Predicting 19F NMR Chemical Shifts: A Combined Computational and Experimental Study of a Trypanosomal Oxidoreductase–Inhibitor Complex

The absence of fluorine from most biomolecules renders it an excellent probe for NMR spectroscopy to monitor inhibitor–protein interactions. However, predicting the binding mode of a fluorinated ligand from a chemical shift (or vice versa) has been challenging due to the high electron density of the fluorine atom. Nonetheless, reliable ¹⁹F chemical‐shift predictions to deduce ligand‐binding modes hold great potential for in silico drug design. Herein, we present a systematic QM/MM study to predict the ¹⁹F NMR chemical shifts of a covalently bound fluorinated inhibitor to the essential oxidoreductase tryparedoxin (Tpx) from African trypanosomes, the causative agent of African sleeping sickness. We include many protein–inhibitor conformations as well as monomeric and dimeric inhibitor–protein complexes, thus rendering it the largest computational study on chemical shifts of ¹⁹F nuclei in a biological context to date. Our predicted shifts agree well with those obtained experimentally and pave the way for future work in this area.     

Predicting 19F NMR Chemical Shifts: A Combined Computational and Experimental Study of a Trypanosomal Oxidoreductase–Inhibitor Complex

The absence of fluorine from most biomolecules renders it an excellent probe for NMR spectroscopy to monitor inhibitor–protein interactions. However, predicting the binding mode of a fluorinated ligand from a chemical shift (or vice versa) has been challenging due to the high electron density of the fluorine atom. Nonetheless, reliable ¹⁹F chemical-shift predictions to deduce ligand-binding modes hold great potential for in silico drug design. Herein, we present a systematic QM/MM study to predict the ¹⁹F NMR chemical shifts of a covalently bound fluorinated inhibitor to the essential oxidoreductase tryparedoxin (Tpx) from African trypanosomes, the causative agent of African sleeping sickness. We include many protein–inhibitor conformations as well as monomeric and dimeric inhibitor–protein complexes, thus rendering it the largest computational study on chemical shifts of ¹⁹F nuclei in a biological context to date. Our predicted shifts agree well with those obtained experimentally and pave the way for future work in this area.     

Biomimetic Synthesis of the Calcineurin Phosphatase Inhibitor Dibefurin

Dibefurin is a C_i‐symmetric natural product that acts as an inhibitor of calcineurin phosphatase. A six‐step synthesis of this compound is reported, which features an oxidative dimerization of the aromatic polyketide epicoccine as the key step. Dibefurin is proposed to be related to epicolactone, a complex yet racemic fungal metabolite that has recently been discovered. Attempts to access epicolactone from epicoccine and epicoccone B resulted in an unusual dimer that is formed through a hetero‐Diels–Alder reaction of a para‐quinone methide with an ortho‐quinone.     

Cellular Synthesis of Protein Catenanes

Direct cellular production of topologically complex proteins is of great interest both in supramolecular chemistry and protein engineering. We describe the first cellular synthesis of protein catenanes through the use of the p53 dimerization domain to guide the intertwining of two protein chains and SpyTag–SpyCatcher chemistry for efficient cyclization. The catenane topology was unambiguously proven by SDS‐PAGE, SEC, and partial digestion experiments and was shown to confer enhanced stability toward trypsin digestion relative to monomeric control mutants. The assembly–reaction synergy enabled by protein folding and genetically encoded protein chemistry offers a convenient yet powerful approach for creating mechanically interlocked, complex protein topologies in vivo.     

Discovery,Characterization, and Analogue Synthesis of Bohemamine Dimers Generated by Non‐enzymatic Biosynthesis

Dibohemamines A–C ( 5 – 7 ), three new dimeric bohemamine analogues dimerized through a methylene group, were isolated from a marine‐derived Streptomyces spinoverrucosus. The structures determined by spectroscopic analysis were confirmed through the semi‐synthetic derivatization of monomeric bohemamines and formaldehyde. These reactions, which could occur under mild conditions, together with the detection of formaldehyde in the culture, revealed that this dimerization is a non‐enzymatic process. In addition to the unique dimerization of the dibohemamines, dibohemamines B and C were found to have nm cytotoxicity against the non‐small cell‐lung cancer cell line A549. In view of the potent cytotoxicity of compounds 6 and 7 , a small library of bohemamine analogues was generated for biological evaluation by utilizing a series of aryl and alkyl aldehydes.     

Synthesis of methacrylate derivatives oligomers by dithiobenzoate‐RAFT‐mediated polymerization

Reversible addition fragmentation chain transfer (RAFT) was used to synthesize methacrylic acid oligomers and oligo(methacrylic acid)‐b‐poly(methyl methacrylate) (PMAA‐b‐PMMA) with targeted degree of polymerization ≈ 10. Characterization is by size‐exclusion chromatography (SEC) and electrospray mass‐spectrometry. SEC data are presented as hydrodynamic volume distributions (HVDs), the only proper means to present comparative and meaningful SEC data when there is no unique relationship between size and molecular weight. The RAFT agent, (4‐cyanopentanoic acid)‐4‐dithiobenzoate (CPADB), produced dithiobenzoic acid as a side product during the polymerization of methacrylate derivatives. Precipitation in diethyl ether proved to be an easy way to remove this impurity from the PMAA‐RAFT oligomers. Both unpurified and purified macro‐RAFT agent were used to prepare amphiphilic PMAA‐b‐PMMA copolymers. Diblock copolymer prepared from the purified PMAA homopolymer had a narrower HVD in comparison to those obtained from the equivalent unpurified macro‐RAFT agent. This work shows that while cyanoisopropyl‐dithiobenzoate or CPADB are good RAFT agents for methacrylate derivatives, they exhibit some instability under typical polymerization conditions, and thus when oligomers are targeted, optimal control requires checking for the degradation product and appropriate purification steps when necessary (the same effect is present for larger polymers but is unimportant). © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 2277–2289, 2008     

Halogen–Aromatic π Interactions Modulate Inhibitor Residence Times

Prolonged drug residence times may result in longer‐lasting drug efficacy, improved pharmacodynamic properties, and “kinetic selectivity” over off‐targets with high drug dissociation rates. However, few strategies have been elaborated to rationally modulate drug residence time and thereby to integrate this key property into the drug development process. Herein, we show that the interaction between a halogen moiety on an inhibitor and an aromatic residue in the target protein can significantly increase inhibitor residence time. By using the interaction of the serine/threonine kinase haspin with 5‐iodotubercidin (5‐iTU) derivatives as a model for an archetypal active‐state (type I) kinase–inhibitor binding mode, we demonstrate that inhibitor residence times markedly increase with the size and polarizability of the halogen atom. The halogen–aromatic π interactions in the haspin–inhibitor complexes were characterized by means of kinetic, thermodynamic, and structural measurements along with binding‐energy calculations.     

Development and validation of a high‐performance liquid chromatography–tandem mass spectrometry method for the determination of the novel proteasome inhibitor CEP‐18770 in human plasma and its application in a clinical pharmacokinetic study

CEP‐18770, [(1R)‐1‐{[(2S,3R)‐3‐hydroxy‐2‐{[(6‐phenyl‐2‐pyridinyl)carbonyl]amino}butanoyl]amino}‐3‐methylbutyl]boronic acid, is a novel proteasome inhibitor, now under early clinical evaluation as an anticancer agent. To investigate its clinical pharmacokinetics, a high‐performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method was developed and validated to measure the drug in human plasma, based on simple protein precipitation with acetonitrile after the addition of irbesartan as internal standard. The method requires a small volume of sample (100 µl) and is rapid and selective, allowing good resolution of peaks in 5 min. It is sensitive, precise and accurate, with overall precision, expressed as coefficient of variation (CV%), always < 10.0%, accuracy in the range 93.8–107.7% and high recovery, close to 100%. The limit of detection is 0.01 ng/ml and the lower limit of quantitation (LLOQ) is 0.20 ng/ml. The assay was validated in the range from the LLOQ up to 50.00 ng/ml. This is the first method developed and validated for analyzing a proteasome inhibitor with a boronic‐acid‐based structure in human plasma. The method was successfully applied to study the pharmacokinetics of CEP‐18770 in cancer patients with solid tumors or multiple myeloma who had received the drug as a short intravenous bolus during the initial Phase I trial. Copyright © 2010 John Wiley & Sons, Ltd.     

Fractionation of ethylene/1‐pentene copolymers using a combination of SEC‐FTIR and SEC‐HPer DSC

The short chain branching distribution (SCBD) and thermal properties of ethylene/1‐pentene copolymers were studied using SEC‐FTIR and SEC‐HPer DSC. The copolymers, synthesized with Cp₂ZrCl₂/MAO, were fractionated using size exclusion chromatography (SEC). The infrared analysis of the fractions showed that the copolymers had—on average—higher 1‐pentene concentration in the low molecular weight range. Furthermore, the thermal properties of the SEC deposits of these copolymers on a Germanium disc were studied using high performance differential scanning calorimetry (HPer DSC). Single SEC separations were used to accumulate fractions in the microgram range that were directly analyzed with regard to their thermal properties, thus allowing us to study SCBD as well as thermal behavior simultaneously. When these fractions (with masses ranging from 10–80 μg) were analyzed using HPer DSC, good melting and crystallization temperature distributions were obtained, proving that HPer DSC can be used as a complementary method to SEC‐FTIR. © 2007 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 45: 2956–2965, 2007     

An Immucillin‐Based Transition‐State‐Analogous Inhibitor of tRNA–Guanine Transglycosylase (TGT)

Shigellosis is one of the most severe diarrheal diseases worldwide without any efficient treatment so far. The enzyme tRNA–guanine transglycosylase (TGT) has been identified as a promising target for small‐molecule drug design. Herein, we report a transition‐state analogue, a small, immucillin‐derived inhibitor, as a new lead structure with a novel mode of action. The complex inhibitor synthesis was accomplished in 18 steps with an overall yield of 3 %. A co‐crystal structure of the inhibitor bound to Z. mobilis TGT confirmed the predicted conformation of the immucillin derivative in the enzyme active site.     

Reductive dimerization and reduction of imines using lanthanum metal

The treatment of N‐benzylideneaniline ( 1a ) with a half‐equivalent of lanthanum metal and a catalytic amount of iodine gave the reductive dimerization product of 1a , a vic‐diamine, in good yield. Various vic‐diamines were synthesized from aldimines in this manner in moderate to good yields. Our findings suggest that electrons of a zero‐valent lanthanum metal were efficiently utilized in this reductive dimerization. In the reaction of ketimines, however, a similar reductive dimerization did not take place, and the corresponding amines were formed as the sole products. © 2002 Wiley Periodicals, Inc. Heteroatom Chem 13:131–135, 2002; Published online in Wiley Interscience (www.interscience.wiley.com). DOI 10.1002/hc.10007     

Interaction of [VIVO(acac)2] with Human Serum Transferrin and Albumin

[VO(acac)₂] is a remarkable vanadium compound and has potential as a therapeutic drug. It is important to clarify how it is transported in blood, but the reports addressing its binding to serum proteins have been contradictory. We use several spectroscopic and mass spectrometric techniques (ESI and MALDI‐TOF), small‐angle X‐ray scattering and size exclusion chromatography (SEC) to characterize solutions containing [VO(acac)₂] and either human serum apotransferrin (apoHTF) or albumin (HSA). DFT and modeling protein calculations are carried out to disclose the type of binding to apoHTF. The measured circular dichroism spectra, SEC and MALDI‐TOF data clearly prove that at least two VO–acac moieties may bind to apoHTF, most probably forming [V^IVO(acac)(apoHTF)] complexes with residues of the HTF binding sites. No indication of binding of [VO(acac)₂] to HSA is obtained. We conclude that V^IVO–acac species may be transported in blood by transferrin. At very low complex concentrations speciation calculations suggest that [(VO)(apoHTF)] species form.     

Drug‐Loaded Elastin‐Like Polypeptide–Collagen Hydrogels with High Modulus for Bone Tissue Engineering

Emphasizing the role of hydrogel stiffness and cellular differentiation, this study develops collagen and elastin‐like polypeptide (ELP)–based bone regenerative hydrogels loaded with recombinant human bone morphogenetic protein‐2 (rhBMP‐2) and doxycycline with mechanical properties suitable for osteogenesis. The drug‐incorporated collagen–ELP hydrogels has significantly higher modulus of 35 ± 5 kPa compared to collagen‐only hydrogels. Doxycycline shows a bi‐phasic release with an initial burst release followed by a gradual release, while rhBMP‐2 exhibits a nearly linear release profile for all hydrogels. The released doxycycline shows anti‐microbial activity against Pseudomonas aeruginosa, Streptococcus sanguinis, and Escherichia coli. Microscopic observation of the hydrogels reveals their interconnected, macroporous, 3D open architecture with pore diameters between 160 and 400 µm. This architecture supports human adipose–derived stem cell attachment and proliferation from initial days of cell seeding, forming a thick cellular sheath by day 21. Interestingly, in collagen and collagen–ELP hydrogels, cell morphology is elongated with stretched slender lamellipodial formation, while cells assemble as spheroidal aggregates in crosslinked as well as drug‐loaded hydrogels. Osteogenic markers, alkaline phosphatase and osteocalcin, are expressed maximally for drug‐loaded hydrogels compared to those without drugs. The drug‐loaded collagen–ELP hydrogels are thus promising for combating bacterial infection and promoting guided bone regeneration.     

Identification of [(GS)2AsSe]− in rabbit bile by size‐exclusion chromatography and simultaneous multielement‐specific detection by inductively coupled plasma atomic emission spectroscopy

An arsenic–selenium metabolite that exhibited the same arsenic and selenium X‐ray absorption near‐edge spectra as the synthetic seleno‐bis(S‐glutathionyl) arsinium ion [(GS)₂AsSe]^? was recently detected in rabbit bile within 25 min after intravenous injection of rabbits with sodium selenite and sodium arsenite. X‐ray absorption spectroscopy did not (and cannot) conclusively identify the sulfur‐donor in the in vivo sample. After similar treatment of rabbits, we analyzed the collected bile samples by size‐exclusion chromatography (SEC) using inductively coupled plasma atomic emission spectroscopy (ICP‐AES) to monitor arsenic, selenium and sulfur simultaneously. The bulk of arsenic and selenium eluted in a single peak, the intensity of which was greatly increased upon spiking of the bile samples with synthethic [(GS)₂AsSe]^?. Hence, we identify [(GS)₂AsSe]^? as the major metabolite in bile after exposure of rabbits to selenite and arsenite. The reported SEC–ICP‐AES method is the first chromatographic procedure to identify this biochemically important metabolite in biological fluids and is thus a true alternative to X‐ray absorption spectroscopy, which is not available to many chemists. Copyright © 2001 John Wiley & Sons, Ltd.     

Nonfitting protein–ligand interaction scoring function based on first‐principles theoretical chemistry methods: Development and application on kinase inhibitors

Targeted therapy is currently a hot topic in the fields of cancer research and drug design. An important requirement for this approach is the development of potent and selective inhibitors for the identified target protein. However, current ways to estimate inhibitor efficacy rely on empirical protein–ligand interaction scoring functions which, suffering from their heavy parameterizations, often lead to a low accuracy. In this work, we develop a nonfitting scoring function, which consists of three terms: (1) gas‐phase protein‐ligand binding enthalpy obtained by the eXtended ONIOM hybrid method based on an integration of density functional theory (DFT) methods (XYG3 and ωB97X‐D) and the semiempirical PM6 method, (2) solvation free energy based on DFT‐SMD solvation model, and (3) entropy effect estimated by using DFT frequency analysis. The new scoring function is tested on a cyclin‐dependent kinase 2 (CDK2) inhibitor database including 76 CDK2 protein inhibitors and a p21‐activated kinase 1 (PAK1) inhibitor database including 20 organometallic PAK1 protein inhibitors. From the results, good correlations are found between the calculated scores and the experimental inhibitor efficacies with the square of correlation coefficient R² of 0.76–0.88. This suggests a good predictive power of this scoring function. To the best of our knowledge, this is the first high level theory‐based nonfitting scoring function with such a good level of performance. This scoring function is recommended to be used in the final screening of lead structure derivatives. © 2013 Wiley Periodicals, Inc.     

A Suite of “Minimalist” Photo‐Crosslinkers for Live‐Cell Imaging and Chemical Proteomics: Case Study with BRD4 Inhibitors

Affinity‐based probes (Af BPs) provide a powerful tool for large‐scale chemoproteomic studies of drug–target interactions. The development of high‐quality probes capable of recapitulating genuine drug–target engagement, however, could be challenging. “Minimalist” photo‐crosslinkers, which contain an alkyl diazirine group and a chemically tractable tag, could alleviate such challenges, but few are currently available. Herein, we have developed new alkyl diazirine‐containing photo‐crosslinkers with different bioorthogonal tags. They were subsequently used to create a suite of Af BPs based on GW841819X (a small molecule inhibitor of BRD4). Through in vitro and in situ studies under conditions that emulated native drug–target interactions, we have obtained better insights into how a tag might affect the probe's performance. Finally, SILAC‐based chemoproteomic studies have led to the discovery of a novel off‐target, APEX1. Further studies showed GW841819X binds to APEX1 and caused up‐regulation of endogenous DNMT1 expression under normoxia conditions.     

A Diene‐Containing Noncanonical Amino Acid Enables Dual Functionality in Proteins: Rapid Diels–Alder Reaction with Maleimide or Proximity‐Based Dimerization

Here, we describe a diene‐containing noncanonical amino acid (ncAA) capable of undergoing fast and selective normal electron‐demand Diels–Alder (DA) reactions following its incorporation into antibodies. A cyclopentadiene derivative of lysine (CpHK) served as the reactive handle for DA transformations and the substrate for genetic incorporation. CpHK incorporated into antibodies with high efficiency and was available for maleimide conjugation or self‐reaction depending on position in the amino acid sequence. CpHK at position K274 reacted with the maleimide drug‐linker AZ1508 at a rate of ≈79 m ^?1 s^?1 to produce functional antibody–drug conjugates (ADCs) in a one‐step process. Incorporation of CpHK at position S239 resulted in dimerization, which covalently linked antibody heavy chains together. The diene ncAA described here is capable of producing therapeutic protein conjugates with clinically validated and widely available maleimide compounds, while also enabling proximity‐based stapling through a DA dimerization reaction.     

Identification and characterization of fluvastatin metabolites in rats by UHPLC/Q‐TOF/MS/MS and in silico toxicological screening of the metabolites

The present study reports the in vivo and in vitro identification and characterization of metabolites of fluvastatin, the 3‐hydroxy‐3‐methyl‐glutaryl‐coenzyme A reductase inhibitor, using liquid chromatography–mass spectrometry (LC–MS). In vitro studies were conducted by incubating the drug with human liver microsomes and rat liver microsomes. In vivo studies were carried out by administration of the drug in the form of suspension to the Sprague–Dawley rats followed by collection of urine, faeces and blood at different time points up to 24 h. Further, samples were prepared by optimized sample preparation method, which includes freeze liquid extraction, protein precipitation and solid phase extraction. The extracted and concentrated samples were analysed using ultrahigh‐performance liquid chromatography–quadruple time‐of‐flight tandem mass spectrometry. A total of 15 metabolites were observed in urine, which includes hydroxyl, sulphated, desisopropyl, dehydrogenated, dehydroxylated and glucuronide metabolites. A few of the metabolites were also present in faeces and plasma samples. In in vitro studies, a few metabolites were observed that were also present in in vivo samples. All the metabolites were characterized using ultrahigh‐performance liquid chromatography–quadruple time‐of‐flight tandem mass spectrometry in combination with accurate mass measurement. Finally, in silico toxicity studies indicated that some of the metabolites show or possess carcinogenicity and skin sensitization. Several metabolites that were identified in rats are proposed to have toxicological significance on the basis of in silico evaluation. However, these metabolites are of no human relevance. Copyright © 2017 John Wiley & Sons, Ltd.     

Lysine‐Targeting Covalent Inhibitors

Targeted covalent inhibitors have gained widespread attention in drug discovery as a validated method to circumvent acquired resistance in oncology. This strategy exploits small‐molecule/protein crystal structures to design tightly binding ligands with appropriately positioned electrophilic warheads. Whilst most focus has been on targeting binding‐site cysteine residues, targeting nucleophilic lysine residues can also represent a viable approach to irreversible inhibition. However, owing to the basicity of the ϵ ‐amino group in lysine, this strategy generates a number of specific challenges. Herein, we review the key principles for inhibitor design, give historical examples, and present recent developments that demonstrate the potential of lysine targeting for future drug discovery.