Among redundant enolases, the largest fragment had reduced volume

Among redundant enolases, the largest fragment had reduced volume in 4 out of 5 cases. The largest fragment between redundant kinases had reduced volume in 10 out of 10 cases. Because the sequences modeled were selleck inhibitor very similar, the cavities modeled were also very similar and they exhibited Inhibitors,Modulators,Libraries fragments with the template cavity of a similar size. When sequence identity is very high, remodeling frequently enhanced cavity similarity. To evaluate how remodeling can assist in the detec tion of cavities with similar binding preferences, we built statistical models of fragment volume between enolases and tyrosine kinases with similar binding pre ferences. Before remodeling, the volume of the largest fragment between enolase cavities and their template was statistically significant in 40% of the data set.

These cavities would have been incorrectly classified as having different binding preferences. After remodeling, the volume of the largest fragment was statistically signifi Inhibitors,Modulators,Libraries cant in 20% of the dataset. Among tyrosine kinases, the volume of the largest fragment, between the tyrosine kinase cavities and their template, was statistically signif icant in 86% of the dataset. After remodeling, Inhibitors,Modulators,Libraries the volume of the largest fragment was statistically signifi cant in just 7 Inhibitors,Modulators,Libraries of the dataset. These results demonstrate that remodeling can reduce geometric dissimilarities related to conformational change that can that can cause similar binding sites to appear different and be incorrectly classified.

Simple remodeling on proteins with heterogeneous binding preferences Simple remodeling of proteins with similar binding pre ferences may enhance the similarity of their binding sites and mitigate variations caused by conformational change, Inhibitors,Modulators,Libraries but in the context of comparing proteins with similar folds, simple remodeling could make proteins with different binding preferences appear too similar. To evaluate this possibility, we remodeled the members of the heterogeneous enolase superfamily against muco nate cycloisomerase from Sinorhizobium meliloti and Thermotoga maritima. When modeling 2pgw as the template, the volume of largest fragment was greater after remodeling in 7 out of the 9 models and nearly identical in the remaining two. When using 2zad as a template, the volume of the largest fragment was greater in 7 out of the 9 models. These volumes are plotted in Figure 4. All of the largest fragments were statistically significant, and thus pointing to differences in binding preferences, relative to fragments between enolase worldwide distributors proteins with similar binding preferences. We also remodeled the tyrosine kinases with large gatekeeper residues using homo sapiens abelson kinase as a template.

Nonetheless, our sys tematic approach did indicate that truncatio

Nonetheless, our sys tematic approach did indicate that truncations rather than mutations were more effective for MK2 crystallization. The exact position selleck chemicals Imatinib Mesylate of the MK2 N terminus is more impor tant than is the C terminus, and the activation loop could not be deleted. Surface arginine and glutamate residues drive crystallization more strongly than do surface lysine residues prevent it. The pseudoactivation constructs were helpful, likely due to slight modulation of protein surface properties rather than by producing a different protein conformation. These unusual MK2 properties result in many closely related crystal forms. Additional surface mutants would seem to be required to drive MK2 into truly differ ent crystal forms that can be produced Inhibitors,Modulators,Libraries under low ionic strength conditions rather than in high salt.

Conclusion The systematic methods for the design, production Inhibitors,Modulators,Libraries and evaluation of MK2 protein constructs presented here allowed us to reproducibly obtain suitable crystals such that structure based drug design could proceed. Our methods are robust and Inhibitors,Modulators,Libraries allowed rapid evaluation of about fifty constructs. This rapidity enabled the scale up produc tion of selected MK2 constructs in multi milligram quan tities proteins for structural studies. Using rational site directed mutagenesis and in house customized crystalli zation screens, we were able to identify several novel crystallization Inhibitors,Modulators,Libraries conditions. Combined with varia tion of other parameters, such as surface side chain entropy and activation state, these high through put techniques produced five new MK2 crystal forms, most with improved diffraction characteristics.

One such crystal form, grown with a MK2 phosphoryla tion site pseudoactivation mutant, was used to solve our first MK2 lead inhibitor complex. Several key lessons were learned from this exercise. First, setting reasonable criteria for construct performance Inhibitors,Modulators,Libraries and prioritization is essential to identify constructs suitable for further evaluation and possible scale up. In the case of MK2, most constructs gave satisfactory performance in expression and purification. Choices were necessary, how ever. we prioritized higher yielding, more soluble con structs for scale up and more extensive crystallization screening. Implicitly, we assumed that con structs that expressed or purified poorly were less likely to crystallize well. Our assumption appears to be supported by the expression yield, protein melting point, and crystal lization data of Malawski et al. Second, a systematic crystallization screen was required to identify crystallization condi tions in a robust manner. Indeed, the interplay between multiple constructs and multiple, customized crystalliza tion solutions likely contributed greatly to our success.

As a widely used tool for microarray data analysis, dChip can dis

As a widely used tool for microarray data analysis, dChip can display and normalize CEL files with a model based approach. For a given group of CEL files, dChip can be used to calculate the model based expression values and make the qualitative detection calls for each array. The detection call provides a statistical selleck chemical assessment about whether the perfect matches show significantly more hybridization signal than the corresponding mis matches in a probe set. Since the detection call and expression level are computed in different ways, a gene transcript with an Absent call may still be given an expression value. from different studies, it might not be applied to an expression dataset with various tissue types.

Owing to the biological variation of gene expression across different tis sues, a baseline array should be used to normalize the microarray profiles of each tissue type. Finally, the normalized microarray profiles were inte grated into a single dataset after outlier array exclusion and global median transformation. When fitting the sta tistical model to a probe set, dChip used an outlier Inhibitors,Modulators,Libraries detec tion algorithm to identify array outliers whose response pattern for the probe set was significantly different from the consensus probe response pattern in the other arrays. After the model was Inhibitors,Modulators,Libraries fitted for all probe sets, the per centage of probe Inhibitors,Modulators,Libraries sets detected as array outliers was cal culated for each array. If the percentage exceeded 15%, the array was discarded as an outlier array. In this study, only 62 outlier arrays were detected for all the 3,030 selected expression profiles.

Global median transformation was then applied to the remaining pro files. Each expression value in a profile was divided by the profiles median value. The transformation was necessary because the expression profiles from different normalization groups often had different median values. Thus, the integrated dataset had 2,968 expression profiles Inhibitors,Modulators,Libraries with the same median value. Genome wide identification of tissue selective genes In this study, a new computational method has been designed to analyze the integrated microarray data for identifying tissue selective genes, which Inhibitors,Modulators,Libraries refers to the genes specifically or preferentially expressed in a parti cular tissue. The computational task is not trivial for the following reasons.

First, the expression profiles have been compiled from various studies, in which tissues at different ages and in different conditions were used for microarray Trichostatin A (TSA) profiling. Thus, the microarray profiles of the same tissue type should not be considered as biolo gical replicates. Second, some tissue selective genes can be expressed at certain developmental stages or in speci fic conditions, and their expression may not be consis tently detected in all the microarray profiles of a tissue type. Third, microarray data are inherently noisy.

P 0 05 were considered statistically

P 0. 05 were considered statistically selleckchem significant. Real time quantitative Inhibitors,Modulators,Libraries RT PCR validation of mRNA and miRNA The data for mRNA and miRNA were selectively corro borated with real time PCR to ascertain their expression trends. For mRNA, 5ng total RNA was reverse tran scribed using oligo d and Superscript III followed by RNase H treatment, per manufacturers instructions. PCR primers were designed for all the 11 genes selected on the basis of the microarray data as well as for the control genes, using the online software Primer 3. All primer pairs were optimized to ensure the specific amplification and the absence of any primer dimer. Quantitative PCR standard curves were set up for all. The cDNA was then subjected to real time quantitative PCR with defined pri mers and SYBR Green using Mx3000p Stratagene real time thermal cycler.

The data were analysed using the MxPro QPCR software version 4. 0. 1. For miRNA, expression levels of six DE miRNAs were validated by quantitative real time RT PCR using Inhibitors,Modulators,Libraries the Qiagen miScript PCR system according to the manufactures protocol. Hs RNU6B 3 was used as the endogenous control to normalize the data. All the experiements were performed in duplicate and relative expression levels of these mRNAs miRNAs were determined by the 2 Ct method. The data then were further analysed by Student t test to check the statistical significance between HAD and HIV non dementia patients brains. Transfection of microRNA mimic SH SY5Y cultures were maintained as Inhibitors,Modulators,Libraries confluent mono layers at 37 C with 5% CO2 and 90% humidity in SH SY5Y media foetal calf serum, 20 mM HEPES, 2 mM L glutamine.

For differ entiation cells were seeded at 4 �� 104 cells cm2 and trea ted with all trans retinoic acid media for five days, followed by treatment in brain derived Inhibitors,Modulators,Libraries neurotrophic factor media for Inhibitors,Modulators,Libraries three days. Cells were then harvested and nucleofected using Amaxa Nucleofector Kit V according to manufacturers instructions. Each nucleofection contained 4 �� 106 cells and 0. 1 nmol miR 137 or mirVanaTM miRNA mimic Negative Control 1, with experiments performed in duplicate. Nucleofected cells were seeded at 5 �� 104 cm2 in BDNF media and grown for 24 hrs before being harvested with TRIzol and RNA isolated as described. Functional validation of proteins using western blot Western blot was employed to validate part of the microarray data.

4 HAD patients and 4 HIV non dementia patients brain samples were used for valid ation of the microarray study by western blot analysis. Total cellular proteins were extracted as described be fore. 40 ug proteins were separated by 12% SDS polyacrylamide gels and then transferred to PVDF membranes or nitrocellulose mem branes using Bio Rad selleck screening library apparatus. Membranes were blocked in 5% skim milk powder or 5% BSA in tris buffered saline for 1 hour at room temperature. Following that, they were incubated for 2 hours at room temperature with each of the fol lowing primary antibodies, Rabbit anti MEK2 and JNK1.

We found that the mRNA expression levels of all members of the TN

We found that the mRNA expression levels of all members of the TNFAIP1 POLDIP2 SFGM were significantly correlated Perifosine with those Inhibitors,Modulators,Libraries of at least two or more members of the ERBB2 CR. ERBB2 and C17orf37 were significantly correlated with almost all members of the TNFAIP1 POLDIP2 SFGM in both cohorts. Similarly, in 38 breast cancer cell lines, the expression of the ERBB2 gene was Inhibitors,Modulators,Libraries significantly correlated with the expression of all the 5 members of the TNFAIP1 POLDIP2 SFGM, although the total num ber of observed significant correlations was less than for the breast cancer patients. Therefore, the expression profiles of the genes of the TNFAIP1 POLDIP2 SFGM and the ERBB2 CR are correlated in breast cancer and this fact could probably be explained by co amplification of their genomic regions.

However, alternative mechanisms could be considered, including similar epigenetic modifications of chromatin as well as common upstream regulatory transcription factors. Genes of the TNFAIP1 POLDIP2 SFGM are co regulated not only through changes in DNA copy number but also by transcription activation and chromatin Inhibitors,Modulators,Libraries remodelling Figure 2B illustrates several findings that could indicate histone modification as Inhibitors,Modulators,Libraries a possible mechanism of the observed transcriptional co regulatory pattern of the TNFAIP1 POLDIP2 SFGM genes. Custom tracks in the UCSC Genome Browser for trimethylated histones H3K4me3 and H3K27me3 modification confirmed the transcriptional activation of the genes involved in the TNFAIP1 POLDIP2 SFGM. All three CpG rich putative promoters in the TNFAIP1 POLDIP2 SFGM showed clear signal for H3K4me3 as well as a lack of signal for H3K27me3.

Nevertheless, putative promoters for the neighbouring genes SEBOX, VTN, and SARM did not show any signal of H3K4me3. A similar situation is observed with the GIS Chip PET track of the UCSC Browser. A strong signal for H3K4me3 is observed in all three putative promoter regions Inhibitors,Modulators,Libraries of the TNFAIP1 POLDIP2 SFGM and only weak signal of H3K27me3 is detected for the TMEM97 and TMEM199 POLDIP2 putative promoters. Alternatively, the putative promoter for the SEBOX gene does not show any signal for both H3K4me3 and H3K27me3. the regulatory region of the VTN and SARM1 genes reveals moderate signal for H3K4me3 and H3K27me3 of the same intensity. Additional custom tracks in the UCSC selleck inhibitor browser for STAT1 binding in HeLa S3 cells clearly demonstrate the presence of two functional STAT1 binding sites in the IFT20 TNFAIP1 bidirectional promoter. Moreover, upon stimulation by interferon gamma, the binding signal intensity for STAT1 increased at least seven fold. Recently, Liu at al. reported that another transcription factor, Sp1, is directly associated with the TNFAIP1 promoter in HeLa cells.

In our data set, we identified 164 genes that were sig nificantly

In our data set, we identified 164 genes that were sig nificantly up regulated selleck chemicals Sorafenib after NGF with drawal and the expression of 48 of these genes increased by more than 2 fold. Conversely, 379 genes were down regulated when the significance threshold was set at p 0. 01 and the expression of 86 of these genes decreased by 2 fold or more. We performed Gene Ontology analysis and functional enrichment ana lysis to identify specific annotations that were enriched following NGF withdrawal. Whilst this type of analysis depends upon a controlled vocabulary and therefore has its limitations, it also represents a powerful method for extracting potentially useful biological information from our gene expression data.

In an analysis of transcription dependent neuronal apoptosis Inhibitors,Modulators,Libraries proceeding via the mitochondrial pathway, functional categories such as intracellular signaling cas cades, transcription and mitochondrial changes might be expected to be enriched. Whilst these categories are indeed enriched after NGF withdrawal, other categories that contain genes which could suggest additional hypotheses about the mechanisms of neuronal death were also highlighted. The significance of the induction of ER stress associated genes, for example, may offer new insights into the cell death process, especially since a similar response was observed in cerebellar granule neurons undergoing apoptosis and experiments in other systems suggest a role for interactions between the mitochondria and the ER.

On the other hand, the down regulation of genes associated with cholesterol and fatty acid biosynthesis may be associated with an inhibition of cell growth since cholesterol and fatty acids are required for the synthesis Inhibitors,Modulators,Libraries of membranes. Cluster analysis allowed us to group the genes accord ing to their pattern of expression, especially in the pre sence of the MLK inhibitor, CEP 11004. The expression of many of the genes induced after NGF Inhibitors,Modulators,Libraries withdrawal is reduced by CEP 11004, suggesting that they may be tar gets of the MLK JNK c Jun pathway. This group includes c jun, dp5 and mkp1 whose promoters contain ATF sites that bind c Jun and which are important for their induction after NGF withdrawal. The induction of a few genes, such as egln3, is not affected by CEP 11004, suggesting that Inhibitors,Modulators,Libraries the tran scription of these genes may be regulated by other tran scription factors that are activated Inhibitors,Modulators,Libraries after NGF withdrawal, but not regulated by the JNK pathway, for example, FOXO3a or Myb.

Interestingly, CEP 11004 reverses the decrease in the level of expression of some of the genes that are down regulated after NGF withdrawal. Many of these genes encode proteins involved in fatty acid metabolism and cholesterol selleck chem inhibitor meta bolism, e. g. insig1, sqle, hmgcr, and hmgcs1, and their transcription is activated by sterol regulatory element binding proteins.

93, 0 92 and 0 91 for, respec tively, the SVM, PLS,

93, 0. 92 and 0. 91 for, respec tively, the SVM, PLS, better and k NN model. Interestingly, the models built on only 10% of the kinases also show good classification performance, the ROC areas being, respec tively, 0. 83, 0. 82, and 0. 79 for SVM, PLS, and k NN mod els. This finding Inhibitors,Modulators,Libraries indicates that even in the cases when quantitative models do not possess very high predictive ability in terms of P2, they may still be able to separate active and inactive kinase inhibitor combinations. Accordingly, our models should be useful for virtual large scale screening to select the promising objects prior to their experimental testing, while sorting away objects with a less probability of having the properties sought for in a development project.

Discussion Design of selective and multiselective medications requires Inhibitors,Modulators,Libraries understanding of the properties of the biological targets that distinguish the chosen target from numer ous similar anti targets encoded in the human genome. Contemporary drug design has to a large extent been focused to structure based methods where ligands are designed to fit into a binding pocket of the target. This requires knowledge of the exact 3 D structures of the tar gets and anti targets, which is a problem for protein kinases as X ray structures have been solved for only 124 human protein kinase domains. Proteochemometrics, on the other hand, has a distinct advantage when the studied proteins share the same structural organization since primary amino acid sequences can then be used without the need to have high resolution 3 D structures of the targets.

Proteoch emometrics has also the advantage that multiple targets and anti targets can be encompassed in one single model. Structural alignments of protein kinases have shown that they all contain universal conserved subdomains whereas their amino acid sequences still show quite notable varia tion. In fact, there is generally a much higher degree of conservation Inhibitors,Modulators,Libraries of the 3D structures among protein families than of their primary sequences. The average pair wise sequence identity over the kinase domains falls below 30%, and only a small fraction of residues are markedly Inhibitors,Modulators,Libraries conserved across the entire superfamily. Use of sequence Inhibitors,Modulators,Libraries derived descriptions can hence be con sidered to be a rational approach for kinase representa tion in multivariate modelling, stated that the sequence descriptions are made in such a way that they are relevant for the structural and functional organization of the kinases.

Descriptions can be derived based on kinase inhibitor Enzastaurin prior sequence alignments or in alignment independent ways, the latter approaches are advantageous for less similar sequences, when unambiguous alignments are impossible to obtain. In the first phase of this study we performed PCA and PLS DA, using one set of alignment based and five sets of alignment independent descriptors of protein kinase amino acid sequences.

CD4 CCR5 cells, whereas the infection with NL4 3 and NL4 3 based

CD4. CCR5 cells, whereas the infection with NL4 3 and NL4 3 based chi meric virus was performed in Sup T1 cells. The chi meric subtype C based strain carrying the pol gene fragment from NL4 3, 1084 polL, demonstrated productive infection with increasing p24CA level more info and a high cytopathic effect, in contrast to the control wild type 1084i isolate which resulted in poor viral replica tion and low cytopathogenicity. The NL polL viral strain containing subtype C pol frag ment in the subtype B backbone displayed an overall threefold lower p24CA level than the wild type NL4 3 isolate. The tested chimeric virus strains were not absolutely identical. The presence of 52 AA sequence of RT connection domain from NL4 3 in sub type C based virus 1084 pol could affect the overall level of virus replication.

However, the data that both subtype B and C based viruses containing the pol gene sequences from the subtype C displayed decreased repli cation level indicate that the subtype C Pol domains to poor viral replication Inhibitors,Modulators,Libraries regardless of the subtype B or C viral backbones. Taken together, our results Inhibitors,Modulators,Libraries indicate that the presence of the polymerase domain or the connection and RNase H domains of RT, integrase and Vif from subtype C iso lates correlates with slower or low efficiency replication of chimeric viruses. The presence of both the whole RT and integrase products of pol gene from subtype C iso lates in subtype B backbone virus strongly decreases the level of viral replication. This lower replica tion suggests that the polymerase and C terminal domains of RT, and likely the integrase protein all con tributed to the slower replicative kinetics of the subtype C viruses.

On the other hand, the presence of the pro tease and RT polymerase domain from subtype C isolate 1084i in NL4 3 virus led to a three fold decrease in viral replication by the 27th day of infection. Whereas the clone NL RTpd, Inhibitors,Modulators,Libraries containing the same RT sequence without subtype Inhibitors,Modulators,Libraries C protease, displayed only slower replication kinetics and reached a similar p24CA level to the NL4 3 backbone by the 21st day of infection. These data suggest that subtype C protease may also Inhibitors,Modulators,Libraries affect the replication of the recombinant viruses.

The presence of GagPol domains from HIV 1 subtype C does not affect incorporation of viral genomic RNA and maturation of the virions We quantitatively analyzed the incorporation of viral RNA into the virions and processing of GagPol polypro tein precursor in the virus particles to test the potential effect of the subtype selleck products C protease and C terminal domains of Gag in GagPol chimeras on the precursor protein sta bility and processing, Gag RNA binding, and compatibil ity between the pol sequences. Virus particles were harvested from 297T17 cells transfected with the pro viral clones, DNase I treated, and purified through a 30% sucrose cushion. To quantify viral RNA in the particles, we performed real time RT PCR using a primer set recognizing U5 �� region of HIV 1 LTR.

In this regard, NADPH inhibitors suppress LPS induced expression

In this regard, NADPH inhibitors suppress LPS induced expression of iNOS, IL 6, IL 1? and TNF ? in glial cells in vitro and NADPH oxi dase has been shown to regulate COX 2 mediated PGE2 production in cultured microglia. The JAKSTAT pathway is a key selleck chemical Crizotinib player Inhibitors,Modulators,Libraries in the intracellular response to cytokines. SOCS3 is a potent inhibitor of the JAKSTAT signaling cascade, negatively regulating signal transduction pathways mediated by a variety of cytokines. SOCS3 has been suggested to play a critical role in inte grating the neuroimmunoendocrine Inhibitors,Modulators,Libraries circuits . Although NF ?B p65 expression were similar in COX 2 and COX 2 mice after LPS, we found that the mRNA expression of STAT3 and the levels of phosphorylated STAT 3 were significantly higher in the COX 2 mice compared to wild type mice.

SOCS3 was also upregulated in the COX 2 mice compared to COX 2 mice after LPS. SOCS3 is a negative modulator of inflammatory cytokine signaling and can be induced by inflammatory stim Inhibitors,Modulators,Libraries uli such as LPS, TNF ? and IL 6. SOCS3 mRNA up regulation in the COX 2 deficient mice can thus be viewed as the consequence of the higher cytokine produc tion in these mice after LPS. In this regard, SOCS3 overex Inhibitors,Modulators,Libraries pression has been shown to lead to neuroblastoma cell death. Overall, our data indicate a dysregulation of the cytokine signaling pathway in the COX 2 mice, which may mediate the increased neuroinflammatory response.

While independent epidemiological studies indicate that non steroidal anti inflammatory drugs admin istration prevents or delays the onset and risk of develop ing Alzheimers disease, clinical Inhibitors,Modulators,Libraries trials using COX 2 selective inhibitors in patients with mild to severe cog nitive impairment, have been unsuccessful to date, with the exception of a small double blind, placebo controlled study with indomethacin, a preferential COX 1 inhibitor. We have recently demonstrated that genetic deletion or pharmacological inhibition selleck compound of COX 1 significantly attenuates glial cells activation and the neu roinflammatory response, oxidative stress and neuronal damage in response to icv injected LPS. Our results show that while COX 1 selective inhibition may be bene ficial, selective inhibition of COX 2 appears not to be ben eficial in neurodegenerative diseases with a marked inflammatory component and may explain the failure of selective COX 2 inhibitors to protect AD patients from cognitive decline in clinical trials. Conclusion These findings altogether indicate that the two COX iso forms display opposite roles in the brain during the acute neuroinflammatory process and that COX 2 inhibition worsens the inflammatory response to LPS, suggesting a neuroprotective function of COX 2 derived products.

Patient selection Patients must have had histologically confirmed

Patient selection Patients must have had histologically confirmed melan oma with evidence for metastatic disease, either regional in transit metastases not amenable to complete surgical resection or distant metastases. Treating physicians were required to discuss available standard therapies including DTIC and IL 2 prior to enrolling patients. Eligibility cri teria included Wortmannin msds the presence of at least two accessible lesions amenable to excisional biopsy for correlative assays. measurable disease in addition to the lesions planned Inhibitors,Modulators,Libraries for biopsy. absence of brain metastases. no al lergies to azoles. no more than one prior immunotherapy for metastatic disease. no prior chemotherapy for any stage of disease. ECOG perform ance status of at least 1. at least 18 years of age. non pregnant and non nursing.

laboratory parameters within the following range absolute neutrophil count 1500 ul. platelet count 100,000ul. bilirubin 1. 5 mgdL. creatinine 2. 0 mgdL. Treatment plan R115777 was administered Inhibitors,Modulators,Libraries orally at a dose of 300 mg twice per day for 21 days of a 28 day cycle. Disease re staging was performed every 2 cycles. Patients could re main on treatment until unacceptable toxicity or disease progression occurred. Prior to initiation of treatment, and again during week 7, an excisional biopsy was required to be performed for biologic correlates. At the same time points, heparinized blood was obtained for analysis of effects on T cells. Evaluation of response and toxicities Disease assessment was performed using RECIST criteria every two cycles. Toxicity evaluation was performed at least once per cycle.

Dose reductions were allowed, with dose level ?1 at 200 mg BID, dose level ?2 at 100 mg BID, and Inhibitors,Modulators,Libraries dose level ?3 being permanent discontinuation. For neurologic toxicity grade 2, drug was held until reso lution to grade 1 Inhibitors,Modulators,Libraries and continued at a 1 level dose reduc tion. If the toxicity did not resolve within one week, then drug was permanently discontinued. For hematologic toxicities, if a grade 4 toxicity was observed then drug was held for up to 2 weeks. If resolution occurred to grade 1, then drug was resumed at a 1 level dose reduction. For other toxicities, if a grade 3 event was attributed to drug, then treatment was held up to 2 weeks. If toxicity resolved to grade 1, then drug was resumed at a 1 level dose re duction. If toxicity did not resolve within 2 weeks then drug was permanently discontinued.

For any grade 4 non hematologic toxicity attributed to drug, treatment was permanently discontinued. Statistical considerations A 3 stage design was used to allow for early termination if the drug appeared ineffective in this patient popula tion. A maximum of 40 patients was targeted for enroll ment, with the null hypothesis that the response rate is less than or equal to 0. 05 versus Inhibitors,Modulators,Libraries the alternative hypothesis that the response rate is greater than or equal to 0. 20.