Combined Targeting of NAD Biosynthesis and the NAD-dependent Transcription Factor C-terminal Binding Protein as a Promising Novel Therapy for Pancreatic Cancer.

Cancer therapies targeting metabolic derangements unique to cancer cells are emerging as a key strategy to address refractory solid tumors such as pancreatic ductal adenocarcinomas (PDAC) that exhibit resistance to extreme nutrient deprivation in the tumor microenvironment. Nicotinamide adenine dinucleotide (NAD) participates in multiple metabolic pathways and nicotinamide phosphoribosyl transferase (NAMPT) is one of the key intracellular enzymes that facilitate the synthesis of NAD. C-terminal binding proteins 1 and 2 (CtBP) are paralogous NAD-dependent oncogenic transcription factors and dehydrogenases that nucleate an epigenetic complex regulating a cohort of genes responsible for cancer proliferation and metastasis. As adequate intracellular NAD is required for CtBP to oligomerize and execute its oncogenic transcriptional coregulatory activities, we hypothesized that NAD depletion would synergize with CtBP inhibition, improving cell inhibitory efficacy. Indeed, depletion of cellular NAD via the NAMPT inhibitor GMX1778 enhanced growth inhibition induced by either RNAi-mediated CtBP1/2 knockdown or the CtBP dehydrogenase inhibitor 4-chlorophenyl-2-hydroxyimino propanoic acid as much as 10-fold in PDAC cells, while untransformed pancreatic ductal cells were unaffected. The growth inhibitory effects of the NAMPT/CtBP inhibitor combination correlated pharmacodynamically with on-target disruption of CtBP1/2 dimerization, CtBP2 interaction with the CoREST epigenetic regulator, and transcriptional activation of the oncogenic target gene TIAM1. Moreover, this same therapeutic combination strongly attenuated growth of PDAC cell line xenografts in immunodeficient mice, with no observable toxicity. Collectively, our data demonstrate that targeting CtBP in combination with NAD depletion represents a promising therapeutic strategy for PDAC. SIGNIFICANCE: Effective precision therapies are lacking in PDAC. We demonstrate that simultaneous inhibition of NAD metabolism and the oncoprotein CtBP is potently effective at blocking growth of both PDAC cells in culture and human PDAC-derived tumors in mice and should be explored further as a potential therapy for patients with PDAC.

Merging cultures and disciplines to create a drug discovery ecosystem at Virginia commonwealth university: Medicinal chemistry, structural biology, molecular and behavioral pharmacology and computational chemistry.

The Department of Medicinal Chemistry, together with the Institute for Structural Biology, Drug Discovery and Development, at Virginia Commonwealth University (VCU) has evolved, organically with quite a bit of bootstrapping, into a unique drug discovery ecosystem in response to the environment and culture of the university and the wider research enterprise. Each faculty member that joined the department and/or institute added a layer of expertise, technology and most importantly, innovation, that fertilized numerous collaborations within the University and with outside partners. Despite moderate institutional support with respect to a typical drug discovery enterprise, the VCU drug discovery ecosystem has built and maintained an impressive array of facilities and instrumentation for drug synthesis, drug characterization, biomolecular structural analysis and biophysical analysis, and pharmacological studies. Altogether, this ecosystem has had major impacts on numerous therapeutic areas, such as neurology, psychiatry, drugs of abuse, cancer, sickle cell disease, coagulopathy, inflammation, aging disorders and others. Novel tools and strategies for drug discovery, design and development have been developed at VCU in the last five decades; e.g., fundamental rational structure-activity relationship (SAR)-based drug design, structure-based drug design, orthosteric and allosteric drug design, design of multi-functional agents towards polypharmacy outcomes, principles on designing glycosaminoglycans as drugs, and computational tools and algorithms for quantitative SAR (QSAR) and understanding the roles of water and the hydrophobic effect.

Automated detection of lameness in sheep using machine learning approaches: novel insights into behavioural differences among lame and non-lame sheep.

Lameness in sheep is the biggest cause of concern regarding poor health and welfare among sheep-producing countries. Best practice for lameness relies on rapid treatment, yet there are no objective measures of lameness detection. Accelerometers and gyroscopes have been widely used in human activity studies and their use is becoming increasingly common in livestock. In this study, we used 23 datasets (10 non-lame and 13 lame sheep) from an accelerometer- and gyroscope-based ear sensor with a sampling frequency of 16 Hz to develop and compare algorithms that can differentiate lameness within three different activities (walking, standing and lying). We show for the first time that features extracted from accelerometer and gyroscope signals can differentiate between lame and non-lame sheep while standing, walking and lying. The random forest algorithm performed best for classifying lameness with an accuracy of 84.91% within lying, 81.15% within standing and 76.83% within walking and overall correctly classified over 80% sheep within activities. Both accelerometer- and gyroscope-based features ranked among the top 10 features for classification. Our results suggest that novel behavioural differences between lame and non-lame sheep across all three activities could be used to develop an automated system for lameness detection.

CtBP-a targetable dependency for tumor-initiating cell activity and metastasis in pancreatic adenocarcinoma.

Ctbp2 is a uniquely targetable oncogenic transcriptional coregulator, exhibiting overexpression in most common solid tumors, and critical to the tumor-initiating cell (TIC) transcriptional program. In the "CKP" mouse pancreatic ductal adenocarcinoma (PDAC) model driven by mutant K-Ras, Ctbp2 haploinsufficiency prolonged survival, abrogated peritoneal metastasis, and caused dramatic downregulation of c-Myc, a known critical dependency for TIC activity and tumor progression in PDAC. A small-molecule inhibitor of CtBP2, 4-chloro-hydroxyimino phenylpyruvate (4-Cl-HIPP) phenocopied Ctbp2 deletion, decreasing tumor burden similarly to gemcitabine, and the combination of 4-Cl-HIPP and gemcitabine further synergistically suppressed tumor growth. Pharmacodynamic monitoring revealed that the 4-Cl-HIPP/gemcitabine combination induced robust and synergistic tumor apoptosis and marked downregulation of the TIC marker CD133 in CKP PDAC tumors. Collectively, our data demonstrate that targeting CtBP represents a fruitful avenue for development of highly active agents in PDAC that cooperate with standard therapy to limit both primary and metastatic tumor burden.

A Combined Offline and Online Algorithm for Real-Time and Long-Term Classification of Sheep Behaviour: Novel Approach for Precision Livestock Farming.

Real-time and long-term behavioural monitoring systems in precision livestock farming have huge potential to improve welfare and productivity for the better health of farm animals. However, some of the biggest challenges for long-term monitoring systems relate to "concept drift", which occurs when systems are presented with challenging new or changing conditions, and/or in scenarios where training data is not accurately reflective of live sensed data. This study presents a combined offline algorithm and online learning algorithm which deals with concept drift and is deemed by the authors as a useful mechanism for long-term in-the-field monitoring systems. The proposed algorithm classifies three relevant sheep behaviours using information from an embedded edge device that includes tri-axial accelerometer and tri-axial gyroscope sensors. The proposed approach is for the first time reported in precision livestock behavior monitoring and demonstrates improvement in classifying relevant behaviour in sheep, in real-time, under dynamically changing conditions.

Active-Site Tryptophan, the Target of Antineoplastic C-Terminal Binding Protein Inhibitors, Mediates Inhibitor Disruption of CtBP Oligomerization and Transcription Coregulatory Activities.

C-terminal binding proteins (CtBP1/2) are oncogenic transcriptional coregulators and dehydrogenases often overexpressed in multiple solid tumors, including breast, colon, and ovarian cancer, and associated with poor survival. CtBPs act by repressing expression of genes responsible for apoptosis (e.g., PUMA, BIK) and metastasis-associated epithelial-mesenchymal transition (e.g., CDH1), and by activating expression of genes that promote migratory and invasive properties of cancer cells (e.g., TIAM1) and genes responsible for enhanced drug resistance (e.g., MDR1). CtBP's transcriptional functions are also critically dependent on oligomerization and nucleation of transcriptional complexes. Recently, we have developed a family of CtBP dehydrogenase inhibitors, based on the parent 2-hydroxyimino-3-phenylpropanoic acid (HIPP), that specifically disrupt cancer cell viability, abrogate CtBP's transcriptional function, and block polyp formation in a mouse model of intestinal polyposis that depends on CtBP's oncogenic functions. Crystallographic analysis revealed that HIPP interacts with CtBP1/2 at a conserved active site tryptophan (W318/324; CtBP1/2) that is unique among eukaryotic D2-dehydrogenases. To better understand the mechanism of action of HIPP-class inhibitors, we investigated the contribution of W324 to CtBP2's biochemical and physiologic activities utilizing mutational analysis. Indeed, W324 was necessary for CtBP2 self-association, as shown by analytical ultracentrifugation and in vivo cross-linking. Additionally, W324 supported CtBP's association with the transcriptional corepressor CoREST, and was critical for CtBP2 induction of cell motility. Notably, the HIPP derivative 4-chloro-HIPP biochemically and biologically phenocopied mutational inactivation of CtBP2 W324. Our data support further optimization of W318/W324-interacting CtBP dehydrogenase inhibitors that are emerging as a novel class of cancer cell-specific therapeutic.

Feature Selection and Comparison of Machine Learning Algorithms in Classification of Grazing and Rumination Behaviour in Sheep.

Grazing and ruminating are the most important behaviours for ruminants, as they spend most of their daily time budget performing these. Continuous surveillance of eating behaviour is an important means for monitoring ruminant health, productivity and welfare. However, surveillance performed by human operators is prone to human variance, time-consuming and costly, especially on animals kept at pasture or free-ranging. The use of sensors to automatically acquire data, and software to classify and identify behaviours, offers significant potential in addressing such issues. In this work, data collected from sheep by means of an accelerometer/gyroscope sensor attached to the ear and collar, sampled at 16 Hz, were used to develop classifiers for grazing and ruminating behaviour using various machine learning algorithms: random forest (RF), support vector machine (SVM), k nearest neighbour (kNN) and adaptive boosting (Adaboost). Multiple features extracted from the signals were ranked on their importance for classification. Several performance indicators were considered when comparing classifiers as a function of algorithm used, sensor localisation and number of used features. Random forest yielded the highest overall accuracies: 92% for collar and 91% for ear. Gyroscope-based features were shown to have the greatest relative importance for eating behaviours. The optimum number of feature characteristics to be incorporated into the model was 39, from both ear and collar data. The findings suggest that one can successfully classify eating behaviours in sheep with very high accuracy; this could be used to develop a device for automatic monitoring of feed intake in the sheep sector to monitor health and welfare.

An intestinal stem cell niche in Apc mutated neoplasia targetable by CtBP inhibition.

C-terminal binding protein 2 (CtBP2) drives intestinal polyposis in the Apcmin mouse model of human Familial Adenomatous Polyposis. As CtBP2 is targetable by an inhibitor of its dehydrogenase domain, understanding CtBP2's role in adenoma formation is necessary to optimize CtBP-targeted therapies in Apc mutated human neoplasia. Tumor initiating cell (TIC) populations were substantially decreased in ApcminCtbp2+/- intestinal epithelia. Moreover, normally nuclear Ctbp2 was mislocalized to the cytoplasm of intestinal crypt stem cells in Ctbp2+/- mice, both Apcmin and wildtype, correlating with low/absent CD133 expression in those cells, and possibly explaining the lower burden of polyps in Apcmin Ctbp2+/- mice. The CtBP inhibitor 4-chloro-hydroxyimino phenylpyruvate (4-Cl-HIPP) also robustly downregulated TIC populations and significantly decreased intestinal polyposis in Apcmin mice. We have therefore demonstrated a critical link between polyposis, intestinal TIC's and Ctbp2 gene dosage or activity, supporting continued efforts targeting CtBP in the treatment or prevention of Apc mutated neoplasia.

Kinetics and inhibition studies of the L205R mutant of cAMP-dependent protein kinase involved in Cushing's syndrome.

Overproduction of cortisol by the hypothalamus-pituitary-adrenal hormone system results in the clinical disorder known as Cushing's syndrome. Genomics studies have identified a key mutation (L205R) in the α-isoform of the catalytic subunit of cAMP-dependent protein kinase (PKACα) in adrenal adenomas of patients with adrenocorticotropic hormone-independent Cushing's syndrome. Here, we conducted kinetics and inhibition studies on the L205R-PKACα mutant. We have found that the L205R mutation affects the kinetics of both Kemptide and ATP as substrates, decreasing the catalytic efficiency (kcat/KM) for each substrate by 12-fold and 4.5-fold, respectively. We have also determined the IC 50 and Ki for the peptide substrate-competitive inhibitor PKI(5-24) and the ATP-competitive inhibitor H89. The L205R mutation had no effect on the potency of H89, but causes a > 250-fold loss in potency for PKI(5-24). Collectively, these data provide insights for the development of L205R-PKACα inhibitors as potential therapeutics.

Evaluation of sampling frequency, window size and sensor position for classification of sheep behaviour.

Automated behavioural classification and identification through sensors has the potential to improve health and welfare of the animals. Position of a sensor, sampling frequency and window size of segmented signal data has a major impact on classification accuracy in activity recognition and energy needs for the sensor, yet, there are no studies in precision livestock farming that have evaluated the effect of all these factors simultaneously. The aim of this study was to evaluate the effects of position (ear and collar), sampling frequency (8, 16 and 32 Hz) of a triaxial accelerometer and gyroscope sensor and window size (3, 5 and 7 s) on the classification of important behaviours in sheep such as lying, standing and walking. Behaviours were classified using a random forest approach with 44 feature characteristics. The best performance for walking, standing and lying classification in sheep (accuracy 95%, F-score 91%-97%) was obtained using combination of 32 Hz, 7 s and 32 Hz, 5 s for both ear and collar sensors, although, results obtained with 16 Hz and 7 s window were comparable with accuracy of 91%-93% and F-score 88%-95%. Energy efficiency was best at a 7 s window. This suggests that sampling at 16 Hz with 7 s window will offer benefits in a real-time behavioural monitoring system for sheep due to reduced energy needs.

Chelation-directed C-H activation/C-C bond forming reactions catalyzed by Pd(ii) nanoparticles supported on multiwalled carbon nanotubes.

Chelation-directed C-H activation/C-C bond forming reactions utilizing homogeneous palladium(ii) and the Pd(ii)/Pd(iv) catalytic cycle have been previously reported. Here we report the first use of a solid-supported Pd(ii) catalyst [Pd(ii) nanoparticles on multiwalled carbon nanotubes, Pd(ii)/MWCNT] to carry out C-H activation/C-C bond forming reactions. The results presented demonstrate that the solid-supported Pd(ii)/MWCNT catalyst can effectively catalyze these arylation reactions using the Pd(ii)/Pd(iv) catalytic cycle. We also show that the solid-supported catalyst is recyclable, has turnover frequencies up to 2.9-fold higher than the homogeneous catalyst, and results in low levels of residual palladium contamination in the products.

Characterization of PKACα enzyme kinetics and inhibition in an HPLC assay with a chromophoric substrate.

Here we describe a convenient, inexpensive, and non-hazardous method for the measurement of the kinase activity of the catalytic subunit of cAMP-dependent protein kinase (PKACα). The assay is based on the separation of a substrate peptide labeled with a strong chromophore from the phosphorylated product peptide by high-performance liquid chromatograph (HPLC) and quantification of the product ratiometrically at a wavelength in the visual spectrum (Vis). The utility and reliability of the HPLC-Vis assay were demonstrated by characterizing the kinetic parameters (KM, Vmax) of the new Rh-MAB-Kemptide substrate, a commercially prepared TAMRA-Kemptide substrate, and ATP as well as the potency (IC50, Ki) of the known PKACα inhibitors H89 and PKI(5-24). The advantages of this assay are that it is convenient and inexpensive, uses readily synthesized or commercially available substrates that are shelf-stable, uses a common piece of laboratory equipment, and does not require any hazardous materials such as radioactive γ-32P-ATP. The assay format is also highly flexible and could be adapted for the testing of many different kinases by changing the peptide substrate sequence.

CtBP- an emerging oncogene and novel small molecule drug target: Advances in the understanding of its oncogenic action and identification of therapeutic inhibitors.

C-terminal Binding Proteins (CtBP) 1 and 2 are oncogenic transcriptional co-regulators overexpressed in many cancer types, with their expression level correlating to worse prognostic outcomes and aggressive tumor features. CtBP negatively regulates the expression of many tumor suppressor genes, while coactivating genes that promote proliferation, epithelial-mesenchymal transition, and cancer stem cell self-renewal activity. In light of this evidence, the development of novel inhibitors that mitigate CtBP function may provide clinically actionable therapeutic tools. This review article focuses on the progress made in understanding CtBP structure, role in tumor progression, and discovery and development of CtBP inhibitors that target CtBP's dehydrogenase activity and other functions, with a focus on the theory and rationale behind the designs of current inhibitors. We provide insight into the future development and use of rational combination therapy that may further augment the efficacy of CtBP inhibitors, specifically addressing metastasis and cancer stem cell populations within tumors.

Design, synthesis, and biological evaluation of substrate-competitive inhibitors of C-terminal Binding Protein (CtBP).

C-terminal Binding Protein (CtBP) is a transcriptional co-regulator that downregulates the expression of many tumor-suppressor genes. Utilizing a crystal structure of CtBP with its substrate 4-methylthio-2-oxobutyric acid (MTOB) and NAD(+) as a guide, we have designed, synthesized, and tested a series of small molecule inhibitors of CtBP. From our first round of compounds, we identified 2-(hydroxyimino)-3-phenylpropanoic acid as a potent CtBP inhibitor (IC50=0.24µM). A structure-activity relationship study of this compound further identified the 4-chloro- (IC50=0.18µM) and 3-chloro- (IC50=0.17µM) analogues as additional potent CtBP inhibitors. Evaluation of the hydroxyimine analogues in a short-term cell growth/viability assay showed that the 4-chloro- and 3-chloro-analogues are 2-fold and 4-fold more potent, respectively, than the MTOB control. A functional cellular assay using a CtBP-specific transcriptional readout revealed that the 4-chloro- and 3-chloro-hydroxyimine analogues were able to block CtBP transcriptional repression activity. This data suggests that substrate-competitive inhibition of CtBP dehydrogenase activity is a potential mechanism to reactivate tumor-suppressor gene expression as a therapeutic strategy for cancer.

Design, synthesis, and in vitro evaluation of a fluorescently labeled irreversible inhibitor of the catalytic subunit of cAMP-dependent protein kinase (PKACα).

The design and development of irreversible kinase inhibitors is an expanding frontier of kinase drug discovery. The current approach to develop these inhibitors utilizes ATP-competitive inhibitor scaffolds to target non-catalytic cysteines in the kinase ATP-binding site. However, this approach is limited as not all kinases have a cysteine in the ATP-binding site that can be targeted. In this work, we report a complementary approach to developing irreversible kinase inhibitors that utilizes the substrate-binding site. Using the catalytic subunit of cAMP-dependent protein kinase (PKACα) as a model system, we have designed and synthesized an irreversible inhibitor based on the substrate-competitive inhibitor scaffold PKI(14-22) that covalently modifies non-catalytic Cys199 in the PKACα substrate-binding site. The new compound inhibits PKACα (IC50 = 11.8 ± 1.1 nM), is ∼100-fold selective for PKACα in a kinase panel, and covalently labels the kinase as demonstrated by fluorescence, mass spectrometry, and kinetics experiments. This study demonstrates the feasibility of utilizing this new approach to develop irreversible inhibitors for any of the eighty-nine kinases that possess a similar non-catalytic cysteine in their substrate-binding sites.

Selective N-chelation-directed C-H activation reactions catalyzed by Pd(II) nanoparticles supported on multiwalled carbon nanotubes.

N-Chelation-directed C-H activation reactions that utilize the Pd(II)/Pd(IV) catalytic cycle have been previously reported. To date, these reactions employ only homogeneous palladium catalysts. The first use of a solid-supported Pd(II) catalyst [Pd(II) nanoparticles on multiwalled carbon nanotubes, Pd(II)/MWCNT] to carry out N-chelation-directed C-H to C-O, C-Cl, and C-Br transformations is reported. The results presented demonstrate that the solid-supported Pd(II)/MWCNT catalyst can effectively catalyze C-H activation reactions using the Pd(II)/Pd(IV) catalytic cycle.

Structure-guided design of a high affinity inhibitor to human CtBP.

Oncogenic transcriptional coregulators C-terminal Binding Protein (CtBP) 1 and 2 possess regulatory d-isomer specific 2-hydroxyacid dehydrogenase (D2-HDH) domains that provide an attractive target for small molecule intervention. Findings that the CtBP substrate 4-methylthio 2-oxobutyric acid (MTOB) can interfere with CtBP oncogenic activity in cell culture and in mice confirm that such inhibitors could have therapeutic benefit. Recent crystal structures of CtBP 1 and 2 revealed that MTOB binds in an active site containing a dominant tryptophan and a hydrophilic cavity, neither of which are present in other D2-HDH family members. Here, we demonstrate the effectiveness of exploiting these active site features for the design of high affinity inhibitors. Crystal structures of two such compounds, phenylpyruvate (PPy) and 2-hydroxyimino-3-phenylpropanoic acid (HIPP), show binding with favorable ring stacking against the CtBP active site tryptophan and alternate modes of stabilizing the carboxylic acid moiety. Moreover, ITC experiments show that HIPP binds to CtBP with an affinity greater than 1000-fold over that of MTOB, and enzymatic assays confirm that HIPP substantially inhibits CtBP catalysis. These results, thus, provide an important step, and additional insights, for the development of highly selective antineoplastic CtBP inhibitors.

Phenylalanine-Based Inactivator of AKT Kinase: Design, Synthesis, and Biological Evaluation.

Strategies to inhibit kinases by targeting the substrate binding site offer many advantages, including naturally evolved selectivity filters, but normally suffer from poor potency. In this work we propose a strategy to design and prepare covalent substrate-competitive kinase inhibitors as a method to improve potency. We have chosen AKT as the model kinase for this work. Using the AKT-GSK3ß cocrystal structure and a reactive cysteine near the substrate binding site, we have identified phenylalanine (Phe) as an appropriate scaffold for the covalent inactivator portion of these inhibitors. By synthesizing compounds that incorporate cysteine-reactive electrophiles into phenylalanine and testing these compounds as AKT inhibitors, we have identified Boc-Phe-vinyl ketone as a submicromolar inactivator of AKT. We also show that Boc-Phe-vinyl ketone (1) potently inhibits AKT1 and inhibits cell growth in HCT116 and H460 cells nearly as well as AKT inhibitors GSK690693 and MK-2206, (2) is selective for kinases that possess an activation loop cysteine such as AKT, (3) requires the vinyl ketone for inactivation, (4) has inactivation that is time-dependent, and (5) alkylates Cys310 of AKT as shown by mass spectrometry. Identification of Boc-Phe-vinyl ketone as a covalent inactivator of AKT will allow the development of peptide and small-molecule substrate-competitive covalent kinase inhibitors that incorporate additional substrate binding elements to increase selectivity and potency. This proof-of-principle study also provides a basis to apply this strategy to other kinases of the AGC and CAMK families.

Spin equilibrium and O2-binding kinetics of Mycobacterium tuberculosis CYP51 with mutations in the histidine-threonine dyad.

The acidic residues of the "acid-alcohol pair" in CYP51 enzymes are uniformly replaced with histidine. Herein, we adopt the Mycobacterium tuberculosis (mt) enzyme as a model system to investigate these residues' roles in finely tuning the heme conformation, iron spin state, and formation and decay of the oxyferrous enzyme. Properties of the mtCYP51 and the T260A, T260V, and H259A mutants were interrogated using UV-Vis and resonance Raman spectroscopies. Evidence supports that these mutations induce comprehensive changes in the heme environment. The heme iron spin states are differentially sensitive to the binding of the substrate, dihydrolanosterol (DHL). DHL and clotrimazole perturb the local environments of the heme vinyl and propionate substituents. Molecular dynamics (MD) simulations of the DHL-enzyme complexes support that the observed perturbations are attributable to changes in the DHL binding mode. Furthermore, the rates of the oxyferrous formation were measured using stopped-flow methods. These studies demonstrate that both HT mutations and DHL modulate the rates of oxyferrous formation. Paradoxically, the binding rate to the H259A mutant-DHL complex was approximately four-fold that of mtCYP51, a phenomenon that is predicted to result from the creation of an additional diffusion channel from loss of the H259-E173 ion pair in the mutant. Oxyferrous enzyme auto-oxidation rates were relatively constant, with the exception of the T260V-DHL complex. MD simulations lead us to speculate that this behavior may be attributed to the distortion of the heme macrocycle by the substrate.

Preparation and evaluation of deconstruction analogues of 7-deoxykalafungin as AKT kinase inhibitors.

The pyranonaphthoquinone (PNQ) lactone natural products, including 7-deoxykalafungin, have been reported to be potent and selective covalent inhibitors of AKT kinase. In this work we seek to identify structural features of the natural product scaffold that are essential for potency and selectivity. Using a deconstruction approach, we designed and prepared simplified analogues of 7-deoxykalafungin. Testing of the compounds for their ability to inhibit AKT and the closely related kinase PKA revealed that the 3,6-dihydro-2H-pyran ring of the PNQ lactones is required for potent and selective inhibition of AKT. We have also unexpectedly identified a new submicromolar inhibitor of PKA.