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Bromodomains are acetyllysine recognition domains present in a variety of human proteins. Bromodomains also bind small molecules that compete with acetyllysine, and therefore bromodomains have been targets for drug discovery efforts. Highly potent and selective ligands with good cellular permeability have been proposed as chemical probes for use in exploring the functions of many of the bromodomain proteins. We report here the discovery of a class of such inhibitors targeting the family VIII bromodomains of SMARCA2 (BRM) and SMARCA4 (BRG1), and PBRM1 (polybromo-1) bromodomain 5. We propose one example from this series, GNE-064, as a chemical probe for the bromodomains SMARCA2, SMARCA4, and PBRM1(5) with the potential for

SPI1 (PU.1) is an ETS family transcription factor that plays a critical role in hematopoietic development and differentiation. Regulation of SPI1 expression has also been implicated in Acute Myeloid Leukemia (AML) oncogenesis, though its mechanism is incompletely understood. Previously we have identified and characterized a series of novel dual inhibitors of the SMARCA4/SMARCA2 ATPases (also referred to as BRG1/BRM), critical components of the BAF (mSWI/SNF) family of chromatin remodeling complexes; and FHD-286, a related SMARCA4/SMARCA2 ATPase inhibitor, is currently being explored clinically for the treatment of metastatic uveal melanoma and AML (NCT04879017 and NCT04891757). Here we show that AML cell lines are sensitive to BAF ATPase inhibition, resulting in both cell cycle arrest and apoptosis. We demonstrate that BAF ATPase inhibition primarily affects the SPI1 transcriptional profile by regulating SPI1 genomic occupancy at various enhancer elements, resulting in downregulation of key target genes. Using in vivo mouse models of AML, we demonstrate dose-dependent tumor growth inhibition and pharmacodynamic modulation of SPI1 target genes. Together, these data suggest that modulation of SPI1 function may, in part, provide a mechanistic basis for the clinical development of FHD-286 in AML. Citation Format: Gabriel J. Sandoval, Katharine Feldman, Sal Topal, Ammar Adam, Hsin-Jung Wu, Darshan Sappal, Luis M. Soares, David L. Lahr, Lan Xu, Rishi G. Vaswani, Jordana Muwanguzi, Liyue Huang, Jessica Piel, Mike Collins, Ho Man Chan, Michael J. Thomenius, Steven F. Bellon, Ryan G. Kruger, Carl P. Decicco, Richard C. Centore, Martin Hentemann. Modulation of SPI1 transcriptional program contributes to the preclinical anti-tumor activity of SMARCA4/SMARCA2 ATPase inhibitors in AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB190.

Contaminants monitoring in wild, large cetaceans may be performed using internal organs of stranded animals. Although extremely valuable, data obtained from dead animals can be biased by poor health conditions, modifying the concentrations of pollutants. Blubber biopsies represent the most feasible sampling technique from free ranging Mysticetes but are little used for trace elements quantification. Few data exist on trace elements in blubber, being this tissue not considered as a site of accumulation. Anyway, accumulation does occur in this tissue. Detected values are in the same range of magnitude of those observed in toothed whales (bottlenose, striped and common dolphins, harbour and Dall’s porpoises). Hg, Cd and As values are among the lowest determined, while Pb is the highest oneSome correlations have been detected among trace elements, excepted for Cd. These correlations can reflect different exposure levels and/or accumulation capacity and elimination pathways.

Polycomb repressive complex 2 (PRC2) has been shown to play a major role in transcriptional silencing in part by installing methylation marks on lysine 27 of histone 3. Dysregulation of PRC2 function correlates with certain malignancies and poor prognosis. EZH2 is the catalytic engine of the PRC2 complex and thus represents a key candidate oncology target for pharmacological intervention. Here we report the optimization of our indole-based EZH2 inhibitor series that led to the identification of CPI-1205, a highly potent (biochemical IC50 = 0.002 μM, cellular EC50 = 0.032 μM) and selective inhibitor of EZH2. This compound demonstrates robust antitumor effects in a Karpas-422 xenograft model when dosed at 160 mg/kg BID and is currently in Phase I clinical trials. Additionally, we disclose the co-crystal structure of our inhibitor series bound to the human PRC2 complex.

Deregulation of the receptor tyrosine kinase mesenchymal epithelial transition factor (MET) has been implicated in several human cancers and is an attractive target for small molecule drug discovery. Herein, we report the discovery of compound 23 (AMG 337), which demonstrates nanomolar inhibition of MET kinase activity, desirable preclinical pharmacokinetics, significant inhibition of MET phosphorylation in mice, and robust tumor growth inhibition in a MET-dependent mouse efficacy model.

The structure-based design and optimization of a novel series of selective PERK inhibitors are described resulting in the identification of 44 as a potent, highly selective, and orally active tool compound suitable for PERK pathway biology exploration both in vitro and in vivo.

The paper describes the measurement procedures used to estimate the parameters of a C40-ST-09 Actidyn Systemes centrifuge by independent external devices. A specific feature of the proposed procedures is that angular errors in compensation for the rotation of the main axis by the satellite table are determined with the use of an autocollimator, which makes it possible to gain information about the angular position of the article under test with a frequency of up to 10 kHz. The test results are discussed.

The single bromodomain of the closely related transcriptional regulators CBP/EP300 is a target of much recent interest in cancer and immune system regulation. A co-crystal structure of a ligand-efficient screening hit and the CBP bromodomain guided initial design targeting the LPF shelf, ZA loop, and acetylated lysine binding regions. Structure–activity relationship studies allowed us to identify a more potent analogue. Optimization of permeability and microsomal stability and subsequent improvement of mouse hepatocyte stability afforded 59 (GNE-272, TR-FRET IC50 = 0.02 μM, BRET IC50 = 0.41 μM, BRD4(1) IC50 = 13 μM) that retained the best balance of cell potency, selectivity, and in vivo PK. Compound 59 showed a marked antiproliferative effect in hematologic cancer cell lines and modulates MYC expression in vivo that corresponds with antitumor activity in an AML tumor model.

Lysine to methionine (K-to-M) mutations in genes encoding histone H3 are thought to drive a subset of pediatric brain and bone cancers. These high-frequency K-to-M mutations occur at sites of methylation on histone H3, and tumors containing the mutant histones exhibit a global loss of specific histone methylation marks. Previous studies showed that K-to-M mutant histones, also known as oncohistones, are potent orthosteric inhibitors of specific Su(var)3-9, Enhancer-of-zeste, Trithorax (SET) domain methyltransferases. However, the biochemical and biophysical details of the interaction between K-to-M mutant histones and the respective SET domain methyltransferases are currently unknown. Here, we use the histone H3K9-directed methyltransferase G9a as a model to explore the mechanism of inhibition by K-to-M oncohistones. X-ray cocrystal structures revealed that the K9M residue of histone H3 occupies the active site cavity of G9a, and kinetic analysis indicates competitive inhibition of G9a by histone H3K9M. Additionally, we find that the cofactor S-adenosyl methionine (SAM) is necessary for stable interaction between G9a and H3K9M histone. Consistent with the formation of a ternary complex, we find that the inhibitory peptide is uncompetitive with regard to SAM. These data and others indicate that K-to-M oncohistones promote global loss of specific lysine methylation through sequestration and inhibition of SAM-bound SET domain methyltransferases.

This communication describes the identification and optimization of a series of pan-KDM5 inhibitors derived from compound 1, a hit initially identified against KDM4C. Compound 1 was optimized to afford compound 20, a 10 nM inhibitor of KDM5A. Compound 20 is highly selective for the KDM5 enzymes versus other histone lysine demethylases and demonstrates activity in a cellular assay measuring the increase in global histone 3 lysine 4 tri-methylation (H3K4me3). In addition compound 20 has good ADME properties, excellent mouse PK, and is a suitable starting point for further optimization.

Deregulation of the receptor tyrosine kinase c-Met has been implicated in several human cancers and is an attractive target for small molecule drug discovery. Herein, we report the discovery of a structurally diverse series of carbon-linked quinoline triazolopyridinones, which demonstrates nanomolar inhibition of c-Met kinase activity. This novel series of inhibitors exhibits favorable pharmacokinetics as well as potent inhibition of HGF-mediated c-Met phosphorylation in a mouse liver pharmacodynamic model.

Deregulation of the receptor tyrosine kinase c-Met has been implicated in several human cancers and is an attractive target for small molecule drug discovery. We previously showed that O-linked triazolopyridazines can be potent inhibitors of c-Met. Herein, we report the discovery of a related series of N-linked triazolopyridazines which demonstrate nanomolar inhibition of c-Met kinase activity and display improved pharmacodynamic profiles. Specifically, the potent time-dependent inhibition of cytochrome P450 associated with the O-linked triazolopyridazines has been eliminated within this novel series of inhibitors. N-linked triazolopyridazine 24 exhibited favorable pharmacokinetics and displayed potent inhibition of HGF-mediated c-Met phosphorylation in a mouse liver PD model. Once-daily oral administration of 24 for 22days showed significant tumor growth inhibition in an NIH-3T3/TPR-Met xenograft mouse efficacy model.

c-Met and RON are receptor tyrosine kinases (RTK) that are closely related, both from a homology as well as from a functional stand point. Both receptors can induce cell migration, invasion, proliferation and survival in response to their respective ligand. Moreover, both possess oncogenic activity in vitro, in animal models in vivo and are often deregulated in human cancers. c-Met attracted a lot of interest shortly after its discovery in the mid-1980s because of its unusual role in cell motility. Moreover, a causal role for c-Met activating mutations in human cancer propelled an intensive drug discovery effort throughout the research and pharmaceutical communities to find inhibitors of c-Met. While c-Met is now a well-accepted target for an anti-cancer drug, less is known about the role of RON in cancer. Interestingly, despite their many common attributes, c-Met and RON are activated by different mechanisms in cancer cells. Because of the homology between the two RTKs, some small molecule kinase inhibitors of c-Met have inhibitory activity on RON, opening the door to exploring the role of both receptors in human cancers. In this review we will discuss the relevance of both c-Met and RON deregulation in human cancers and the progress so far in identifying small molecule kinase inhibitors that can block the activity of these targets in vitro and lead to anti-tumor effects in animal models.

c-Met is a receptor tyrosine kinase that plays a key role in several cellular processes but has also been found to be overexpressed and mutated in different human cancers. Consequently, targeting this enzyme has become an area of intense research in drug discovery. Our studies began with the design and synthesis of novel pyrimidone 7, which was found to be a potent c-Met inhibitor. Subsequent SAR studies identified 22 as a more potent analog, whereas an X-ray crystal structure of 7 bound to c-Met revealed an unexpected binding conformation. This latter finding led to the development of a new series that featured compounds that were more potent both in vitro and in vivo than 22 and also exhibited different binding conformations to c-Met. Novel c-Met inhibitors have been designed, developed, and found to be potent in vitro and in vivo.

Deregulation of the receptor tyrosine kinase c-Met has been implicated in human cancers. Pyrazolones with N-1 bearing a pendent hydroxyalkyl side chain showed selective inhibition of c-Met over VEGFR2. However, studies revealed the generation of active, nonselective metabolites. Blocking this metabolic hot spot led to the discovery of 17 (AMG 458). When dosed orally, 17 significantly inhibited tumor growth in the NIH3T3/TPR-Met and U-87 MG xenograft models with no adverse effect on body weight.

Primary and acquired resistance to kinase inhibitors due to pre-existing mutations of the target or to mutations that arise as a result of selection by therapy is now a common theme in cancer patients treated with these drugs. Different classes of inhibitors for the same target have been successful in overcoming, at least temporarily, these resistance mechanisms because of their ability to interact with the mutated receptor. Therefore, having different classes of inhibitors for a given target might offer more treatment options for cancer patients. c-Met inhibitors are emerging as potentially important new cancer drugs and profiling these agents against several mutant receptors has begun. We have recently identified c-Met inhibitors that are active against wild-type and mutated c-Met variants. X-ray crystallography revealed that this class of inhibitors binds c-Met very differently than another c-Met inhibitor that shows primary resistance to some c-Met mutants. Our results suggested that it is possible to identify c-Met inhibitors that will be active against a range of c-Met mutations.

Tumorigenesis is a multistep process in which oncogenes play a key role in tumor formation, growth, and maintenance. MET was discovered as an oncogene that is activated by its ligand, hepatocyte growth factor. Deregulated signaling in the c-Met pathway has been observed in multiple tumor types. Herein we report the discovery of potent and selective triazolopyridazine small molecules that inhibit c-Met activity.

c-Met is a receptor tyrosine kinase often deregulated in human cancers, thus making it an attractive drug target. One mechanism by which c-Met deregulation leads to cancer is through gain-of-function mutations. Therefore, small molecules capable of targeting these mutations could offer therapeutic benefits for affected patients. SU11274 was recently described and reported to inhibit the activity of the wild-type and some mutant forms of c-Met, whereas other mutants are resistant to inhibition. We identified a novel series of c-Met small molecule inhibitors that are active against multiple mutants previously identified in hereditary papillary renal cell carcinoma patients. AM7 is active against wild-type c-Met as well as several mutants, inhibits c-Met-mediated signaling in MKN-45 and U-87 MG cells, and inhibits tumor growth in these two models grown as xenografts. The crystal structures of AM7 and SU11274 bound to unphosphorylated c-Met have been determined. The AM7 structure reveals a novel binding mode compared with other published c-Met inhibitors and SU11274. The molecule binds the kinase linker and then extends into a new hydrophobic binding site. This binding site is created by a significant movement of the C-helix and so represents an inactive conformation of the c-Met kinase. Thus, our results demonstrate that it is possible to identify and design inhibitors that will likely be active against mutants found in different cancers.

Deregulation of the receptor tyrosine kinase mesenchymal epithelial transition factor (MET) has been implicated in several human cancers and is an attractive target for small molecule drug discovery. Herein, we report the discovery of compound 23 (AMG 337), which demonstrates nanomolar inhibition of MET kinase activity, desirable preclinical pharmacokinetics, significant inhibition of MET phosphorylation in mice, and robust tumor growth inhibition in a MET-dependent mouse efficacy model.

The overexpression of c-Met and/or hepatocyte growth factor (HGF), the amplification of the MET gene, and mutations in the c-Met kinase domain can activate signaling pathways that contribute to cancer progression by enabling tumor cell proliferation, survival, invasion, and metastasis. Herein, we report the discovery of 8-fluorotriazolopyridines as inhibitors of c-Met activity. Optimization of the 8-fluorotriazolopyridine scaffold through the combination of structure-based drug design, SAR studies, and metabolite identification provided potent (cellular IC50 < 10 nM), selective inhibitors of c-Met with desirable pharmacokinetic properties that demonstrate potent inhibition of HGF-mediated c-Met phosphorylation in a mouse liver pharmacodynamic model.

The crystal structure of the dimerization domain of the V(D)J recombination-activating protein, RAG1, was solved using zinc anomalous scattering. The structure reveals an unusual combination of multi-class zinc-binding motifs, including a zinc RING finger and a C2H2 zinc finger, that together from a single structural domain. The domain also contains a unique zinc binuclear cluster in place of a normally mononuclear zinc site in the RING finger. Together, four zinc ions help organize the entire domain, including the two helices that form the dimer interface.

As part of our effort toward developing an effective therapeutic agent for c-Met-dependent tumors, a pyrazolone-based class II c-Met inhibitor, N-(4-((6,7-dimethoxyquinolin-4-yl)oxy)-3-fluorophenyl)-1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide (1), was identified. Knowledge of the binding mode of this molecule in both c-Met and VEGFR-2 proteins led to a novel strategy for designing more selective analogues of 1. Along with detailed SAR information, we demonstrate that the low kinase selectivity associated with class II c-Met inhibitors can be improved significantly. This work resulted in the discovery of potent c-Met inhibitors with improved selectivity profiles over VEGFR-2 and IGF-1R that could serve as useful tools to probe the relationship between kinase selectivity and in vivo efficacy in tumor xenograft models. Compound 59e (AMG 458) was ultimately advanced into preclinical safety studies.

Deregulation of c-Met receptor tyrosine kinase activity leads to tumorigenesis and metastasis in animal models. More importantly, the identification of activating mutations in c-Met, as well as MET gene amplification in human cancers, points to c-Met as an important target for cancer therapy. We have previously described two classes of c-Met kinase inhibitors (class I and class II) that differ in their binding modes and selectivity profiles. The class II inhibitors tend to have activities on multiple kinases. Knowledge of the binding mode of these molecules in the c-Met protein led to the design and evaluation of several new class II c-Met inhibitors that utilize various 5-membered cyclic carboxamides to conformationally restrain key pharmacophoric groups within the molecule. These investigations resulted in the identification of a potent and novel class of pyrazolone c-Met inhibitors with good in vivo activity.

The DNA unwinding produced by specific adducts of the antitumor drug cis-diamminedi-chloroplatinum(II) has been quantitatively determined. Synthetic DNA duplex oligonucleotides of varying lengths with two base pair cohesive ends were synthesized and characterized that contained site-specific intrastrand N7-purine/N7-purine cross-links. Included are cis-(Pt(NH{sub 3}){sub 2}(d(GpG))), cis-(Pt(NH){sub 3}{sub 2}(d(ApG))), and cis-(Pt(NH{sub 3}){sub 2}(d(GpTpG))) adducts, respectively referred to as cis-GG, cis-AG, and cis-GTG. Local DNA distortions at the site of platination were amplified by polymerization of these monomers and quantitatively evaluated by using polyacrylamide gel electrophoresis. The extent of DNA unwinding was determined by systematically varying the interplatinum distance, or phasing, in polymers containing the adducts. The multimer that migrates most slowly gives the optimal phasing for cooperative bending, from which the degree of unwinding can be obtained. The authors find that the cis-GG and cis-AG adducts both unwind DNA by 13{degrees}, while the cis-GTG adduct unwinds DNA by 23{degrees}. In addition, experiments are presented that support previous studies revealing that a hinge joint forms at the sites of platination in DNA molecules containing trans-GTG adducts. On the basis of an analysis of the present and other published studies of site-specifically modified DNA. The authors propose that local duplex unwinding is a majormore » determinant in the recognition of DNA damage by the Escherichia coli (A)BC excinuclease. In addition, local duplex unwinding of 13{degrees} and bending by 35{degrees} are shown to correlate well with the recognition of platinated DNA by a previously identified damage recognition protein (DRP) in human cells.« less

Duplex oligonucleotides containing a single intrastrand [Pt(NH3)2]2+ cross-link or monofunctional adduct and either 15 or 22 bp in length were synthesized and chemically characterized. The platinum-modified and unmodified control DNAs were polymerized in the presence of DNA ligase and the products studied on 8% native polyacrylamide gels. The extent of DNA bending caused by the various platinum-DNA adducts was revealed by their gel mobility shifts relative to unplatinated controls. The bifunctional adducts cis-[Pt(NH3)2[d(GpG)]]+, cis-[Pt(NH3)2[d(ApG)]]+, and cis-[Pt(NH3)2[d(G*pTpG*)]], where the asterisks denote the sites of platinum binding, all bend the double helix, whereas the adduct trans-[Pt(NH3)2[d(G*pTpG*)]] imparts a degree of flexibility to the duplex. When modified by the monofunctional adduct cis-[Pt(NH3)2(N3-cytosine)(dG)]Cl the helix remains rod-like. These results reveal important structural differences in DNAs modified by the antitumor drug cisplatin and its analogs that could be important in the biological processing of the various adducts in vivo.

Intrathecal immunoglobulin synthesis is observed in more than 90% of all cases of multiple sclerosis, producing a specific CSF IgG oligoclonal electrophoretic pattern. The consensual method used as reference is isoelectric focusing (IEF). We developed a new CSF Ig analysis method by immunofixation (IF). The method includes an immunoenzymatic detection step performed directly on the gel allowing the use of unconcentrated CSF and avoiding the blotting step. The reliability of this method was established by the analysis of 210 CSF/serum pairs including defined, probable and possible MS, other inflammatory CNS diseases and controls (noninflammatory CNS diseases and peripheral nervous system diseases). Intrathecal IgG synthesis was detected in 95.5% of defined MS cases. The specificity for CNS inflammatory diseases including MS diagnosis, evaluated by comparison with controls, was 98.8%. This new method is quicker and visual interpretation is easier than with IEF. It is a semi-automated method that should be considered for standardization of CSF IgG analysis.

The transcription factor c-Myc regulates diverse biological processes including growth, proliferation, differentiation, quiescence, and senescence or apoptosis. c-Myc proto-oncogene overexpression has been implicated in the genesis of many human malignancies. Identification of compounds that selectively and potently inhibit the expression of c-Myc hence could be beneficial for the treatment of these diseases. We and others have recently shown that inhibiting the binding of bromodomain-containing proteins such as Brd4 to their acetylated histone substrates leads to the specific suppression of c-Myc transcription. We report here the characterization of CPI-267203, a potent, selective and competitive inhibitor of BET domain proteins (Brd4 IC50 = 26 nM and CBP IC50 = 6.0 uM). In vitro, this molecule inhibits IL-6 production (EC50 = 11 nM) induced by lipopolysaccharide (LPS) in acute monocytic leukemia cells (THP1) and c-Myc mRNA production (EC50 = 27 nM) in Burkitt lymphoma cells (Raji). c-Myc mRNA and protein levels decline as early as 4hrs after treatment with CPI-267203, leading to cell cycle arrest and apoptosis. In vivo, CPI-267203 has 84% oral bioavailability and a plasma half-life of 1.9 h. In a xenograft model of human acute myelogenous leukemia (MV4:11), CPI-267203 inhibits the production of c-Myc mRNA and protein, inhibits tumor growth, and triggers tumor apoptosis. Reduction of c-Myc protein levels can also be detected in the normal skin of treated animals, suggesting that the level of c-Myc in the skin might be a useful surrogate pharmacodynamic marker to aid clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-237. doi:1538-7445.AM2012-LB-237

Chemical and biological studies are presented for a new series of platinum(II) antitumor agents that violate the classical structure-activity relationships established for platinum complexes. These new agents, which have demonstrated activity against murine and human tumor systems, are cis-[Pt(NH3)2(Am)Cl]+ cations, in which Am is a derivative of pyridine, pyrimidine, purine, or aniline. Members from this series block simian virus 40 DNA replication in vitro and inhibit the action of DNA polymerases at individual guanine residues in replication mapping experiments. Monoclonal antibodies that bind selectively to cisplatin lesions on calf thymus DNA were used in a competitive enzyme-linked immunosorbent assay study to show that the platinum-triamine complexes do not produce the type of intrastrand cross-links on DNA that are characteristics of cisplatin and analogues with the general formula cis-[Pt(amine)2X2]. These results indicate that cis-[Pt(NH3)2(Am)Cl]+ cations form monofunctional adducts on DNA rather than eliminate NH3 or Am to afford bifunctional lesions. This conclusion is further supported by nuclear magnetic resonance spectroscopic and enzymatic digestion analyses of the products of the reactions of these triamine complexes with d(GpG) and dG, which also reveal monofunctional binding. When cis-[Pt(NH3)2(4-Br-pyridine)Cl]+ was allowed to stand in phosphate-buffered saline at 37 degrees C for 14 days, however, NH4+ was released and trans-[Pt(NH3)(4-Br-pyridine)Cl2] formed concomitantly. This compound was characterized by a single crystal X-ray diffraction study, the details of which are reported. The fact that trans-[Pt(NH3)(4-Br-pyridine)Cl2] displays no anticancer activity, however, indicates that its formation from cis-[Pt(NH3)2(4-Br-pyridine)Cl]+ is not a significant component of the mechanism of action of this platinum-triamine complex. Taken together, these findings indicate that the cytotoxicity of cis-[Pt(NH3)2(Am)Cl]+ complexes most likely arises from the formation of monofunctional adducts. The DNA binding properties associated with this new class of antitumor agents suggest that they may display an activity profile different from that of cisplatin and related analogues.

The anticancer drug cis-diamminedichloroplatinum(II) (cis-DDP or cisplatin), is believed to kill cells by blocking DNA replication or transcription (Roberts and Thomson, 1979; Sorenson and Eastman, 1988a, b). cis-DDP-DNA adducts have been shown to block DNA replication in vitro (Pinto and Lippard, 1985b; Heiger-Bernays et al., 1990) and in vivo (Harder and Rosenberg, 1970; Howie and Gale, 1970; Ciccarelli et al., 1985). Similar recent studies have shown that selected platinum adducts also inhibit RNA polymerases (Corda et al., 1991).

A factor has been identified in extracts from human HeLa and hamster V79 cells that retards the electrophoretic mobility of several DNA restriction fragments modified with the antitumor drug cis-diamminedichloroplatinum(II) (cisplatin). Binding of the factor to cisplatin-modified DNA was sensitive to pretreatment with proteinase K, establishing that the factor is a protein. Gel mobility shifts were observed with probes containing as few as seven Pt atoms per kilobase of duplex DNA. By competition experiments the dissociation constant, Kd, of the protein from cisplatin-modified DNA was estimated to be (1-20) X 10(-10) M. Protein binding is selective for DNA modified with cisplatin, [Pt(en)Cl2] (en, ethylenediamine), and [Pt(dach)Cl2] (dach, 1,2-diaminocyclohexane) but not with chemotherapeutically inactive trans-diamminedichloroplatinum(II) or monofunctionally coordinating [Pt(dien)Cl]Cl (dien, diethylenetriamine) complexes. The protein also does not bind to DNA containing UV-induced photoproducts. The protein binds specifically to 1,2-intrastrand d(GpG) and d(ApG) cross-links formed by cisplatin, as determined by gel mobility shifts with synthetic 110-bp duplex oligonucleotides; these modified oligomers contained five equally spaced adducts of either cis-[Pt(NH3)2d(GpG) or cis-[Pt(NH3)2d(ApG)]. Oligonucleotides containing the specific adducts cis-[Pt(NH3)2d(GpTpG)], trans-[Pt(NH3)2d(GpTpG)], or cis-[Pt(NH3)2(N3-cytosine)d(G)] were not recognized by the protein. The apparent molecular weight of the protein is 91,000, as determined by sucrose gradient centrifugation of a preparation partially purified by ammonium sulfate fractionation. Binding of the protein to platinum-modified DNA does not require cofactors but is sensitive to treatment with 5 mM MnCl2, CdCl2, CoCl2, or ZnCl2 and with 1 mM HgCl2.(ABSTRACT TRUNCATED AT 250 WORDS)

1. ‘Inhibitor fragment’ isolated from human serum albumin degraded by rabbit cathepsin D is composed of one peptide chain with two intrachain disulphide bonds. There are two kinds of inhibitor molecules having different N-terminal amino acids: one is threonine and the other glutamine. 2. Fragment F1, isolated from inhibitor degraded by trypsin, is composed of two chains linked by a disulphide bond. There are three kinds of fragment F1. All have one alpha chain in common, which has an intrachain disulphide bond. They differ by the nature of the chain, which is linked to the alpha chain by a disulphide bond. The epsilon chain is present in trace amounts. The two other chains, beta and gamma, differ by their C-terminal amino acid, which is respectively arginine and lysine. 3. Inhibitor is composed of the last 92 or 89 residues of the human albumin molecule and fragment F1 is composed of two parts of this C-terminal portion of the albumin molecule.

Resume L'activiteimmunologique de fragments d'albumine humaine aetemesuree par une methode utilisant des anticorps anti-albumine marquesal'125I. La methode consisteafixer les anticorps marques sur un immunoadsorbant insoluble etadeplacer ces anticorps par les fragments d'albumine. On peut ainsi, d'une part mesurer le pourcentage d'anticorps anti-albumine specifiques d'un fragment et, d'autre part, comparer la capacitede combinaison de l'albumine et d'un fragment avec les anticorps specifiques du ou des sites portes par le fragment.

Crystalline formic acid has been investigated by IR-spectroscopy and differential thermal analysis from 78°K to 300°K. A first-order phase transition, showing hysteresis between 207°K and 218°K has been observed, and tentative assignments are given.

The antigenic structure of a fragment (F1) of human serum albumin (HSA), carrying one of the antigenic sites of the whole molecule, has been studied. Previous studies have shown that this fragment is made of two peptide chains linked by a disulfide bond. One of these chains named alpha has 28 residues and one intrachain disulfide bond giving rise to a loop of 9 or 10 residues. The chain occurs under two forms: the beta chain made of 25 residues and the gamma chain which differs from the beta chain by the absence of the two last C-terminal residues (F. Bellon and C. Lapresle, Biochem. J., 1975, 147, 585-592). The alpha and beta chains both inhibit independantly at 100% the agglutination by an anti-albumin serum of red cells sensitized with F1. Chains alpha, beta and gamma displace anti-HSA antibodies fixed upon an immunoadsorbent prepared with F1. Chain alpha is more active than chain beta which in turn is more active than chain gamma. Reduction and alkylation of alpha and beta chains abolish almost completely their immunological activity. Immunoadsorbents prepared with alpha or beta chains adsorb all of the anti-F1 antibodies from an anti-HSA serum. Antibodies isolated from each chain are inhibited by both chains. Chains alpha and beta induce in rabbits the formation of antibodies reacting with F1 and HSA, alpha chain being a better immunogen than beta chain.

Using an iodine-vapor cell and a single-mode argon–ion laser, we have constructed a spectrometer for low-shifted light scattering, which has resolution better than 0.3 GHz (0.01 cm−1). A method has been proposed to deconvolute the spectra.