cells. Moreover, it showed antitumor activity in imatinib-resistant K562-R and KCL22-R cells at concentrations similar to those active in the respective sensitive cells. Further, TR120 induced a marked decrease in signal transducer and activator of transcription 5 (STAT5) expression in K562 cells. Consistent with this effect, it determined a block of cells in the G0-G1 phase of the cell cycle, a decrease in the level of cyclin D1, and a reduction in Bcl-xL expression; however, it did not cause modifications in the Bcl-2 level. Of interest, TR120 had synergistic effects when used in combination with imatinib in both sensitive and resistant cells. Considering that STAT5 is a BCR-ABL molecular target that plays a key role in the pathogenesis of CML as well as in BCR-ABL-mediated resistance to apoptosis, TR120 could potentially be a useful novel agent in the treatment of imatinib-resistant CML. Anti-Cancer Drugs 24: 384-393 (C) 2013 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins. Anti-Cancer Drugs 2013, 24:384-393

Hemiasterlins are cytotoxic tripeptides with antimicrotubule activity originally isolated from marine sponges. We have developed new hemiasterlin derivatives BF65 and BF78 that are highly potent to induce cancer cell death in the low nanomolar range. Examination of their mechanisms of cell cycle arrest and disruption of microtubules revealed an unusual characteristic in addition to anti-tubulin effect. Immunofluorescence staining revealed that A549 lung carcinoma cells treated with BF65 or BF78 exhibited both monopolar and multipolar mitotic spindles. Centrosomes were separated with short spindle microtubules in cells with multipolar spindles. In vitro tubulin polymerization assay confirmed that both BF65 and BF78 were highly potent to inhibit tubulin polymerization. These two compounds induced the formation of monoastral spindles suggesting that they might be inhibitors of mitotic kinesins such as KSP/Eg5. However, kinetic measurement of microtubule activated kinesin ATPase activity demonstrated that unlike the positive control monastrol, neither BF65 nor BF78 suppressed KSP/Eg5 activity. Hence the effect may be a variant form of tubulin inhibition. Similar to vinca alkaloids, BF compounds synergized with a colchicine site microtubule inhibitor stilbene 5c both in vitro and in vivo, which may provide a potential drug combination in the future clinical application.

The introduction of the isoxazole ring as bioisosteric replacement of the acetyl group of anatoxin-a led to a new series of derivatives binding to nicotinic acetylcholine receptors. Bulkier substitutions than methyl at the 3 position of isoxazole were shown to be detrimental for the activity. The binding potency of the most interesting compounds with α1, α7 and α3β4 receptor subtypes, was, anyway, only at micromolar level. Moreover, differently from known derivatives with pyridine, isoxazole condensed to azabicyclo ring led to no activity.

After developing a blood disorder, Yale Nemerson became interested in hematology. This led to his lifelong study of thrombogenic tissue factor and to his contributions to developing the modern theory of blood coagulation. The two Classic papers reprinted here detail some of Nemerson's studies on coagulation factors IX and VII.

Edmond H. Fischer was awarded the 1992 Nobel Prize in Physiology or Medicine for his joint research with Edwin G. Krebs on reversible protein phosphorylation. The two Classics reprinted here relate some of Fischer and Krebs' early discoveries in their phosphorylase research Edmond H. Fischer was awarded the 1992 Nobel Prize in Physiology or Medicine for his joint research with Edwin G. Krebs on reversible protein phosphorylation. The two Classics reprinted here relate some of Fischer and Krebs' early discoveries in their phosphorylase research Phosphorylase Activity of Skeletal Muscle Extracts (Krebs, E. G., and Fischer, E. H. (1955) J. Biol. Chem. 216, 113-120) Conversion of Phosphorylase b to Phosphorylase a in Muscle Extracts (Fischer, E. H., and Krebs, E. G. (1955) J. Biol. Chem. 216, 121-132) Edmond H. Fischer was born in Shanghai, China in 1920. He was sent to boarding school in Switzerland at age 7, and in 1935, he entered Geneva's Collège de Calvin. There, he became friends with his classmate Wilfried Haudenschild, and together, they decided that one of them should go into the sciences and the other into medicine so they could cure the world of all ills. Fischer chose science. Just before the start of World War II, Fischer completed high school and entered the School of Chemistry at the University of Geneva. He earned two Licences ès Sciences, one in biology, the other in chemistry, and 2 years later, he was awarded a Diploma of “Ingénieur Chimiste.” For his thesis, he worked with Kurt H. Meyer on the purification of amylase from hog and human pancreas, as well as saliva and several strains of bacteria. In 1950, Fischer went to the United States to do a postdoctoral fellowship with Paul Karrer at CalTech. However, when he arrived in Pasadena he received a letter from Journal of Biological Chemistry (JBC) Classic author Hans Neurath (1JBC Classics Schwert G.W. Neurath H. Kaufman S. Snoke J.E. J. Biol. Chem. 1948; 172: 221-239Abstract Full Text PDF PubMed Google ScholarDavie E.W. Neurath H. J. Biol. Chem. 1955; 212 (http://www.jbc.org/cgi/content/full/280/2/e1): 515-530Abstract Full Text PDF PubMed Google Scholar), chairman of the department of biochemistry at the University of Washington, offering him an assistant professorship in his department. Fischer visited Seattle and accepted the offer, in part because the surrounding mountains, forests, and lakes reminded him of his native Switzerland. Within 6 months of his arrival, Fischer started working on glycogen phosphorylase with Edwin G. Krebs, who was featured in a previous JBC Classic (2JBC Classics Walsh D.A. Perkins J.P. Krebs E.G. J. Biol. Chem. 1968; 243: 3763-3765Abstract Full Text PDF PubMed Google ScholarAhn N.G. Seger R. Bratlien R.L. Diltz C.D. Tonks N.K. Krebs E.G. J. Biol. Chem. 1991; 266 (http://www.jbc.org/cgi/content/full/280/43/e40): 4220-4227Abstract Full Text PDF PubMed Google Scholar). Krebs had trained with JBC Classic authors Carl and Gerty Cori who had discovered that muscle phosphorylase exists in two forms, phosphorylase a, which was easily crystallized and was active without the addition of AMP, and phosphorylase b, a more soluble protein, which was inactive without AMP (3JBC Classics Cori C.F. Cori G.T. J. Biol. Chem. 1928; 79: 321-341Abstract Full Text PDF Google ScholarCori G.T. Colowick S.P. Cori C.F. J. Biol. Chem. 1938; 124: 543-555Abstract Full Text PDF Google ScholarCori G.T. Colowick S.P. Cori C.F. J. Biol. Chem. 1939; 127: 771-782Abstract Full Text PDF Google ScholarGreen A.A. Cori G.T. J. Biol. Chem. 1943; 151: 21-29Abstract Full Text PDF Google ScholarCori G.T. Green A.A. J. Biol. Chem. 1943; 151 (http://www.jbc.org/cgi/content/full/277/29/e18): 31-38Abstract Full Text PDF Google Scholar). They believed that AMP served some kind of co-factor function for the enzyme, facilitating its transition between the two forms. However, in Geneva, Fischer had purified potato phosphorylase, which had no AMP requirement. Because it seemed unlikely that muscle phosphorylase but not potato phosphorylase would require AMP as a co-factor, Fischer and Krebs decided to try to elucidate the role of AMP in the phosphorylase reaction. They never discovered what the nucleotide was doing (this problem was solved several years later when Jacques Monod proposed his allosteric model for the regulation of enzymes), but they did discover that muscle phosphorylase was regulated by an enzyme-catalyzed phosphorylation-dephosphorylation reaction. The two JBC Classics reprinted here relate some of Fischer and Krebs' early discoveries in their phosphorylase research. In the first Classic, the pair performed experiments to determine whether environmental temperature affects the phosphorylase content of skeletal muscle. They were unable to detect any temperature effects, but they did make the surprising discovery that the muscle extracts contained mainly phosphorylase b rather than phosphorylase a. The pair concluded that “If resting muscle contains mainly phosphorylase b… then pronounced activation of the phosphorylase reaction under various conditions is possible.” The second JBC Classic was printed back-to-back with the first. In it, Krebs and Fischer examine the requirements for the phosphorylase conversion and present evidence that the conversion of phosphorylase b to a in cell-free muscle extracts requires a nucleotide containing high energy phosphate and a divalent metal ion. However, they state that “whether this implies that during conversion there is a direct phosphorylation of the enzyme or the formation of an ‘active’ intermediate cannot be stated at this time. It is also possible that the function of ATP is concerned with the synthesis of a prosthetic group.” Similar work was being carried out on liver phosphorylase at approximately the same time by Earl Sutherland. As discussed in a previous JBC Classic (4JBC Classics Rall T.W. Sutherland E.W. J. Biol. Chem. 1958; 232: 1065-1076Abstract Full Text PDF PubMed Google ScholarSutherland E.W. Rall T.W. J. Biol. Chem. 1958; 232 (http://www.jbc.org/cgi/content/full/280/42/e39): 1065-1076Abstract Full Text PDF PubMed Google Scholar), Sutherland discovered the second messenger cyclic AMP (cAMP), which he showed promoted the phosphorylation and activation of phosphorylase. The way in which cAMP promoted phosphorylase activation was eventually elucidated when Krebs and Fischer discovered phosphorylase kinase, which was responsible for phosphorylating phosphorylase. Phosphorylase kinase itself existed in a highly activated phosphorylated form and a less active nonphosphorylated form. As a result of the significance of their studies, Krebs and Fischer were awarded the 1992 Nobel Prize in Physiology or Medicine “for their discoveries concerning reversible protein phosphorylation as a biological regulatory mechanism.” In addition to the Nobel Prize, Fischer has received many awards and honors in recognition of his contributions to science. These include the Werner Medal from the Swiss Chemical Society, the Lederle Medical Faculty Award, the Prix Jaubert from the University of Geneva, and jointly with Krebs, the Senior Passano Award and the Steven C. Beering Award from Indiana University. Fischer was elected to the American Academy of Arts and Sciences in 1972 and to the National Academy of Sciences in 1973. 1Biographical information on Edmond H. Fischer was taken from Ref. 5Fischer E. Edmond H. Fischer—Autobiography. Les Prix Nobel.in: Frängsmyr T. The Nobel Prizes 1992. Nobel Foundation, Stockholm1993Google Scholar.1Biographical information on Edmond H. Fischer was taken from Ref. 5Fischer E. Edmond H. Fischer—Autobiography. Les Prix Nobel.in: Frängsmyr T. The Nobel Prizes 1992. Nobel Foundation, Stockholm1993Google Scholar.

A structural investigation on the isoxazole scaffold led to the discovery of 3,4-isoxazolediamide compounds endowed with potent Hsp90 inhibitory properties. We have found that compounds possessing a nitrogen atom directly attached to the C-4 heterocycle ring possess in vitro Hsp90 inhibitory properties at least comparable to those of the structurally related 4,5-diarylisoxazole derivatives. A group of compounds from this series of diamides combine potent binding affinity and cell growth inhibitory activity in both series of alkyl- and aryl- or heteroarylamides, with IC50 in the low nanomolar range. The 3,4-isoxazolediamides were also very effective in causing dramatic depletion of the examined client proteins and, as expected for the Hsp90 inhibitors, always induced a very strong increase in the expression levels of the chaperone Hsp70. In vivo studies against human epidermoid carcinoma A431 showed an antitumor effect of morpholine derivative 73 comparable to that induced by the reference compound 10.

During his career, Christian Raetz has characterized many enzymes responsible for synthesizing or modifying lipid molecules, including the entire nine-enzyme pathway for the biosynthesis of lipid A, an essential part of bacterial outer membranes that plays a role in making many Gram-negative bacteria toxic. The findings from the two Journal of Biological Chemistry (JBC) Classic articles reprinted here were the start of Raetz' elucidation of the enzymology, genetics, and structural biology of lipid A assembly.