Lawrence C. Fritz

A high-affinity conformation of Hsp90 confers tumour selectivity on Hsp90 inhibitors.

Heat shock protein 90 (Hsp90) is a molecular chaperone that plays a key role in the conformational maturation of oncogenic signalling proteins, including HER-2/ErbB2, Akt, Raf-1, Bcr-Abl and mutated p53. Hsp90 inhibitors bind to Hsp90, and induce the proteasomal degradation of Hsp90 client proteins. Although Hsp90 is highly expressed in most cells, Hsp90 inhibitors selectively kill cancer cells compared to normal cells, and the Hsp90 inhibitor 17-allylaminogeldanamycin (17-AAG) is currently in phase I clinical trials. However, the molecular basis of the tumour selectivity of Hsp90 inhibitors is unknown. Here we report that Hsp90 derived from tumour cells has a 100-fold higher binding affinity for 17-AAG than does Hsp90 from normal cells. Tumour Hsp90 is present entirely in multi-chaperone complexes with high ATPase activity, whereas Hsp90 from normal tissues is in a latent, uncomplexed state. In vitro reconstitution of chaperone complexes with Hsp90 resulted in increased binding affinity to 17-AAG, and increased ATPase activity. These results suggest that tumour cells contain Hsp90 complexes in an activated, high-affinity conformation that facilitates malignant progression, and that may represent a unique target for cancer therapeutics.

Rationally designed high-affinity 2-amino-6-halopurine heat shock protein 90 inhibitors that exhibit potent antitumor activity

Heat shock protein 90 (Hsp90) is a molecular chaperone protein implicated in stabilizing the conformation and maintaining the function of many cell-signaling proteins. Many oncogenic proteins are more dependent on Hsp90 in maintaining their conformation, stability, and maturation than their normal counterparts. Furthermore, recent data show that Hsp90 exists in an activated form in malignant cells but in a latent inactive form in normal tissues, suggesting that inhibitors selective for the activated form could provide a high therapeutic index. Hence, Hsp90 is emerging as an exciting new target for the treatment of cancer. We now report on a novel series of 2-amino-6-halopurine Hsp90 inhibitors exemplified by 2-amino-6-chloro-9-(4-iodo-3,5-dimethylpyridin-2-ylmethyl)purine (30). These highly potent inhibitors (IC50 of 30 = 0.009 microM in a HER-2 degradation assay) also display excellent antiproliferative activity against various tumor cell lines (IC50 of 30 = 0.03 microM in MCF7 cells). Moreover, this class of inhibitors shows higher affinity for the activated form of Hsp90 compared to our earlier 8-sulfanylpurine Hsp90 inhibitor series. When administered orally to mice, these compounds exhibited potent tumor growth inhibition (>80%) in an N87 xenograft model, similar to that observed with 17-allylamino-17-desmethoxygeldanamycin (17-AAG), which is a compound currently in phase I/II clinical trials.

Studies on the Mechanism of Action of Avermectin B1a: Stimulation of Release of γ‐Aminobutyric Acid from Brain Synaptosomes

Avermectin B1a, a novel macrocyclic lactone antiparasitic agent, causes a marked and sustained increase of γ‐aminobutyric acid release from rat brain synaptosomes. A concentration of 8‐10 μM of avermectin B1a produced the maximal effect (310 ± 30% of the control), while the half‐maximal level was achieved at 2‐3 μM. The drug also stimulated γ‐aminobutyric acid release (251 ± 11% of the basal level) from synaptosomes in calcium‐free medium, which was 28 ± 4% lower than that in the 1.8 mm‐Ca2+ medium. The compound did not, however, affect the synaptosomal release of glutamate. At the lobster neuromuscular junction, avermectin B1a reduced the input resistance of muscle fibers in control Ringers solution as well as in Ringers solution in which Co2+ was substituted for Ca2+. This observation is in accord with the Ca2+ independent stimulation of γ‐aminobutyric acid release seen with synaptosomes. A good correlation between antiparasitic activity and γ‐aminobutyric acid‐releasing activity has been found among various derivatives of avermectin B1a, which suggests that the ability of the drug to release this neurotransmitter may be the basis of its antiparasitic action.

The heat-shock protein 90 inhibitor 17-allylamino-17-demethoxygeldanamycin suppresses glial inflammatory responses and ameliorates experimental autoimmune encephalomyelitis

The heat‐shock response (HSR), a highly conserved cellular response, is characterized by rapid expression of heat‐shock proteins (HSPs), and inhibition of other synthetic activities. The HSR can attenuate inflammatory responses, via suppression of transcription factor activation. A HSR can be induced pharmacologically by HSP90 inhibitors, through activation of the transcription factor Heat Shock Factor 1 (HSF1). In the present study we characterized the effects of 17‐allylamino‐17‐demethoxygeldanamycin (17‐AAG), a less toxic derivative of the naturally occurring HSP90 inhibitor geldanamycin, on glial inflammatory responses and the development of experimental autoimmune encephalomyelitis. In primary enriched glial cultures, 17‐AAG dose dependently reduced lipopolysaccharide‐dependent expression and activity of inducible nitric oxide synthase, attenuated interleukin (IL)‐1β expression and release, increased inhibitor of κB protein levels, and induced HSP70 expression. 17‐AAG administration to mice immunized with myelin oligodendrocyte glycoprotein peptide prevented disease onset when given at an early time, and reduced clinical symptoms when given during ongoing disease. T cells from treated mice showed a reduced response to immunogen re‐stimulation, and 17‐AAG reduced CD3‐ and CD28‐dependent IL‐2 production. Together, these data suggest that HSP90 inhibitors could represent a new approach for therapeutic intervention in autoimmune diseases such as multiple sclerosis.

Analysis and quantitation of the beta-amyloid precursor protein in the cerebrospinal fluid of Alzheimer's disease patients with a monoclonal antibody-based immunoassay.

Abstract: One of the major clinical findings in Alzheimers disease (AD) is the formation of deposits of β‐amyloid protein in amyloid plaques, derived from the β‐amyloid precursor protein (β‐APP). To determine the possible use of β‐APP as a diagnostic marker for AD in CSF, a monoclonal antibody‐based immunoassay specific for this protein was developed. The assay does not differentiate between β‐APP695 and β‐APP751 forms but does preferentially recognize β‐APP751 complexed with a protease. Of the two sets of CSF samples tested, one set, obtained from living patients, gave a slightly lower level of β‐APP in AD and Parkinsons disease patients relative to controls, whereas the other set, composed of postmortem samples, showed no significant differences between the AD and control groups.

The effect of diamide on transmitter release and on synaptic vesicle population at vertebrate synapses

Diamide, a sulfhydryl-oxidizing agent, has previously been shown to cause acetylcholine release in two preparations in the absence of added Ca2+. Similarities in action between diamide and alpha-latrotoxin, a component of black widow spider venom which causes transmitter release with no added Ca2+, and which seems to require a disulfide bond for its action, led us to study further the transmitter-releasing properties of diamide. In rat cerebral cortical slices we show that diamide, like alpha-latrotoxin, released all transmitters studied; GABA, acetylcholine, norepinephrine and dopamine. The response reached a peak after a delay (5-15 min), in contrast to the much faster release evoked by high K+ (within 3 min). Diamide-induced GABA release was found to occur equally well in the absence of added Ca2+, and was blocked when diamide was reduced prior to addition. Our ultrastructural studies of the frog neuromuscular junction showed that whereas alpha-latrotoxin caused the elimination of synaptic vesicles, diamide did not. Dithiothreitol, a disulfide-reducing agent, also caused GABA release, but this effect was Ca2+-dependent, blocked by high Mg2+, and occurred without delay. These observations comparing the 3 transmitter-releasing agents have further delineated the sulfhydryl/disulfide-group involvement in transmitter release and have demonstrated that dithiothreitol is operating at a different site from either alpha-latrotoxin or diamide.