New Strategies For Drug Discovery Research Articles

Design and Characterization of a Novel eEF2K Degrader with Potent Therapeutic Efficacy Against Triple-Negative Breast Cancer.

Dysregulated eEF2K expression is implicated in the pathogenesis of many human cancers, including triple-negative breast cancer (TNBC), making it a plausible therapeutic target. However, specific eEF2K inhibitors with potent anti-cancer activity have not been available so far. Targeted protein degradation has emerged as a new strategy for drug discovery. In this study, a novel small molecule chemical is designed and synthesized, named as compound C1, which shows potent activity in degrading eEF2K. C1 selectively binds to F8, L10, R144, C146, E229, and Y236 of the eEF2K protein and promotes its proteasomal degradation by increasing the interaction between eEF2K and the ubiquitin E3 ligase βTRCP in the form of molecular glue. C1 significantly inhibits the proliferation and metastasis of TNBC cells both in vitro and in vivo and in TNBC patient-derived organoids, and these antitumor effects are attributed to the degradation of eEF2K by C1. Additionally, combination treatment of C1 with paclitaxel, a commonly used chemotherapeutic drug, exhibits synergistic anti-tumor effects against TNBC. This study not only generates a powerful research tool to investigate the therapeutic potential of targeting eEF2K, but also provides a promising lead compound for developing novel drugs for the treatment of TNBC and other cancers.

UDock2: interactive real-time multi-body protein-protein docking software.

Protein-protein docking aims at predicting the geometry of protein interactions to gain insights into the mechanisms underlying these processes and develop new strategies for drug discovery. Interactive and user-oriented manipulation tools can support this task complementary to automated software. This article presents an interactive multi-body protein-protein docking software, UDock2, designed for research but also usable for teaching and popularization of science purposes due to its high usability. In UDock2, the users tackle the conformational space of protein interfaces using an intuitive real-time docking procedure with on-the-fly scoring. UDock2 integrates traditional computer graphics methods to facilitate the visualization and to provide better insight into protein surfaces, interfaces, and properties. UDock2 is open-source, cross-platform (Windows and Linux), and available at http://udock.fr. The code can be accessed at https://gitlab.com/Udock/Udock2.

From fragment screening to fragment growing – new strategies for drug discovery

From fragment screening to fragment growing – new strategies for drug discovery

Elucidation and Application of Novel Action of Therapeutic Agents for Diabetic Neuropathy

Epalrestat is the only aldose reductase inhibitor that is currently available for diabetic peripheral neuropathy. Oxidative stress impairs endothelial cells, thereby leading to numerous pathological conditions. Increasing antioxidative ability is important to prevent cellular toxicity induced by reactive oxygen species. Epalrestat increases antioxidant defense factors such as glutathione and γ-glutamylcysteine ligase in vascular endothelial cells through activation of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2). This increases suppression of oxidative stress-induced cellular toxicity. Cadmium is an industrial and environmental pollutant that targets the vascular endothelium. The vascular system is critically affected by cadmium toxicity. Therapeutic treatment against cadmium toxicity is chelation therapy that promotes metal excretion; however, cadmium chelators can cause renal toxicity. Therefore, safe and efficient therapeutic agents are required. Epalrestat suppresses cadmium-induced cytotoxicity in vascular endothelial cells through activation of Nrf2. In addition, epalrestat affects the intracellular levels of cadmium, cadmium transporter Zrt-Irt-like protein 8 (ZIP8), and metallothionein (MT). The upregulation of ZIP8 and MT may be involved in the suppression of cadmium-induced cytotoxicity by epalrestat. Drug repurposing is a new strategy for drug discovery in which the pharmacological action of existing medicines whose safety and pharmacokinetics have already been confirmed clinically and whose use has been approved is examined comprehensively at the molecular level. The results can be applied to the development of existing drugs for use as medicines for the treatment of other diseases. This review provides useful findings for future expansion of indications as research leading to drug repurposing of epalrestat.

Targeting Lysosomal Degradation Pathways: New Strategies and Techniques for Drug Discovery.

A series of tools for targeted protein degradation are inspiring scientists to develop new drugs with advantages over traditional small-molecule drugs. Among these tools, proteolysis-targeting chimeras (PROTACs) are most representative of the technology based on proteasomes. However, the proteasome has little degradation effect on certain macromolecular proteins or aggregates, extracellular proteins, and organelles, which limits the application of PROTACs. Additionally, lysosomes play an important role in protein degradation. Therefore, lysosome-induced protein degradation drugs can directly regulate protein levels in vivo, achieve the goal of treating diseases, and provide new strategies for drug discovery. Lysosome-based degradation technology has the potential for clinical translation. In this review, strategies targeting lysosomal pathways and lysosome-based degradation techniques are summarized. In addition, lysosome-based degrading drugs are described, and the advantages and challenges are listed. Our efforts will certainly promote the design, discovery, and clinical application of drugs associated with this technology.

Out of breath, out of time: interactions between HIF and circadian rhythms.

Humans have internal circadian clocks that ensure that important physiological functions occur at specific times of the day. These molecular clocks are regulated at the genomic level and exist in most cells of the body. Multiple circadian resetting cues have been identified, including light, temperature, and food. Recently, oxygen has been identified as a resetting cue, and emerging science indicates that this occurs through interactions at the cellular level between the circadian transcription-translation feedback loop and the hypoxia-inducible pathway (hypoxia-inducible factor; subject of the 2019 Nobel Prize in Physiology or Medicine). This review will cover recently identified relationships between HIF and proteins of the circadian clock. Interactions between the circadian clock and hypoxia could have wide-reaching implications for human diseases, and understanding the molecular mechanisms regulating these overlapping pathways may open up new strategies for drug discovery.

Analysis of SARS-CoV-2 RNA-dependent RNA polymerase as a potential therapeutic drug target using a computational approach

BackgroundThe Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) outbreak originating in Wuhan, China, has raised global health concerns and the pandemic has now been reported on all inhabited continents. Hitherto, no antiviral drug is available to combat this viral outbreak.MethodsKeeping in mind the urgency of the situation, the current study was designed to devise new strategies for drug discovery and/or repositioning against SARS-CoV-2. In the current study, RNA-dependent RNA polymerase (RdRp), which regulates viral replication, is proposed as a potential therapeutic target to inhibit viral infection.ResultsEvolutionary studies of whole-genome sequences of SARS-CoV-2 represent high similarity (> 90%) with other SARS viruses. Targeting the RdRp active sites, ASP760 and ASP761, by antiviral drugs could be a potential therapeutic option for inhibition of coronavirus RdRp, and thus viral replication. Target-based virtual screening and molecular docking results show that the antiviral Galidesivir and its structurally similar compounds have shown promise against SARS-CoV-2.ConclusionsThe anti-polymerase drugs predicted here—CID123624208 and CID11687749—may be considered for in vitro and in vivo clinical trials.

Innovative omics-based approaches for prioritisation and targeted isolation of natural products - new strategies for drug discovery.

Covering: 2013 to 2019 The exploration of the chemical diversity of extracts from various biological sources has led to major drug discoveries. Over the past two decades, despite the introduction of advanced methodologies for natural product (NP) research (e.g., dereplication and high content screening), successful accounts of the validation of NPs as lead therapeutic candidates have been limited. In this context, one of the main challenges faced is related to working with crude natural extracts because of their complex composition and the inadequacies of classical bioguided isolation studies given the pace of high-throughput screening campaigns. In line with the development of metabolomics, genomics and chemometrics, significant advances in metabolite profiling have been achieved and have generated high-quality massive genome and metabolome data on natural extracts. The unambiguous identification of each individual NP in an extract using generic methods remains challenging. However, the establishment of structural links among NPs via molecular network analysis and the determination of common features of extract composition have provided invaluable information to the scientific community. In this context, new multi-informational-based profiling approaches integrating taxonomic and/or bioactivity data can hold promise for the discovery and development of new bioactive compounds and return NPs back to an exciting era of development. In this article, we examine recent studies that have the potential to improve the efficiency of NP prioritisation and to accelerate the targeted isolation of key NPs. Perspectives on the field's evolution are discussed.

Green Pharma: A New Strategy for Drug Discovery in Academia by Targeting Glial Cells and ATP Receptors

Neuropathic pain associated with cancer, diabetic neuropathy, and postherpetic neuralgia is a type of intractable chronic pain characterized by mechanical allodynia and abnormal pain hypersensitivity evoked by innocuous stimuli. However, this disorder has no specific treatment. We previously showed that the purinergic receptor P2X4 (P2X4R), a subtype of ATP-gated nonselective cation channels, is highly upregulated in spinal microglia after peripheral nerve injury, and blocking the function of P2X4R reverses mechanical allodynia. In the present study, we screened a chemical library of 1979 clinically approved compounds (a gift from the Drug Discovery Initiative at the University of Tokyo) aimed at achieving "Eco-Pharma," which refers to seeking new effects of existing drugs. We demonstrated that duloxetine, a serotonin and noradrenaline reuptake inhibitor, has an inhibitory effect on rat and human P2X4R. In rat primary cultured microglial cells, duloxetine also inhibited P2X4R-mediated responses. Moreover, intrathecal administration of duloxetine in a model of neuropathic pain reversed nerve injury-induced mechanical allodynia. Based on those results, we suggest that the inhibition of P2X4R expressed in microglial cells may be involved in the antiallodynic effect of duloxetine in neuropathic pain. Furthermore, in this review, we discuss a new strategy for drug discovery called "Green Pharma" (a merger of "Eco-Pharma" and "Green chemistry" and referring to the development of eco-friendly pharmaceuticals).

Protein conformational flexibility modulates kinetics and thermodynamics of drug binding

Structure-based drug design has often been restricted by the rather static picture of protein–ligand complexes presented by crystal structures, despite the widely accepted importance of protein flexibility in biomolecular recognition. Here we report a detailed experimental and computational study of the drug target, human heat shock protein 90, to explore the contribution of protein dynamics to the binding thermodynamics and kinetics of drug-like compounds. We observe that their binding properties depend on whether the protein has a loop or a helical conformation in the binding site of the ligand-bound state. Compounds bound to the helical conformation display slow association and dissociation rates, high-affinity and high cellular efficacy, and predominantly entropically driven binding. An important entropic contribution comes from the greater flexibility of the helical relative to the loop conformation in the ligand-bound state. This unusual mechanism suggests increasing target flexibility in the bound state by ligand design as a new strategy for drug discovery.

New strategy for drug discovery by large-scale association analysis of molecular networks of different species.

The development of modern omics technology has not significantly improved the efficiency of drug development. Rather precise and targeted drug discovery remains unsolved. Here a large-scale cross-species molecular network association (CSMNA) approach for targeted drug screening from natural sources is presented. The algorithm integrates molecular network omics data from humans and 267 plants and microbes, establishing the biological relationships between them and extracting evolutionarily convergent chemicals. This technique allows the researcher to assess targeted drugs for specific human diseases based on specific plant or microbe pathways. In a perspective validation, connections between the plant Halliwell-Asada (HA) cycle and the human Nrf2-ARE pathway were verified and the manner by which the HA cycle molecules act on the human Nrf2-ARE pathway as antioxidants was determined. This shows the potential applicability of this approach in drug discovery. The current method integrates disparate evolutionary species into chemico-biologically coherent circuits, suggesting a new cross-species omics analysis strategy for rational drug development.

Abstract OT3-2-02: MINT I: Multi-Institutional Neo-adjuvant Therapy, MammaPrint Project I

Abstract Background: Women with locally advanced breast cancer (LABC) are often treated with neo-adjuvant chemotherapy to reduce the size of the tumor prior to surgery, to enable breast conserving surgery and to observe the clinical effect of therapy in real time. Studies have shown that the 25–27% of individuals who have a pathologic complete response (pCR) to neoadjuvant therapy have a survival advantage of 80% in 5 years, which is double the expected survival of the remaining patients without pCR. If patients who are likely to show a pCR could be identified prior to initiation of therapy, it would enable more informed treatment decisions – patients likely to respond would be served well by current neoadjuvant chemotherapy protocols, while those unlikely to respond may be better suited to innovative new strategies for drug discovery [von Minckwitz et al. JCO 2006]. Genomic assays, which are widely used to provide prognostic and predictive information in early breast cancer, have the potential to provide information on the likelihood of a patient with LABC responding to neo-adjuvant therapy [Glück et al. BRCRT2013]. Trial design: MINT I is a prospective study designed to test the ability of molecular profiling, as well as traditional pathologic and clinical prognostic factors, to predict response to neo-adjuvant chemotherapy in patients with LABC. MammaPrint risk profile, BluePrint molecular subtyping profile, TargetPrint estrogen receptor (ER), progesterone receptor (PR) and HER2 single gene readout, and TheraPrint Research Gene Panel will be analyzed on a fresh tumor specimen using the whole genome array. Patients will receive neo-adjuvant chemotherapy pre-specified in the protocol. Response will be measured centrally. pCR is defined as the absence of invasive carcinoma in both the breast and axilla at microscopic examination of the resection specimen, regardless of the presence of carcinoma in situ. Eligibility: The study will include women ≥18 years with histologically-proven invasive breast cancer T2 (≥3.5cm)-T4, N0M0 or T2-T4N1M0, adequate bone marrow reserves and normal renal and hepatic function who signed an IRB approved informed consent. Objectives: The objectives of the study are to: 1. Determine the predictive power of MammaPrint and BluePrint for sensitivity to neo-adjuvant chemotherapy as measured by pCR. 2. Compare TargetPrint ER, PR and HER2 with local and centralized IHC and/or CISH/FISH assessment. 3. Identify correlations between TheraPrint and response to neo-adjuvant chemotherapy. 4. Identify and/or validate predictive gene expression profiles of clinical response or resistance to neo-adjuvant chemotherapy. 5. Compare BluePrint with IHC-based subtype classification. Statistical methods: Standard statistical tests such as the Pearson Chi-square test will be used to characterize and evaluate the relationship between chemoresponsiveness and gene expression patterns. Accrual: A total of 226 eligible patients will be enrolled from multiple institutions. To date (June 06, 2014), 103 patients have been enrolled. Citation Format: Charles E Cox, Peter Blumencranz, Ruben Saez, Robert Wesolowski, William Dooley, Lisette Stork-Sloots, Femke de Snoo, Sarah Untch, Eli Avisar. MINT I: Multi-Institutional Neo-adjuvant Therapy, MammaPrint Project I [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr OT3-2-02.

Minimum Bayesian error probability-based gene subset selection.

Sifting functional genes is crucial to the new strategies for drug discovery and prospective patient-tailored therapy. Generally, simply generating gene subset by selecting the top k individually superior genes may obtain an inferior gene combination, for some selected genes may be redundant with respect to some others. In this paper, we propose to select gene subset based on the criterion of minimum Bayesian error probability. The method dynamically evaluates all available genes and sifts only one gene at a time. A gene is selected if its combination with the other selected genes can gain better classification information. Within the generated gene subset, each individual gene is the most discriminative one in comparison with those that classify cancers in the same way as this gene does and different genes are more discriminative in combination than in individual. The genes selected in this way are likely to be functional ones from the system biology perspective, for genes tend to co-regulate rather than regulate individually. Experimental results show that the classifiers induced based on this method are capable of classifying cancers with high accuracy, while only a small number of genes are involved.

Targeting the cis-dimerization of LINGO-1 with low MW compounds affects its downstream signalling.

The transmembrane protein LINGO-1 is a negative regulator in the nervous system mainly affecting axonal regeneration, neuronal survival, oligodendrocyte differentiation and myelination. However, the molecular mechanisms regulating its functions are poorly understood. In the present study, we investigated the formation and the role of LINGO-1 cis-dimers in the regulation of its biological activity. LINGO-1 homodimers were identified in both HEK293 and SH-SY5Y cells using co-immunoprecipitation experiments and BRET saturation analysis. We performed a hypothesis-driven screen for identification of small-molecule protein-protein interaction modulators of LINGO-1 using a BRET-based assay, adapted for screening. The compound identified was further assessed for effects on LINGO-1 downstream signalling pathways using Western blotting analysis and AlphaScreen technology. LINGO-1 was present as homodimers in primary neuronal cultures. LINGO-1 interacted homotypically in cis-orientation and LINGO-1 cis-dimers were formed early during LINGO-1 biosynthesis. A BRET-based assay allowed us to identify phenoxybenzamine as the first conformational modulator of LINGO-1 dimers. In HEK-293 cells, phenoxybenzamine was a positive modulator of LINGO-1 function, increasing the LINGO-1-mediated inhibition of EGF receptor signalling and Erk phosphorylation. Our data suggest that LINGO-1 forms constitutive cis-dimers at the plasma membrane and that low MW compounds affecting the conformational state of these dimers can regulate LINGO-1 downstream signalling pathways. We propose that targeting the LINGO-1 dimerization interface opens a new pharmacological approach to the modulation of its function and provides a new strategy for drug discovery.

RNA polymerase genetic engineering (RPGE) as a new strategy for drug discovery and improvement of industrial microorganisms

RNA polymerase genetic engineering (RPGE) as a new strategy for drug discovery and improvement of industrial microorganisms

Identification and pharmacological induction of autophagy in the larval stages of Echinococcus granulosus: an active catabolic process in calcareous corpuscles

Autophagy is a fundamental catabolic pathway conserved from yeast to mammals, but which remains unknown in parasite cestodes. In this work, the pharmacological induction of autophagy was cellularly and molecularly analysed in the larval stages of Echinococcus granulosus. Metacestode sensitivity to rapamycin and TORC1 expression in protoscoleces and metacestodes were shown. Ultrastructural studies showed that treated parasites had an isolation membrane, autophagosomes and autolysosomes, all of which evidenced the autophagic flux. Genes coding for key autophagy-related proteins were also identified in the Echinococcus genome. These genes were involved in autophagosome formation and transcriptional over-expression of Eg-atg5, Eg-atg6, Eg-atg8, Eg-atg12, Eg-atg16 and Eg-atg18 was shown in presence of rapamycin or arsenic trioxide. Thus, Echinococcus autophagy could be regulated by non-transcriptional inhibition through TOR and by transcription-dependent up-regulation via FoxO-like transcription factors and/or TFEB proteins. An increase in the punctate pattern and Eg-Atg8 polypeptide level in the tegument, parenchyma cells and excretory system of protoscoleces and in vesicularised parasites was detected after rapamycin treatment. This suggests the occurrence of basal autophagy in the larval stages and during vesicular development. In arsenic-treated protoscoleces, high Eg-Atg8 polypeptide levels within the free cytoplasmic matrix of calcareous corpuscles were observed, thus verifying the occurrence of autophagic events. These experiments also confirmed that the calcareous corpuscles are sites of arsenic trioxide accumulation. The detection of the autophagic machinery in this parasite represents a basic starting point to unravel the role of autophagy under both physiological and stress conditions which will allow identification of new strategies for drug discovery against neglected parasitic diseases caused by cestodes.

Abstract OT1-2-01: MINT I: Multi-institutional neo-adjuvant therapy, MammaPrint project I

Abstract Background: Women with locally advanced breast cancer (LABC) are often treated with neo-adjuvant chemotherapy to reduce the size of the tumor prior to surgery, to enable breast conserving surgery and to observe the clinical effect of therapy in real time. Studies have shown that the 25–27% of individuals who have a pathologic complete response (pCR) to neoadjuvant therapy have a survival advantage of 80% in 5 years, which is double the expected survival of the remaining patients without pCR. If patients who are likely to show a pCR could be identified prior to initiation of therapy, it would enable more informed treatment decisions – patients likely to respond would be served well by current neoadjuvant chemotherapy protocols, while those unlikely to respond may be better suited to innovative new strategies for drug discovery [von Minckwitz et al. JCO 2006]. Genomic assays, which are widely used to provide prognostic and predictive information in early breast cancer, have the potential to provide information on the likelihood of a patient with LABC responding to neo-adjuvant therapy [Glück et al. ASCO 2012]. Trial design: MINT I is a prospective study designed to test the ability of molecular profiling, as well as traditional pathologic and clinical prognostic factors, to predict response to neo-adjuvant chemotherapy in patients with LABC. MammaPrint risk profile, BluePrint molecular subtyping profile, TargetPrint estrogen receptor (ER), progesterone receptor (PR) and HER2 single gene readout, and TheraPrint Research Gene Panel will be analyzed on a fresh tumor specimen using the whole genome array. Patients will receive neo-adjuvant chemotherapy pre-specified in the protocol. Response will be measured centrally. pCR is defined as the absence of invasive carcinoma in both the breast and axilla at microscopic examination of the resection specimen, regardless of the presence of carcinoma in situ. Eligibility: The study will include women ≥18 years with histologically-proven invasive breast cancer T2 (≥3.5cm)-T4, N0M0 or T2-T4N1M0, adequate bone marrow reserves and normal renal and hepatic function who signed an IRB approved informed consent. Objectives: The objectives of the study are to: 1. Determine the predictive power of MammaPrint and BluePrint for sensitivity to neo-adjuvant chemotherapy as measured by pCR. 2. Compare TargetPrint ER, PR and HER2 with local and centralized IHC and/or CISH/FISH assessment. 3. Identify correlations between TheraPrint and response to neo-adjuvant chemotherapy. 4. Identify and/or validate predictive gene expression profiles of clinical response or resistance to neo-adjuvant chemotherapy. 5. Compare BluePrint with IHC-based subtype classification. Statistical methods: Standard statistical tests such as the Pearson Chi-square test will be used to characterize and evaluate the relationship between chemoresponsiveness and gene expression patterns. Accrual: A total of 226 eligible patients will be enrolled from multiple institutions. To date (June 06, 2013), 57 patients have been enrolled. Clinical trial registry number: NCT01501487. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr OT1-2-01.

Pharmaceutical innovation and public policy: The case for a new strategy for drug discovery and development

The major research-intensive pharmaceutical companies (big pharma), are facing a profound crisis. Their R&D expenditures have grown at a staggering rate, while the creation of drugs that represent truly novel advances in therapy, so-called new molecular entities (NMEs), has stagnated. Here I present a comprehensive analysis of the NME crisis and discuss possible solutions. This includes proposals to fix the problem by increasing the efficiency of corporate R&D, as well as several alternative strategies for pharmaceutical research, including academic drug discovery centers and the public--private partnerships that have been so successful in attacking neglected diseases. Finally, a new policy initiative, the creation of non-profit drug development corporations (NPDDCs) designed to bring key aspects of early-stage drug development into the pre-competitive public arena is proposed. The creation of effective NPDDCs will be challenging, but promises to provide the health care system with less costly and more effective medicines. Copyright The Author 2013. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com, Oxford University Press.

Novel insights which may translate into treatments for irritable bowel syndrome

Novel insights which may translate into treatments for irritable bowel syndrome

Abstract OT3-4-02: MINT I: Multi-Institutional Neo-adjuvant Therapy, MammaPrint Project I

Abstract Background: Women with locally advanced breast cancer (LABC) are often treated with neo-adjuvant chemotherapy to reduce the size of the tumor prior to surgery, to enable breast conserving surgery and to observe the clinical effect of therapy in real time. Studies have shown that the 25–27% of individuals who have a pathologic complete response (pCR) to neoadjuvant therapy have a survival advantage of 80% in 5 years, which is double the expected survival of the remaining patients without pCR. If patients who are likely to show a pCR could be identified prior to initiation of therapy, it would enable more informed treatment decisions – patients likely to respond would be served well by current neoadjuvant chemotherapy protocols, while those unlikely to respond may be better suited to innovative new strategies for drug discovery [von Minckwitz et al. JCO 2006]. Genomics assays, which are widely used to provide prognostic and predictive information in early breast cancer, have the potential to provide information on the likelihood of a patient with LABC responding to neo-adjuvant therapy [Glück et al. ASCO 2012]. Trial design: MINT I is a prospective study designed to test the ability of molecular profiling, as well as traditional pathologic and clinical prognostic factors, to predict responsiveness to neo-adjuvant chemotherapy in patients with LABC. MammaPrint risk profile, BluePrint molecular subtyping profile, TargetPrint estrogen receptor (ER), progesterone receptor (PR) and HER2 single gene readout, and the 56-gene TheraPrint Research Gene Panel will be analyzed on a fresh tumor specimen using the whole genome array. Patients will receive neo-adjuvant chemotherapy pre-specified in the protocol. Response will be measured centrally. pCR is defined as the absence of invasive carcinoma in both the breast and axilla at microscopic examination of the resection specimen, regardless of the presence of carcinoma in situ. Eligibility: The study will include women ≥18 years with histologically-proven invasive breast cancer T2 (≥3.5cm)-T4, N0M0 or T2-T4N1M0, adequate bone marrow reserves and normal renal and hepatic function who signed an IRB approved informed consent. Objectives: The objectives of the study are to: 1. Determine the predictive power of MammaPrint and BluePrint for sensitivity to neo-adjuvant chemotherapy as measured by pCR.2. Compare TargetPrint ER, PR and HER2 with local and centralized IHC and/or CISH/FISH assessment.3. Identify correlations between TheraPrint and response to neo-adjuvant chemotherapy.4. Identify and/or validate predictive gene expression profiles of clinical response or resistance to neo-adjuvant chemotherapy.5. Compare BluePrint with IHC-based subtype classification. Statistical methods: Standard statistical tests such as the Pearson Chi-square test will be used to characterize and evaluate the relationship between chemoresponsiveness and gene expression patterns. Accrual: A total of 226 eligible patients will be enrolled from multiple institutions. To date (June 06, 2012), 31 patients have been enrolled. Clinical trial registry number: NCT01501487. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr OT3-4-02.