Palladium-peptide oxidative addition complexes for bioconjugation.

The synthesis of palladium oxidative addition complexes derived from unprotected peptides is described. Incorporation of 4-halophenylalanine into a peptide during solid phase peptide synthesis allows for subsequent oxidative addition at this position upon treatment with a palladium precursor and suitable ligand. The resulting palladium-peptide complexes are solid, storable, water-soluble, and easily purified via high-performance liquid chromatography. These complexes react with thiols in aqueous buffer, offering an efficient method for bioconjugation. Using this strategy, peptides can be functionalized with small molecules to prepare modified aryl thioether side-chains at low micromolar concentrations. Additionally, peptide-peptide and peptide-protein ligations are demonstrated under dilute aqueous conditions.

Quantitative Interpretation of Protein Diffusion Coefficients in Mixed Protiated-Deuteriated Aqueous Solvents.

Diffusion-ordered nuclear magnetic resonance (NMR) spectroscopy is widely used for the analysis of mixtures, dispersing the signals of different species in a two-dimensional spectrum according to their diffusion coefficients. However, interpretation of these diffusion coefficients is typically purely qualitative, for example, to deduce which species are bigger or smaller. In studies of proteins in solution, important questions concern the molecular weight of the proteins, the presence or absence of aggregation, and the degree of folding. The Stokes-Einstein Gierer-Wirtz estimation (SEGWE) method has been previously developed to simplify the complex relationship between diffusion coefficient and molecular mass, allowing the prediction of a species' diffusion coefficient in a pure solvent based on its molecular weight. Here, we show that SEGWE can be extended to successfully predict both peptide and protein diffusion coefficients in mixed protiated-deuteriated water samples and, hence, distinguish effectively between globular and disordered proteins.

Immunomodulatory drugs activate NK cells via both Zap-70 and cereblon-dependent pathways.

Immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide show remarkable antitumor activity in multiple myeloma (MM) via directly inhibiting MM-cell growth in the bone marrow (BM) microenvironment and promoting immune effector cell function. They are known to bind to the ubiquitin 3 ligase CRBN complex and thereby triggering degradation of IKZF1/3. In this study, we demonstrate that IMiDs also directly bind and activate zeta-chain-associated protein kinase-70 (Zap-70) via its tyrosine residue phosphorylation in T cells. IMiDs also triggered phosphorylation of Zap-70 in natural killer (NK) cells. Importantly, increased granzyme-B (GZM-B) expression and NK-cell activity triggered by IMiDs is associated with Zap-70 activation and inhibited by Zap-70 knockdown (KD), independent of CRBN. We also demonstrate a second mechanism whereby IMiDs trigger GZM-B and NK cytotoxicity which is CRBN and IKZF3 mediated, and inhibited or enhanced by KD of CRBN or IKZF3, respectively, independent of Zap-70. Our studies therefore show that IMiDs can enhance NK and T-cell cytotoxicity in (1) ZAP-70-mediated CRBN independent, as well as (2) CRBN-mediated ZAP-70 independent mechanisms; and provide the framework for developing novel therapeutics to activate Zap-70 and thereby enhance T and NK anti-MM cytotoxicity.

Arsenic targets Pin1 and cooperates with retinoic acid to inhibit cancer-driving pathways and tumor-initiating cells.

Arsenic trioxide (ATO) and all-trans retinoic acid (ATRA) combination safely cures fatal acute promyelocytic leukemia, but their mechanisms of action and efficacy are not fully understood. ATRA inhibits leukemia, breast, and liver cancer by targeting isomerase Pin1, a master regulator of oncogenic signaling networks. Here we show that ATO targets Pin1 and cooperates with ATRA to exert potent anticancer activity. ATO inhibits and degrades Pin1, and suppresses its oncogenic function by noncovalent binding to Pin1's active site. ATRA increases cellular ATO uptake through upregulating aquaporin-9. ATO and ATRA, at clinically safe doses, cooperatively ablate Pin1 to block numerous cancer-driving pathways and inhibit the growth of triple-negative breast cancer cells and tumor-initiating cells in cell and animal models including patient-derived orthotopic xenografts, like Pin1 knockout, which is substantiated by comprehensive protein and microRNA analyses. Thus, synergistic targeting of Pin1 by ATO and ATRA offers an attractive approach to combating breast and other cancers.

Structural and Atropisomeric Factors Governing the Selectivity of Pyrimido-benzodiazipinones as Inhibitors of Kinases and Bromodomains.

Bromodomains have been pursued intensively over the past several years as emerging targets for the development of anticancer and anti-inflammatory agents. It has recently been shown that some kinase inhibitors are able to potently inhibit the bromodomains of BRD4. The clinical activities of PLK inhibitor BI-2536 and JAK2-FLT3 inhibitor TG101348 have been attributed to this unexpected polypharmacology, indicating that dual-kinase/bromodomain activity may be advantageous in a therapeutic context. However, for target validation and biological investigation, a more selective target profile is desired. Here, we report that benzo[e]pyrimido-[5,4- b]diazepine-6(11H)-ones, versatile ATP-site directed kinase pharmacophores utilized in the development of inhibitors of multiple kinases, including several previously reported kinase chemical probes, are also capable of exhibiting potent BRD4-dependent pharmacology. Using a dual kinase-bromodomain inhibitor of the kinase domains of ERK5 and LRRK2, and the bromodomain of BRD4 as a case study, we define the structure-activity relationships required to achieve dual kinase/BRD4 activity, as well as how to direct selectivity toward inhibition of either ERK5 or BRD4. This effort resulted in identification of one of the first reported kinase-selective chemical probes for ERK5 (JWG-071), a BET selective inhibitor with 1 µM BRD4 IC50 (JWG-115), and additional inhibitors with rationally designed polypharmacology (JWG-047, JWG-069). Co-crystallography of seven representative inhibitors with the first bromodomain of BRD4 demonstrate that distinct atropisomeric conformers recognize the kinase ATP-site and the BRD4 acetyl lysine binding site, conformational preferences supported by rigid docking studies.

[18F]Fluorocholine and [18F]Fluoroacetate PET as Imaging Biomarkers to Assess Phosphatidylcholine and Mitochondrial Metabolism in Preclinical Models of TSC and LAM.

PURPOSE: Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by inactivating mutations of the TSC1 or TSC2 gene, characterized by neurocognitive impairment and benign tumors of the brain, skin, heart, and kidneys. Lymphangioleiomyomatosis (LAM) is a diffuse proliferation of α-smooth muscle actin-positive cells associated with cystic destruction of the lung. LAM occurs almost exclusively in women, as a TSC manifestation or a sporadic disorder (TSC1/TSC2 somatic mutations). Biomarkers of whole-body tumor burden/activity and response to rapalogs or other therapies remain needed in TSC/LAM. EXPERIMENTAL DESIGN: These preclinical studies aimed to assess feasibility of [18F]fluorocholine (FCH) and [18F]fluoroacetate (FACE) as TSC/LAM metabolic imaging biomarkers. RESULTS: We previously reported that TSC2-deficient cells enhance phosphatidylcholine synthesis via the Kennedy pathway. Here, we show that TSC2-deficient cells exhibit rapid uptake of [18F]FCH in vivo and can be visualized by PET imaging in preclinical models of TSC/LAM, including subcutaneous tumors and pulmonary nodules. Treatment with rapamycin (72 hours) suppressed [18F]FCH standardized uptake value (SUV) by >50% in tumors. Interestingly, [18F]FCH-PET imaging of TSC2-deficient xenografts in ovariectomized mice also showed a significant decrease in tumor SUV. Finally, we found rapamycin-insensitive uptake of FACE by TSC2-deficient cells in vitro and in vivo, reflecting its mitochondrial accumulation via inhibition of aconitase, a TCA cycle enzyme. CONCLUSIONS: Preclinical models of TSC2 deficiency represent informative platforms to identify tracers of potential clinical interest. Our findings provide mechanistic evidence for testing the potential of [18F]FCH and [18F]FACE as metabolic imaging biomarkers for TSC and LAM proliferative lesions, and novel insights into the metabolic reprogramming of TSC tumors.

The Antibiotic Novobiocin Binds and Activates the ATPase That Powers Lipopolysaccharide Transport.

Novobiocin is an orally active antibiotic that inhibits DNA gyrase by binding the ATP-binding site in the ATPase subunit. Although effective against Gram-positive pathogens, novobiocin has limited activity against Gram-negative organisms due to the presence of the lipopolysaccharide-containing outer membrane, which acts as a permeability barrier. Using a novobiocin-sensitive Escherichia coli strain with a leaky outer membrane, we identified a mutant with increased resistance to novobiocin. Unexpectedly, the mutation that increases novobiocin resistance was not found to alter gyrase, but the ATPase that powers lipopolysaccharide (LPS) transport. Co-crystal structures, biochemical, and genetic evidence show novobiocin directly binds this ATPase. Novobiocin does not bind the ATP binding site but rather the interface between the ATPase subunits and the transmembrane subunits of the LPS transporter. This interaction increases the activity of the LPS transporter, which in turn alters the permeability of the outer membrane. We propose that novobiocin will be a useful tool for understanding how ATP hydrolysis is coupled to LPS transport.

Allosteric sensitization of proapoptotic BAX.

BCL-2-associated X protein (BAX) is a critical apoptotic regulator that can be transformed from a cytosolic monomer into a lethal mitochondrial oligomer, yet drug strategies to modulate it are underdeveloped due to longstanding difficulties in conducting screens on this aggregation-prone protein. Here, we overcame prior challenges and performed an NMR-based fragment screen of full-length human BAX. We identified a compound that sensitizes BAX activation by binding to a pocket formed by the junction of the α3-α4 and α5-α6 hairpins. Biochemical and structural analyses revealed that the molecule sensitizes BAX by allosterically mobilizing the α1-α2 loop and BAX BH3 helix, two motifs implicated in the activation and oligomerization of BAX, respectively. By engaging a region of core hydrophobic interactions that otherwise preserve the BAX inactive state, the identified compound reveals fundamental mechanisms for conformational regulation of BAX and provides a new opportunity to reduce the apoptotic threshold for potential therapeutic benefit.

Rapid convergence of optimal control in NMR using numerically-constructed toggling frames.

We present a numerical method for rapidly solving the Bloch equation for an arbitrary time-varying spin-1/2 Hamiltonian. The method relies on fast, vectorized computations such as summation and quaternion multiplication, rather than slow computations such as matrix exponentiation. A toggling frame is constructed in which the Hamiltonian is time-invariant, and therefore has a simple analytical solution. The key insight is that constructing this frame is faster than solving the system dynamics in the original frame. Rapidly solving the Bloch equations for an arbitrary Hamiltonian is particularly useful in the context of NMR optimal control. Optimal control theory can be used to design pulse shapes for a range of tasks in NMR spectroscopy. However, it requires multiple simulations of the Bloch equations at each stage of the algorithm, and for each relevant set of parameters (e.g. chemical shift frequencies). This is typically time consuming. We demonstrate that by working in an appropriate toggling frame, optimal control pulses can be generated much faster. We present a new alternative to the well-known GRAPE algorithm to continuously update the toggling-frame as the optimal pulse is generated, and demonstrate that this approach is extremely fast. The use and benefit of rapid optimal pulse generation is demonstrated for 19F fragment screening experiments.

p53-related protein kinase confers poor prognosis and represents a novel therapeutic target in multiple myeloma.

p53-related protein kinase (TP53RK, also known as PRPK) is an upstream kinase that phosphorylates (serine residue Ser15) and mediates p53 activity. Here we show that TP53RK confers poor prognosis in multiple myeloma (MM) patients, and, conversely, that TP53RK knockdown inhibits p53 phosphorylation and triggers MM cell apoptosis, associated with downregulation of c-Myc and E2F-1-mediated upregulation of pro-apoptotic Bim. We further demonstrate that TP53RK downregulation also triggers growth inhibition in p53-deficient and p53-mutant MM cell lines and identify novel downstream targets of TP53RK including ribonucleotide reductase-1, telomerase reverse transcriptase, and cyclin-dependent kinase inhibitor 2C. Our previous studies showed that immunomodulatory drugs (IMiDs) downregulate p21 and trigger apoptosis in wild-type-p53 MM.1S cells, Importantly, we demonstrate by pull-down, nuclear magnetic resonance spectroscopy, differential scanning fluorimetry, and isothermal titration calorimetry that IMiDs bind and inhibit TP53RK, with biologic sequelae similar to TP53RK knockdown. Our studies therefore demonstrate that either genetic or pharmacological inhibition of TP53RK triggers MM cell apoptosis via both p53-Myc axis-dependent and axis-independent pathways, validating TP53RK as a novel therapeutic target in patients with poor-prognosis MM.

Rapid Evaluation of Platelet Function With T2 Magnetic Resonance.

OBJECTIVES: The clinical diagnosis of qualitative platelet disorders (QPDs) based on light transmission aggregometry (LTA) requires significant blood volume, time, and expertise, all of which can be barriers to utilization in some populations and settings. Our objective was to develop a more rapid assay of platelet function by measuring platelet-mediated clot contraction in small volumes (35 µL) of whole blood using T2 magnetic resonance (T2MR). METHODS: We established normal ranges for platelet-mediated clot contraction using T2MR, used these ranges to study patients with known platelet dysfunction, and then evaluated agreement between T2MR and LTA with arachidonic acid, adenosine diphosphate, epinephrine, and thrombin receptor activator peptide. RESULTS: Blood from 21 healthy donors was studied. T2MR showed 100% agreement with LTA with each of the four agonists and their cognate inhibitors tested. T2MR successfully detected abnormalities in each of seven patients with known QPDs, with the exception of one patient with a novel mutation leading to Hermansky-Pudlak syndrome. T2MR appeared to detect platelet function at similar or lower platelet counts than LTA. CONCLUSIONS: T2MR may provide a clinically useful approach to diagnose QPDs using small volumes of whole blood, while also providing new insight into platelet biology not evident using plasma-based platelet aggregation tests.

T2 magnetic resonance: a diagnostic platform for studying integrated hemostasis in whole blood--proof of concept.

BACKGROUND: Existing approaches for measuring hemostasis parameters require multiple platforms, can take hours to provide results, and generally require 1-25 mL of sample. We developed a diagnostic platform that allows comprehensive assessment of hemostatic parameters on a single instrument and provides results within 15 min using 0.04 mL of blood with minimal sample handling. METHODS: T2 magnetic resonance (T2MR) was used to directly measure integrated reactions in whole blood samples by resolving multiple water relaxation times from distinct sample microenvironments. Clotting, clot contraction, and fibrinolysis stimulated by thrombin or tissue plasminogen activator, respectively, were measured. T2MR signals of clotting samples were compared with images produced by scanning electron microscopy and with standard reference methods for the following parameters: hematocrit, prothrombin time, clot strength, and platelet activity. RESULTS: Application of T2MR methodology revealed conditions under which a unique T2MR signature appeared that corresponded with the formation of polyhedral erythrocytes, the dynamics and morphology of which are dependent on thrombin, fibrinogen, hematocrit, and platelet levels. We also showed that the T2MR platform can be used for precise and accurate measurements of hematocrit (%CV, 4.8%, R(2) = 0.95), clotting time (%CV, 3.5%, R(2) = 0.94), clot strength (R(2) = 0.95), and platelet function (93% agreement with light transmission aggregometry). CONCLUSIONS: This proof-of-concept study demonstrates that T2MR has the potential to provide rapid and sensitive identification of patients at risk for thrombosis or bleeding and to identify new biomarkers and therapeutic targets with a single, simple-to-employ analytic approach that may be suitable for routine use in both research and diverse clinical settings.

Clot contraction: compression of erythrocytes into tightly packed polyhedra and redistribution of platelets and fibrin.

Contraction of blood clots is necessary for hemostasis and wound healing and to restore flow past obstructive thrombi, but little is known about the structure of contracted clots or the role of erythrocytes in contraction. We found that contracted blood clots develop a remarkable structure, with a meshwork of fibrin and platelet aggregates on the exterior of the clot and a close-packed, tessellated array of compressed polyhedral erythrocytes within. The same results were obtained after initiation of clotting with various activators and also with clots from reconstituted human blood and mouse blood. Such close-packed arrays of polyhedral erythrocytes, or polyhedrocytes, were also observed in human arterial thrombi taken from patients. The mechanical nature of this shape change was confirmed by polyhedrocyte formation from the forces of centrifugation of blood without clotting. Platelets (with their cytoskeletal motility proteins) and fibrin(ogen) (as the substrate bridging platelets for contraction) are required to generate the forces necessary to segregate platelets/fibrin from erythrocytes and to compress erythrocytes into a tightly packed array. These results demonstrate how contracted clots form an impermeable barrier important for hemostasis and wound healing and help explain how fibrinolysis is greatly retarded as clots contract.

Synthesis of potent and orally efficacious 11ß-hydroxysteroid dehydrogenase type 1 inhibitor HSD-016.

Cortisol and the glucocorticoid receptor (GR) signaling pathway has been linked to the development of diabetes and metabolic syndrome. In vivo, 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) catalyzes the conversion of inactive cortisone to its active form, cortisol. Existing clinical data have supported 11ß-HSD1 as a valid therapeutic target for type 2 diabetes. In our research program, (R)-1,1,1-trifluoro-2-(3-((R)-4-(4-fluoro-2-(trifluoromethyl)phenyl)-2-methylpiperazin-1-ylsulfonyl)phenyl)propan-2-ol (HSD-016) was discovered to be a potent, selective, and efficacious 11ß-HSD1 inhibitor and advanced as a clinical candidate. Herein, a reliable and scalable synthesis of HSD-016 is described. Key transformations include an asymmetric synthesis of a chiral tertiary alcohol via Sharpless dihydroxylation, epoxide formation, and subsequent mild reduction. This route ensured multikilogram quantities of HSD-016 necessary for clinical studies.

An improved method for determining enantiomeric excess by (13)C-NMR in chiral liquid crystal media.

By using a combination of inverse gated (1)H decoupled (13)C-NMR experiments1 with short acquisition times and NMR Cryo-probe technology, the sample requirements and experimental times necessary to accurately measure enantiomeric excess of small chiral molecules has been reduced 16-fold. Quality (13)C-NMR spectra can now be obtained from a 1 to 5 mg sample in 12 minutes. The enantiomeric excess determination achieved from the average integration of all the (13)C-resonances in the spectrum is comparable to enantiomeric excess measured by chiral SFC. The advantage of the NMR method is that enantiomeric excess can rapidly be measured in situ on practical amounts of enantioselective reaction products without the need for chromatographic separation or chemical modification and with substantially less solvent waste.

Solvation of carbohydrates in n,n'-dialkylimidazolium ionic liquids: a multinuclear NMR spectroscopy study.

The solvation of carbohydrates in N, N'-dialkylimidazolium ionic liquids (ILs) was investigated by means of 13C and 35/37Cl NMR relaxation and 1H pulsed field gradient stimulated echo (PFG-STE) diffusion measurements. Solutions of model sugars in 1- n-butyl-3-methylimidazolium chloride ([C4mim]Cl), 1-allyl-3-methylimidazolium chloride ([CC2mim]Cl), and 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]) were studied to evaluate the effects of cation and anion structure on the solvation mechanism. In all cases, the changes in the relaxation times of carbon nuclei of the IL cations as a function of carbohydrate concentration are small and consistent with the variation in solution viscosities. Conversely, the 35/37Cl and 13C relaxation rates of chloride ions and acetate ion carbons, respectively, have a strong dependency on sugar content. For [C2mim][OAc], the correlation times estimated from 13C relaxation data for both ions reveal that, as the carbohydrate concentration increases, the reorientation rate of the anion decreases faster than that of the cation. Although not as marked as the variations observed in the relaxation data, similar trends were obtained from the analysis of cation and, in the case of [C2mim][OAc], anion self-diffusion coefficients of the sugar/IL systems. Our results show that the interactions between the IL cation and the solutes are nonspecific, confirm that the process is governed by the interactions between the IL anion and the carbohydrate, and, more importantly, indicate no change in the solvation mechanism regardless of the structure of the anion.

Capillary scale NMR flow mapping.

Stopped-flow NMR at capillary scale has many advantages over traditional methods of introducing the sample into the probe, particularly when large numbers of samples must be examined. This work describes application of a simple method for direct visualization of a sample inside the flow cell of flow NMR systems to capillary scale analysis. We describe the details of the method and show how it can be used to measure the optimum flow rate for a capillary NMR system and how to determine the optimum sampling efficiency for small samples.

Preparation of alpha-haloacrylate derivatives via dimethyl sulfoxide-mediated selective dehydrohalogenation.

Dimethyl sulfoxide causes alpha,beta-dihalopropanoate derivatives to undergo efficient, selective dehydrohalogenation to form alpha-haloacrylate analogues. A variety of alpha-halo Michael acceptors were prepared in dimethyl sulfoxide under mild, base-free conditions, including the preparation of alpha-bromoacrolein and alpha-chloro- and bromoacrylonitriles. Synthesis of these molecules has been reported in the literature to be difficult. Among all the existing dehydrohalogenation procedures, this protocol is the most facile, practical, and environmentally benign process.

Synthesis of pyrazolo[1,5-alpha]pyrimidinone regioisomers.

This work describes two distinct routes to prepare pyrazolo[1,5-alpha]pyrimidin-7-ones and two distinct routes to prepare pyrazolo[1,5-alpha]pyrimidin-5-ones. Use of 1,3-dimethyluracil as the electrophile in the preparation of the pyrimidin-5-one regioisomer represents a correction of previously reported results. Also, a novel reaction to prepare this isomer was identified and the reaction mechanism elucidated. This work provides the experimentalist with complimentary synthetic pathways that afford either the pyrimidin-7-one or the pyrimidin-5-one regioisomer.

Identification of the degradation product of ezlopitant, a non-peptidic substance P antagonist receptor, by hydrogen deuterium exchange, electrospray ionization tandem mass spectrometry (ESI/MS/MS) and nuclear magnetic resonance (NMR) spectroscopy.

The degradation product of ezlopitant was isolated from low specific activity material and identified by solution phase hydrogen/deuterium (H/D) exchange and electrospray ionization tandem mass spectrometry (ESI/MS/MS) to be an isopropyl peroxide analog of ezlopitant. The structure of the degradant was further confirmed by nuclear magnetic resonance (NMR) spectroscopy utilizing complete 1H and 13C assignments. Studies were also performed to identify the factors responsible for the oxidative degradation of ezlopitant, which included salt form, storage conditions and salt formation solvent. Of all the variable studies over a 3 weeks period, only a change in the salt form prevented this oxidative degradation.