Marrow stromal cell recovery after radiation-induced aplasia in mice.

PURPOSE: The identification of fibroblast-like cells of the marrow stroma by means of alkaline phosphatase (ALP) cytochemistry reveals delicate ALP-positive structures interspersed among haematopoietic cells and arranged in a loosely meshed network. These cells are often referred to as 'reticular' cells and the network they form is known as the 'ALP network'. The purpose was to analyse the evolution of this ALP network in relation to haemopoietic regeneration after whole-body irradiation. MATERIALS AND METHODS: The total surface occupied by ALP-positive processes revealed by means of ALP cytochemistry was expressed as a ratio of the total marrow area. ALP-positive cells were counted using nuclei as the defining unit. Cell proliferation was analysed by the detection of bromodeoxyuridine (BrdU) incorporation. Fat cells were identified by oil red O staining and alpha-glycerophosphate dehydrogenase (alpha-GPDH) activity. RESULTS: The ALP network and ALP-positive cell number began to increase 24 h after 4-Gy irradiation to reach a maximum after 72 h, when the bone marrow was almost completely empty of haemopoietic cells. This increase was in advance of haemopoietic recovery and was not due to cell proliferation. A decrease in the ALP network occurred in parallel with an increase in haemopoiesis and was accompanied by a transient increase in fat cells on day 7. CONCLUSIONS: These data indicate that the recovery of the ALP network, which is partially due to the recruitment of ALP- positive cells, occurs in advance of the haemopoietic recovery and that the equilibrium between fat cells and ALP-positive cells seems to be controlled by haemopoietic cells.

Structure-based design of caspase-1 inhibitor containing a diphenyl ether sulfonamide.

A series of compounds was designed and prepared as inhibitors of interleukin-1beta converting enzyme (ICE), also known as caspase-1. These inhibitors, which employ a diphenyl ether sulfonamide, were designed to improve potency by forming favorable interactions between the diphenyl ether rings and the prime side hydrophobic region. An X-ray crystal structure of a representative member of the diphenyl ether sulfonamide series bound to the active site of caspase-1 was obtained.

Genetic imbalances in preleukemic thymuses.

To understand the molecular mechanisms involved in preleukemia, the suppression subtractive hybridization method was used in a murine radiation-induced thymic lymphoma model. Seventeen mRNAs overexpressed in preleukemic thymuses were identified: mouse laminin binding protein (p40/37LBP), E25 protein, Rattus norvegicus clone BB.1.4.1, profilin, poly(A) binding protein (PABP), mouse high mobility group protein 1, topoisomerase I, clusterin, proteasome RC1 subunit, rat prostatein C3 and C1 subunits; two ESTs and four unknown genes. The overexpression of PABP, clusterin, profilin, and the p40/37LBP mRNAs was confirmed in preleukemic thymuses and can be related to some cellular events observed during the preleukemic period, i.e., alterations of cell cycle and apoptosis properties. The p40/37LBP and 67-kDa laminin receptor proteins were upregulated during the preleukemic period. The data suggest that additional studies on p40/37LBP and 67-kDa laminin receptor regulation are required to evaluate their potential role in the lymphoma prevention by TNF-alpha and IFN-gamma.

X-ray structures of five renin inhibitors bound to saccharopepsin: exploration of active-site specificity.

Saccharopepsin is a vacuolar aspartic proteinase involved in activation of a number of hydrolases. The enzyme has great structural homology to mammalian aspartic proteinases including human renin and we have used it as a model system to study the binding of renin inhibitors by X-ray crystallography. Five medium-to-high resolution structures of saccharopepsin complexed with transition-state analogue renin inhibitors were determined. The structure of a cyclic peptide inhibitor (PD-129,541) complexed with the proteinase was solved to 2.5 A resolution. This inhibitor has low affinity for human renin yet binds very tightly to the yeast proteinase (K(i)=4 nM). The high affinity of this inhibitor can be attributed to its bulky cyclic moiety spanning P(2)-P(3)' and other residues that appear to optimally fit the binding sub-sites of the enzyme. Superposition of the saccharopepsin structure on that of renin showed that a movement of the loop 286-301 relative to renin facilitates tighter binding of this inhibitor to saccharopepsin. Our 2.8 A resolution structure of the complex with CP-108,420 shows that its benzimidazole P(3 )replacement retains one of the standard hydrogen bonds that normally involve the inhibitor's main-chain. This suggests a non-peptide lead in overcoming the problem of susceptible peptide bonds in the design of aspartic proteinase inhibitors. CP-72,647 which possesses a basic histidine residue at P(2), has a high affinity for renin (K(i)=5 nM) but proves to be a poor inhibitor for saccharopepsin (K(i)=3.7 microM). This may stem from the fact that the histidine residue would not bind favourably with the predominantly hydrophobic S(2) sub-site of saccharopepsin.

Bit-string methods for selective compound acquisition

Selective compound acquisition programs need to ensure that the compounds that are chosen do not contain undesirable functionality. This is easy to achieve if a supplier is prepared to provide unambiguous structure representations for the compounds that they have available: this paper discusses selection techniques that can be used when a supplier is prepared to make available only fragment bit-string representations for the compounds in their catalog. Experiments with three databases and three types of bit-string show that a simple k-nearest-neighbor searching method provides a surprisingly effective, although far from perfect, way of selecting compounds when only bit-string representations are available. A second approach, based on the use of a fragment weighting scheme analogous to those used in substructural analysis studies, proved to be noticeably less effective in operation.

Structural basis of the thrombin selectivity of a ligand that contains the constrained arginine mimic (2S)-2-amino-(3S)-3-(1-carbamimidoyl- piperidin-3-yl)-propanoic acid at P1.

We have studied the thrombin and trypsin complexed structures of a pair of peptidomimetic thrombin inhibitors, containing different P1 fragments. The first has arginine as its P1 fragment, and the second contains the constrained arginine mimic (2S)-2-amino-(3S)-3-(1-carbamimidoyl-piperidin-3-yl)-propano ic acid (SAPA), a fragment known to enhance thrombin/trypsin selectivity of inhibitors. On the basis of an analysis of the nonbonded interactions present in the structures of the trypsin and thrombin complexes of the two inhibitors, the calculated accessible surfaces of the enzymes and inhibitors in the four complexes, data on known structures of trypsin complexes of inhibitors, and factor Xa inhibitory potency of these compounds, we conclude that the ability of this arginine mimic to increase thrombin selectivity of an inhibitor is mediated by its differential interaction with the residue at position 192 (chymotrypsinogen numbering). Thrombin has a glutamic acid at residue 192, and trypsin has a glutamine. The analysis also suggests that this constrained arginine mimic, when present in an inhibitor, might enhance selectivity against other trypsin-like enzymes that have a glutamine at residue position 192.

Assessment of tumor angiogenesis in invasive breast carcinomas: absence of correlation with prognosis and pathological factors.

Different retrospective studies have shown that microvessel counting (MVC) is an independent prognostic marker for clinical outcome in breast cancer. The aim of this study was to evaluate the prognostic value of MVC alone or in association with classical clinicopathological parameters, as well as the reproducibility of the technique. We analyzed a retrospective series of 216 cases of breast carcinoma. Tissue sections were stained for Factor-VIII-related antigen. Microvessel quantification was performed at x400 magnification in the three most vascular areas of the tumors (hot spots). Mean and highest values were studied. Furthermore, a semi-quantitative evaluation of MVC was performed by use of an image-analysis system. The effect of multiple factors on survival was tested under a Cox multivariate proportional hazards model. In ten cases, a study of the reproducibility was done by evaluating MVC in different sections of the same block and in different blocks of a same tumor. There was no association between MVC (determined at a microscopic level or by image analysis) and overall survival or relapse-free survival. No association was found with tumor size, tumor grade, and lymph-node status. The study of reproducibility showed a very high intra-tumoral variation of MVC. The intra-individual coefficient of variation (CV) varied between 20 and 80%. This study did not show any significant correlation between angiogenesis, as assessed by MVC, and relapse-free survival or overall survival in infiltrating breast carcinomas. The low reproducibility of the MVC for the same tumors suggests that this technique must still be optimized before routine application.

Analysis of the binding of hydroxamic acid and carboxylic acid inhibitors to the stromelysin-1 (matrix metalloproteinase-3) catalytic domain by isothermal titration calorimetry.

Matrix metalloproteinases (MMPs) are implicated in diseases such as arthritis and cancer. Among these enzymes, stromelysin-1 can also activate the proenzymes of other MMPs, making it an attractive target for pharmaceutical design. Isothermal titration calorimetry (ITC) was used to analyze the binding of three inhibitors to the stromelysin catalytic domain (SCD). One inhibitor (Galardin) uses a hydroxamic acid group (pK(a) congruent with 8.7) to bind the active site zinc; the others (PD180557 and PD166793) use a carboxylic acid group (pK(a) congruent with 4.7). Binding affinity increased dramatically as the pH was decreased over the range 5.5-7.5. Experiments carried out at pH 6.7 in several different buffers revealed that approximately one and two protons are transferred to the enzyme-inhibitor complexes for the hydroxamic and carboxylic acid inhibitors, respectively. This suggests that both classes of inhibitors bind in the protonated state, and that one amino acid residue of the enzyme also becomes protonated upon binding. Similar experiments carried out with the H224N mutant gave strong evidence that this residue is histidine 224. DeltaG, DeltaH, DeltaS, and DeltaC(p) were determined for the three inhibitors at pH 6.7, and DeltaC(p) was used to obtain estimates of the solvational, translational, and conformational components of the entropy term. The results suggest that: (1) a polar group at the P1 position can contribute a large favorable enthalpy, (2) a hydrophobic group at P2' can contribute a favorable entropy of desolvation, and (3) P1' substituents of certain sizes may trigger an entropically unfavorable conformational change in the enzyme upon binding. These findings illustrate the value of complete thermodynamic profiles generated by ITC in discovering binding interactions that might go undetected when relying on binding affinities alone.

Nuclear localization of a new c-cbl related protein, CARP 90, during in vivo thymic apoptosis in mice.

This study investigates the involvement of the c-cbl protooncogene in thymocyte apoptosis occurring in vivo after hydrocortisone treatment. In the thymus of untreated mice, a few medullary and cortical thymocytes expressed p120cbl, mainly in the cytoplasm. In the cortex, their number and distribution resemble that of apoptotic cells evidenced by TUNEL staining. The expression of Cbl is rapidly increased when apoptosis is triggered by hydrocortisone. This Cbl-specific immunostaining was detected in the nucleus and is due to a Cbl-related 90 kDa protein (CARP 90). These results show that a c-cbl product could localize in the nucleus and suggest that it could be involved as a regulator of thymic apoptosis.

X-ray structure of human stromelysin catalytic domain complexed with nonpeptide inhibitors: implications for inhibitor selectivity.

Effective inhibitors of matrix metalloproteinases (MMPs), a family of connective tissue-degrading enzymes, could be useful for the treatment of diseases such as cancer, multiple sclerosis, and arthritis. Many of the known MMP inhibitors are derived from peptide substrates, with high potency in vitro but little selectivity among MMPs and poor bioavailability. We have discovered nonpeptidic MMP inhibitors with improved properties, and report here the crystal structures of human stromelysin-1 catalytic domain (SCD) complexed with four of these inhibitors. The structures were determined and refined at resolutions ranging from 1.64 to 2.0 A. Each inhibitor binds in the active site of SCD such that a bulky diphenyl piperidine moiety penetrates a deep, predominantly hydrophobic S'1 pocket. The active site structure of the SCD is similar in all four inhibitor complexes, but differs substantially from the peptide hydroxamate complex, which has a smaller side chain bound in the S'1 pocket. The largest differences occur in the loop forming the "top" of this pocket. The occupation of these nonpeptidic inhibitors in the S'1 pocket provides a structural basis to explain their selectivity among MMPs. An analysis of the unique binding mode predicts structural modifications to design improved MMP inhibitors.

Nonpeptidic HIV protease inhibitors possessing excellent antiviral activities and therapeutic indices. PD 178390: a lead HIV protease inhibitor.

With the insight generated by the availability of X-ray crystal structures of various 5,6-dihydropyran-2-ones bound to HIV PR, inhibitors possessing various alkyl groups at the 6-position of 5,6-dihydropyran-2-one ring were synthesized. The inhibitors possessing a 6-alkyl group exhibited superior antiviral activities when compared to 6-phenyl analogues. Antiviral efficacies were further improved upon introduction of a polar group (hydroxyl or amino) on the 4-position of the phenethyl moiety as well as the polar group (hydroxymethyl) on the 3-(tert-butyl-5-methyl-phenylthio) moiety. The polar substitution is also advantageous for decreasing toxicity, providing inhibitors with higher therapeutic indices. The best inhibitor among this series, (S)-6-[2-(4-aminophenyl)-ethyl]-(3-(2-tert-butyl-5-methyl-phenylsulfa nyl)-4-hydroxy-6-isopropyl-5,6-dihydro-pyran-2-one (34S), exhibited an EC50 of 200 nM with a therapeutic index of > 1000. More importantly, these non-peptidic inhibitors, 16S and 34S, appear to offer little cross-resistance to the currently marketed peptidomimetic PR inhibitors. The selected inhibitors tested in vitro against mutant HIV PR showed a very small increase in binding affinities relative to wild-type HIV PR. Cmax and absolute bioavailability of 34S were higher and half-life and time above EC95 were longer compared to 16S. Thus 34S, also known as PD 178390, which displays good antiviral efficacy, promising pharmacokinetic characteristics and favorable activity against mutant enzymes and CYP3A4, has been chosen for further preclinical evaluation.

p53, Bax and Bcl-2 in vivo expression in the murine thymus after apoptogenic treatments.

BACKGROUND: Neoplasia can results from a lack of cell elimination by apoptosis. In order to determine if mechanisms controlling apoptosis are disturbed during neoplastic transformation in a model of murine radio-induced thymic lymphomas, we have assessed the kinetics of p53, Bax and Bcl-2 in situ expression after induction of thymic apoptosis by irradiation or glucocorticoids at first in normal mice. MATERIALS AND METHODS: TUNEL method was used for in situ detection of apoptosis and protein expression was determined by indirect immunohistochemistry. RESULTS: After hydrocortisone injection, levels of p53 and Bax, but not Bcl-2, expression were raised. A whole body sublethal irradiation led to an increase of p53 and Bcl-2, but not Bax, expression. CONCLUSIONS: This is the first in vivo report of in situ protein expression in the thymus after apoptogenic treatments of mice. The results suggest that Bax could be involved in glucocorticoid-mediated apoptosis. The increased levels of Bcl-2 expression are discussed.

Apoptosis during the development of radiogenic thymic lymphomas: effects of treatments inhibiting lymphoma development.

INTRODUCTION: Whole body fractionated irradiation induces thymic lymphomas in C57BL/Ka mice after a latent period during which preleukemic cells progressively transform into leukemic cells within an abnormal thymic microenvironment. A bone marrow graft or repeated cytokine injections prevent lymphoma development. We think that these treatments restore altered mechanisms controlling apoptosis. MATERIALS AND METHODS: Apoptosis was analyzed by flow cytometry in thymocytes from different groups of mice (control, preleukemic, prevented mice). RESULTS: The apoptotic rates did not change in freshly isolated thymocytes from different experimental groups. However, after culture, the level of apoptosis increased in preleukemic thymuses; and returned to normal value in cultured thymocytes from irradiated mice after lymphoma preventing treatments. Furthermore, thymic microenvironmental factors can control thymocyte apoptosis. CONCLUSION: We propose that after leukemogenic irradiation, there is an increase of cells with an activated suicide program, but that alterations of thymic environmental factors rescue them from apoptosis, allowing their further neoplastic transformation.

Development of a binding model to protein tyrosine kinases for substituted pyrido[2,3-d]pyrimidine inhibitors.

Previously, our laboratories have reported on a new class of highly potent tyrosine kinase inhibitors based on the pyrido[2, 3-d]pyrimidine core template. To understand the structural basis for the potency and specificity, a model for the binding mode of this class of inhibitors to the tyrosine kinase domains of c-Src, PDGFr, FGFr, and EGFr tyrosine kinases was developed from structural information (principally utilizing the catalytic domain of c-AMP-dependent protein kinase as template) and structure-activity relationship (SAR) information. In the resulting docking mode, the pyrido[2,3-d]pyrimidine template shows a hydrogen-bonding pattern identical to that of olomoucine. The 6-aryl substituent of the heterocycle is located deep in the binding cleft in a pocket not used by ATP, which helps to confer high-affinity binding as well as specificity. The 2-anilino and 2-(dialkylamino)alkylamino substituents as well as the 7-urea substituent of inhibitors within this class are located at the entrance of the binding cleft and make contact with residues in the hinge region between the two kinase lobes. This allows considerable variability and bulk tolerance for C-2 and N-7 substituents. The models presented here are consistent with the SAR seen for the inhibition of a number of isolated enzymes and provide a structural basis to explain their specificity. They have been used successfully to design new highly potent protein kinase inhibitors.

4-hydroxy-5,6-dihydropyrones. 2. Potent non-peptide inhibitors of HIV protease.

The 4-hydroxy-5,6-dihydropyrone template was utilized as a flexible scaffolding from which to build potent active site inhibitors of HIV protease. Dihydropyrone 1c (5,6-dihydro-4-hydroxy-6-phenyl-3-[(2-phenylethyl)thio]-2H-pyran-2-one) was modeled in the active site of HIV protease utilizing a similar binding mode found for the previously reported 4-hydroxybenzopyran-2-ones. Our model led us to pursue the synthesis of 6,6-disubstituted dihydropyrones with the aim of filling S1 and S2 and thereby increasing the potency of the parent dihydropyrone 1c which did not fill S2. Toward this end we attached various hydrophobic and hydrophilic side chains at the 6-position of the dihydropyrone to mimic the natural and unnatural amino acids known to be effective substrates at P2 and P2'. Parent dihydropyrone 1c (IC50 = 2100 nM) was elaborated into compounds with greater than a 100-fold increase in potency [18c, IC50 = 5 nM, 5-(3,6-dihydro-4-hydroxy-6-oxo-2-phenyl-5-[2-phenylethyl)thio] -2H-pyran-2-yl)pentanoic acid and 12c, IC50 = 51 nM, 5,6-dihydro-4-hydroxy-6-phenyl-6-(2-phenylethyl)-3- [(2-phenyl-ethyl)thio]-2H-pyran-2-one]. Optimization of the 3-position fragment to fill S1' and S2' afforded potent HIV protease inhibitor 49 [IC50 = 10 nM, 3-[(2-tert-butyl-5-methylphenyl)sulfanyl]-5,6-dihydro-4 -hydroxy-6-phenyl-6-(2-phenylethyl)-2H-pyran-2-one]. The resulting low molecular weight compounds (< 475) have one or no chiral centers and are readily synthesized.

Stages in the development of radiation-induced thymic lymphomas in C57 BL/Ka mice: preleukemic cells become progressively resistant to the tumor preventing effects of a bone marrow graft.

Fractionated whole body irradiation induces thymic lymphomas in C57 BL/Ka mice after 6-12 months. A graft of normal congenic bone marrow cells immediately after the last irradiation prevents the development of lymphomas by inducing the disappearance of preleukemic cells. When such a graft is performed one month later, it does not inhibit the emergence of tumors. It could be because, one month after irradiation, preleukemic cells become insensitive to the effects of the grafted bone marrow on their leukemogenic potential. To check this hypothesis, we have investigated the capacity of grafted bone marrow cells to prevent the development of lymphomas in mice inoculated with radiation-induced preleukemic cells collected at several time intervals after the completion of the radiation regimen. It was found that the bone marrow graft reduced the incidence of thymic lymphoma at day 2 (10 vs. 43%; p < 0.01) and 10 (39 vs. 86%; p < 0.01) but not at day 15 (64 vs. 80%; NS) or 30 (93 vs. 82%; NS). The inefficacy of the marrow graft was not associated with proliferation of the inoculate in the recipient thymus nor with inhibition by preleukemic cells of thymic repopulation by bone marrow precursors. The data provide evidence that preleukemic cells undergo intrinsic changes which are reflected by the acquisition of resistance to bone marrow grafts.

Three-dimensional molecular models of the hMC1R melanocortin receptor: complexes with melanotropin peptide agonists.

Three-dimensional molecular models of the human melanocortin receptor (hMC1R) have been developed based upon the electron cryo-microscopic structure of bacteriorhodopsin and the electron density footprint of bovine rhodopsin. alpha-Melanocyte-stimulating hormone, Ac-Ser-Tyr-Ser-Met4-Glu-His-Phe7-Arg-Trp-Gly-Lys-Pro-Val-NH2 (alpha-MSH, alpha-melanotropin), and the superpotent, prolonged acting agonists, Ac-Ser-Tyr-Ser-Nle4-Glu-His-DPhe7-Arg-Trp-Gly-Lys-Pro-Val-NH2 (NDP-MSH) and Ac-Nle4-c[Asp5-His6-DPhe7-Arg8-Trp9-Lys10]-NH2 (MTII), have been modeled into the proposed binding sites with specific ligand-receptor interactions identified. The melanotropin sidechain pharmacophores, DPhe7 and Trp9, are proposed to interact with a hydrophobic network of receptor aromatic residues in transmembrane regions 4, 5, 6, and 7. In addition, a hydrophilic network involving the ligand Arg8 and polar receptor residues located in transmembrane regions 2 and 3 were identified. Biological studies on alpha-MSH, NDP-MSH, MTII, and related peptides have been correlated with the proposed hMC1R model in terms of agonism, affinity, and prolongation. Finally, limited MC1R mutagenesis studies comparing alpha-MSH and NDP-MSH are interpreted within the context of the proposed hMC1R models.

In vivo expression of interleukin-1 beta (IL-1 beta), IL-2, IL-4, IL-6, tumour necrosis factor-alpha and interferon-gamma in the fetal murine thymus.

Cytokines are known to play a role in T-cell lymphopoiesis as potent growth or differentiation factors, but many experiments focusing on their role in the thymus have been conducted only in vitro. We have thus used frozen sections obtained from fetal thymuses of normal C57BL 6 mice to investigate by immunohistochemistry the presence of interleukin-1 beta (I4-1 beta), IL-2. IL-4. IL-6. interferon-7 (IFN-7) and tumour necrosis facor-alpha (TNF-alpha). The results reveal that apart from IL-2, which was not detected, all these cytokines display a time-dependent expression pattern in the normal fetal thymus. First, production of IL-4, IL-6 and TNF-alpha is detected around days 13 14; this is followed by a second wave on days 16 17, with a production of IL-1 beta, IL-4 and IL-6, and finally, just before birth (day 19), by a third wave of IL-1 beta, IL-4, IL-6, IFN-7 and TNF-alpha production. This supports the hypothesis that cytokines play a rote in T-cell lymphopoiesis.