Identification of a second aryl phosphate-binding site in protein-tyrosine phosphatase 1B: A paradigm for inhibitor design

The structure of the catalytically inactive mutant (C215S) of the human protein-tyrosine phosphatase 1B (PTP1B) has been solved to high resolution in two complexes. In the first, crystals were grown in the presence of bis-(para-phosphophenyl) methane (BPPM), a synthetic high-affinity low-molecular weight nonpeptidic substrate (Km = 16 μM), and the structure was refined to an R-factor of 18.2% at 1.9 Å resolution. In the second, crystals were grown in a saturating concentration of phosphotyrosine (pTyr), and the structure was refined to an R-factor of 18.1% at 1.85 Å. Difference Fourier maps showed that BPPM binds PTP1B in two mutually exclusive modes, one in which it occupies the canonical pTyr-binding site (the active site), and another in which a phosphophenyl moiety interacts with a set of residues not previously observed to bind aryl phosphates. The identification of a second pTyr molecule at the same site in the PTP1B/C215S–pTyr complex confirms that these residues constitute a low-affinity noncatalytic aryl phosphate-binding site. Identification of a second aryl phosphate binding site adjacent to the active site provides a paradigm for the design of tight-binding, highly specific PTP1B inhibitors that can span both the active site and the adjacent noncatalytic site. This design can be achieved by tethering together two small ligands that are individually targeted to the active site and the proximal noncatalytic site.

Synthesis and characterization of a novel retinylamine analog inhibitor of constitutively active rhodopsin mutants found in patients with autosomal dominant retinitis pigmentosa

Two different mutations of the active-site Lys-296 in rhodopsin, K296E and K296M, have been found to cause autosomal dominant retinitis pigmentosa (ADRP). In vitro studies have shown that both mutations result in constitutive activation of the protein, suggesting that the activated state of the receptor may be responsible for retinal degeneration in patients with these mutations. Previous work has highlighted the potential of retinylamine analogs as active-site directed inactivators of constitutively active mutants of rhodopsin with the idea that these or related compounds might be used therapeutically for cases of ADRP involving mutations of the active-site Lys. Unfortunately, however, amine derivatives of 11-cis-retinal, although highly effective against a K296G mutant of rhodopsin, were without affect on the two naturally occurring ADRP mutants, presumably because of the greater steric bulk of Glu and Met side chains in comparison to Gly. For this reason we synthesized a retinylamine analog one carbon shorter than the parent 11-cis-retinal and show that this compound is indeed an effective inhibitor of both the K296E and K296M mutants. The 11-cis C19 retinylamine analog 1 inhibits constitutive activation of transducin by these mutants and their constitutive phosphorylation by rhodopsin kinase, and it does so in the presence of continuous illumination from room lights.

An amyloid-forming peptide from the yeast prion Sup35 reveals a dehydrated β-sheet structure for amyloid

X-ray diffraction and other biophysical tools reveal features of the atomic structure of an amyloid-like crystal. Sup35, a prion-like protein in yeast, forms fibrillar amyloid assemblies intrinsic to its prion function. We have identified a polar peptide from the N-terminal prion-determining domain of Sup35 that exhibits the amyloid properties of full-length Sup35, including cooperative kinetics of aggregation, fibril formation, binding of the dye Congo red, and the characteristic cross-β x-ray diffraction pattern. Microcrystals of this peptide also share the principal properties of the fibrillar amyloid, including a highly stable, β-sheet-rich structure and the binding of Congo red. The x-ray powder pattern of the microcrystals, extending to 0.9-Å resolution, yields the unit cell dimensions of the well-ordered structure. These dimensions restrict possible atomic models of this amyloid-like structure and demonstrate that it forms packed, parallel-stranded β-sheets. The unusually high density of the crystals shows that the packed β-sheets are dehydrated, despite the polar character of the side chains. These results suggest that amyloid is a highly intermolecularly bonded, dehydrated array of densely packed β-sheets. This dry β-sheet could form as Sup35 partially unfolds to expose the peptide, permitting it to hydrogen-bond to the same peptide of other Sup35 molecules. The implication is that amyloid-forming units may be short segments of proteins, exposed for interactions by partial unfolding.

Juglone, an inhibitor of the peptidyl-prolyl isomerase Pin1, also directly blocks transcription

The C-terminal domain (CTD) of the large subunit of RNA polymerase II plays a role in transcription and RNA processing. Yeast ESS1, a peptidyl-prolyl cis/trans isomerase, is involved in RNA processing and can associate with the CTD. Using several types of assays we could not find any evidence of an effect of Pin1, the human homolog of ESS1, on transcription by RNA polymerase II in vitro or on the expression of a reporter gene in vivo. However, an inhibitor of Pin1, 5-hydroxy-1,4-naphthoquinone (juglone), blocked transcription by RNA polymerase II. Unlike N-ethylmaleimide, which inhibited all phases of transcription by RNA polymerase II, juglone disrupted the formation of functional preinitiation complexes by modifying sulfhydryl groups but did not have any significant effect on either initiation or elongation. Both RNA polymerases I and III, but not T7 RNA polymerase, were inhibited by juglone. The primary target of juglone has not been unambiguously identified, although a site on the polymerase itself is suggested by inhibition of RNA polymerase II during factor-independent transcription of single-stranded DNA. Because of its unique inhibitory properties juglone should prove useful in studying transcription in vitro.

Male fertility defects in mice lacking the serine protease inhibitor protease nexin-1

Understanding infertility and sterility requires knowledge of the molecular mechanisms underlying sexual reproduction. We have found that male mice deficient for the gene encoding the protease inhibitor protease nexin-1 (PN-1) show a marked impairment in fertility from the onset of sexual maturity. Absence of PN-1 results in altered semen protein composition, which leads to inadequate semen coagulation and deficient vaginal plug formation upon copulation. Progressive morphological changes of the seminal vesicles also are observed. Consistent with these findings, abnormal PN-1 expression was found in the semen of men displaying seminal dysfunction. The data demonstrate that the level of extracellular proteolytic activity is a critical element in controlling male fertility.

Local gene transfer of tissue factor pathway inhibitor regulates intimal hyperplasia in atherosclerotic arteries

Tissue factor (TF), the initiator of blood coagulation and thrombosis, is up-regulated after vascular injury and in atherosclerotic states. Systemic administration of recombinant TF pathway inhibitor (TFPI) has been reported to decrease intimal hyperplasia after vascular injury and also to suppress systemic mechanisms of blood coagulation and thrombosis. Here we report that, in heritable hyperlipidemic Watanabe rabbits, adenoviral gene transfer of TFPI to balloon-injured atherosclerotic arteries reduced the extent of intimal hyperplasia by 43% (P < 0.05) compared with a control vector used at identical titer (1 × 1010 plaque-forming units/ml). Platelet aggregation and coagulation studies performed 7 days after local gene transfer of TFPI failed to show any impairment in systemic hemostasis. At time of sacrifice, 4 weeks after vascular injury, the 10 Ad-TFPI treated carotid arteries were free of thrombi, whereas two control-treated arteries were occluded (P, not significant). These findings suggest that TFPI overexpressed in atherosclerotic arteries can regulate hyperplastic response to injury in the absence of changes in the hemostatic system, establishing a role for local TF regulation as target for gene transfer-based antirestenosis therapies.

Suppression of ras-mediated transformation and inhibition of tumor growth and angiogenesis by anthrax lethal factor, a proteolytic inhibitor of multiple MEK pathways

Lethal factor is a protease, one component of Bacillus anthracis exotoxin, which cleaves many of the mitogen-activated protein kinase kinases (MEKs). Given the importance of MEK signaling in tumorigenesis, we assessed the effects of anthrax lethal toxin (LeTx) on tumor cells. LeTx was very effective in inhibiting mitogen-activated protein kinase activation in V12 H-ras-transformed NIH 3T3 cells. In vitro, treatment of transformed cells with LeTx caused them to revert to a nontransformed morphology, and inhibited their abilities to form colonies in soft agar and to invade Matrigel without markedly affecting cell proliferation. In vivo, LeTx inhibited growth of ras-transformed cells implanted in athymic nude mice (in some cases causing tumor regression) at concentrations that caused no apparent animal toxicity. Unexpectedly, LeTx also greatly decreased tumor neovascularization. These results demonstrate that LeTx potently inhibits ras-mediated tumor growth and is a potential antitumor therapeutic.

Protein folding from a highly disordered denatured state: The folding pathway of chymotrypsin inhibitor 2 at atomic resolution

Previous experimental and theoretical studies have produced high-resolution descriptions of the native and folding transition states of chymotrypsin inhibitor 2 (CI2). In similar fashion, here we use a combination of NMR experiments and molecular dynamics simulations to examine the conformations populated by CI2 in the denatured state. The denatured state is highly unfolded, but there is some residual native helical structure along with hydrophobic clustering in the center of the chain. The lack of persistent nonnative structure in the denatured state reduces barriers that must be overcome, leading to fast folding through a nucleation–condensation mechanism. With the characterization of the denatured state, we have now completed our description of the folding/unfolding pathway of CI2 at atomic resolution.

Immucillin H, a powerful transition-state analog inhibitor of purine nucleoside phosphorylase, selectively inhibits human T lymphocytes

Transition-state theory has led to the design of Immucillin-H (Imm-H), a picomolar inhibitor of purine nucleoside phosphorylase (PNP). In humans, PNP is the only route for degradation of deoxyguanosine, and genetic deficiency of this enzyme leads to profound T cell-mediated immunosuppression. This study reports the biological effects and mechanism of action of Imm-H on malignant T cell lines and on normal activated human peripheral T cells. Imm-H inhibits the growth of malignant T cell leukemia lines with the induction of apoptosis. Imm-H also inhibits activated normal human T cells after antigenic stimulation in vitro. However, Imm-H did not inhibit malignant B cells, colon cancer cell lines, or normal human nonstimulated T cells, demonstrating the selective activity of Imm-H. The effects on leukemia cells were mediated by the cellular phosphorylation of deoxyguanosine and the accumulation of dGTP, an inhibitor of ribonucleotide diphosphate reductase. Cells were protected from the toxic effects of Imm-H when deoxyguanosine was absent or when deoxycytidine was present. Guanosine incorporation into nucleic acids was selectively blocked by Imm-H with no effect on guanine, adenine, adenosine, or deoxycytidine incorporation. Imm-H may have clinical potential for treatment of human T cell leukemia and lymphoma and for other diseases characterized by abnormal activation of T lymphocytes. The design of Imm-H from an enzymatic transition-state analysis exemplifies a powerful approach for developing high-affinity enzyme inhibitors with pharmacologic activity.

β-Amyloid peptide blocks the response of α7-containing nicotinic receptors on hippocampal neurons

Alzheimer's disease produces a devastating decline in mental function, with profound effects on learning and memory. Early consequences of the disease include the specific loss of cholinergic neurons in brain, diminished cholinergic signaling, and the accumulation of β-amyloid peptide in neuritic plaques. Of the nicotinic acetylcholine receptors at risk, the most critical may be those containing the α7 gene product (α7-nAChRs), because they are widespread, have a high relative permeability to calcium, and regulate numerous cellular events in the nervous system. With the use of whole-cell patch–clamp recording we show here that nanomolar concentrations of β-amyloid peptides specifically and reversibly block α7-nAChRs on rat hippocampal neurons in culture. The block is noncompetitive, voltage-independent, and use-independent and is mediated through the N-terminal extracellular domain of the receptor. It does not appear to require either calcium influx or G protein activation. β-Amyloid blockade is likely to be a common feature of α7-nAChRs because it applies to the receptors at both somato-dendritic and presynaptic locations on rat hippocampal neurons and extends to homologous receptors on chick ciliary ganglion neurons as well. Because α7-nAChRs in the central nervous system are thought to have numerous functions and recently have been implicated in learning and memory, impaired receptor function in this case may contribute to cognitive deficits associated with Alzheimer's disease.

Commitment to natural killer cells requires the helix–loop–helix inhibitor Id2

We have previously described how T and natural killer (NK) lineage commitment proceeds from common T/NK progenitors (p-T/NK) in the murine fetal thymus (FT), with the use of a clonal assay system capable of discriminating p-T/NK from unipotent T or NK lineage-committed progenitors (p-T and p-NK, respectively). The molecular mechanisms controlling the commitment processes, however, are yet to be defined. In this study, we investigated the progenitor activity of FT cells from Id2−/− mice that exhibit defective NK cell development. In the Id2−/− FT, NK cells were greatly reduced, and a cell population that exclusively contains p-NK in the wild-type thymus was completely missing. Id2−/− FT progenitors were unable to differentiate into NK cells in IL-2-supplemented-FT organ culture. Single progenitor analysis demonstrated that all Id2−/− fetal thymic progenitors are destined for the T cell lineage, whereas progenitors for T/NK, T, and NK cell lineages were found in the control. Interestingly, the total progenitor number was similar between Id2−/− and Id2+/+ embryos analyzed. Expression of Id2 was correlated with p-NK activity. Our results suggest that Id2 is indispensable in thymic NK cell development, where it most probably restricts bipotent T/NK progenitors to the NK cell lineage.

Low cholesterol stimulates the nonamyloidogenic pathway by its effect on the α-secretase ADAM 10

Biochemical, epidemiological, and genetic findings demonstrate a link between cholesterol levels, processing of the amyloid precursor protein (APP), and Alzheimer's disease. In the present report, we identify the α-secretase ADAM 10 (a disintegrin and metalloprotease) as a major target of the cholesterol effects on APP metabolism. Treatment of various peripheral and neural cell lines with either the cholesterol-extracting agent methyl-β-cyclodextrin or the hydroxymethyl glutaryl-CoA reductase inhibitor lovastatin resulted in a drastic increase of secreted α-secretase cleaved soluble APP. This strong stimulatory effect was in the range obtained with phorbol esters and was further increased in cells overexpressing ADAM 10. In cells overexpressing APP, the increase of α-secretase activity resulted in a decreased secretion of Aβ peptides. Several mechanisms were elucidated as being the basis of enhanced α-secretase activity: increased membrane fluidity and impaired internalization of APP were responsible for the effect observed with methyl-β-cyclodextrin; treatment with lovastatin resulted in higher expression of the α-secretase ADAM 10. Our results demonstrate that cholesterol reduction promotes the nonamyloidogenic α-secretase pathway and the formation of neuroprotective α-secretase cleaved soluble APP by several mechanisms and suggest approaches to prevention of or therapy for Alzheimer's disease.