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Chronic infections with Pseudomonas aeruginosa are associated with the formation of bacterial biofilms. The tetrameric P. aeruginosa lectin LecA is a virulence factor and an anti-biofilm drug target. Increasing the overall binding affinity by multivalent presentation of binding epitopes can enhance the weak carbohydrate-ligand interactions. Low-nanomolar divalent LecA ligands/inhibitors with up to 260-fold valency-normalized potency boost and excellent selectivity over human galectin-1 were synthesized from d-galactose pentaacetate and benzaldehyde-based linkers in four linear steps.

Among non-covalent interactions, B-H⋯π and C-H⋯π hydrogen bonding is rather weak and less studied. Nevertheless, since both can affect the energetics of protein-ligand binding, their understanding is an important prerequisite for reliable predictions of affinities. Through a combination of high-resolution X-ray crystallography and quantum-chemical calculations on carbonic anhydrase II/carborane-based inhibitor systems, this paper provides the first example of B-H⋯π hydrogen bonding in a protein-ligand complex. It shows that the B-H⋯π interaction is stabilized by dispersion, followed by electrostatics. Furthermore, it demonstrates that the similar C-H⋯π interaction is twice as strong, with a slightly smaller contribution of dispersion and a slightly higher contribution of electrostatics. Such a detailed insight will facilitate the rational design of future protein ligands, controlling these types of non-covalent interactions.

DNA-binding fluorescent probes are useful tools in cell biology for various applications enabling visualisation and quantification of DNA. Herein, we report on HeliDye1, a novel cationic helicene-like DNA probe, and its versatile usability. HeliDye1 exhibits fluorescence exclusively in the presence of DNA duplexes containing AT base pairs and (contrary to any recent DNA probe) it remains non-fluorescent in the presence of single-stranded DNA and RNA. Favourable chemical properties of HeliDye1 allowed its use in karyotyping and super-resolution microscopy. We present five more applications of HeliDye1. Viscosimetry showed that HeliDye1 is a minor groove binder. HeliDye1 interacts with double-stranded DNA as an induced (P)-enantiomer, which we confirmed by ECD spectroscopy and DFT calculations. ECD spectroscopy suggested the formation of HeliDye1 dimers. MD simulations predicted the structures of (P)-HeliDye1 dimers inside DNA and showed their stability in AT-rich sequences and instability in GC-rich sequences.

The C-type lectin receptor DC-SIGN has been highlighted as co-receptor for the spike protein of the SARS-CoV-2 virus. A multivalent glycomimetic ligand, Polyman26, has been found to inhibit DCSIGN- dependent trans-infection of SARS-CoV-2. The molecular details underlying avidity generation in such systems remain poorly characterized. In an effort to dissect the contribution of the known multivalent effects - chelation, clustering and statistical rebinding – we studied a series of dendrimer constructs related to Polyman26 with a rod core rationally designed to engage simultaneously two binding sites of the tetrameric DC-SIGN. Binding properties of these compounds have been studied with a range of biophysical techniques, including recently developed Surface Plasmon Resonance oriented-surface methodology. Using molecular modelling we addressed, for the first time, the impact of the carbohydrate recognition domains’ flexibility of the DC-SIGN tetramer on the compounds’ avidity. We were able to gain deeper insight into the role of different binding modes, which in combination produce a construct with a nM affinity despite a limited valency. This multi-faceted experimental-theoretical approach provides detailed understanding of multivalent ligand/multimeric protein interactions which can lead to future predictions. This work opens the way to the development of new virus attachment blockers adapted to different C-type lectin receptors of viruses.

The 3H-pyrazolo[4,3-f]quinoline moiety has been recently shown to be a privileged kinase inhibitor core with potent activities against acute myeloid leukemia (AML) cell lines in vitro. Herein, various 3H-pyrazolo[4,3-f]quinoline-containing compounds were rapidly assembled via the Doebner-Povarov multicomponent reaction from the readily available 5-aminoindazole, ketones, and heteroaromatic aldehydes in good yields. The most active compounds potently inhibit the recombinant FLT3 kinase and its mutant forms with nanomolar IC50 values. Docking studies with the FLT3 kinase showed a type I binding mode, where the 3H-pyrazolo group interacts with Cys694 in the hinge region. The compounds blocked the proliferation of AML cell lines harboring oncogenic FLT3-ITD mutations with remarkable IC50 values, which were comparable to the approved FLT3 inhibitor quizartinib. The compounds also inhibited the growth of leukemia in a mouse-disseminated AML model, and hence, the novel 3H-pyrazolo[4,3-f]quinoline-containing kinase inhibitors are potential lead compounds to develop into anticancer agents, especially for kinase-driven cancers.

Pathogenic Candida albicans yeasts frequently cause infections in hospitals. Antifungal drugs lose effectiveness due to other Candida species and resistance. New medications are thus required. Secreted aspartic protease of C. parapsilosis (Sapp1p) is a promising target. We have thus solved the crystal structures of Sapp1p complexed to four peptidomimetic inhibitors. Three potent inhibitors (Ki: 0.1, 0.4, 6.6 nM) resembled pepstatin A (Ki: 0.3 nM), a general aspartic protease inhibitor, in terms of their interactions with Sapp1p. However, the weaker inhibitor (Ki: 14.6 nM) formed fewer nonpolar contacts with Sapp1p, similarly to the smaller HIV protease inhibitor ritonavir (Ki: 1.9 µM), which, moreover, formed fewer H-bonds. The analyses have revealed the structural determinants of the subnanomolar inhibition of C. parapsilosis aspartic protease. Because of the high similarity between Saps from different Candida species, these results can further be used for the design of potent and specific Sap inhibitor-based antimycotic drugs.

Azapeptide nitriles are postulated to reversibly covalently react with the active-site cysteine residue of cysteine proteases and form isothiosemicarbazide adducts. We investigated the interaction of azadipeptide nitriles with the cathepsin B1 drug target (SmCB1) from Schistosoma mansoni, a pathogen that causes the global neglected disease schistosomiasis. Azadipeptide nitriles were superior inhibitors of SmCB1 over their parent carba analogs. We determined the crystal structure of SmCB1 in complex with an azadipeptide nitrile and analyzed the reaction mechanism using quantum chemical calculations. The data demonstrate that azadipeptide nitriles, in contrast to their carba counterparts, undergo a change from E- to Z-configuration upon binding, which gives rise to a highly favorable energy profile of noncovalent and covalent complex formation. Finally, azadipeptide nitriles were considerably more lethal than their carba analogs against the schistosome pathogen in culture, supporting the further development of this chemotype as a treatment for schistosomiasis.

The success of approximate computational methods, such as molecular mechanics, or dispersion-corrected density functional theory, in the description of non-covalent interactions relies on accurate parameterizations. Benchmark data sets are thus required. This area is well developed for organic molecules and biomolecules but practically non-existent for boron clusters, which have been gaining in importance in modern drug as well as material design. To fill this gap, we have introduced two data sets featuring the most common non-covalent interaction of boron clusters, the dihydrogen bond, and calculated reference interaction energies at the "golden standard" CCSD(T)/CBS level. The boron clusters studied interact with formamide, methanol, water and methane at various distances and in two geometrical arrangements. The performance of the tested approximate methods is variable and recommendations for further use are given.

RNA–peptide/protein interactions have been of utmost importance to life since its earliest forms, reaching even before the last universal common ancestor (LUCA). However, the ancient molecular mechanisms behind this key biological interaction remain enigmatic because extant RNA–protein interactions rely heavily on positively charged and aromatic amino acids that were absent (or heavily under-represented) in the early pre-LUCA evolutionary period. Here, an RNA-binding variant of the ribosomal uL11 C-terminal domain was selected from an approximately 1010 library of partially randomized sequences, all composed of ten prebiotically plausible canonical amino acids. The selected variant binds to the cognate RNA with a similar overall affinity although it is less structured in the unbound form than the wild-type protein domain. The variant complex association and dissociation are both slower than for the wild-type, implying different mechanistic processes involved. The profile of the wild-type and mutant complex stabilities along with molecular dynamics simulations uncovers qualitative differences in the interaction modes. In the absence of positively charged and aromatic residues, the mutant uL11 domain uses ion bridging (K+/Mg2+) interactions between the RNA sugar-phosphate backbone and glutamic acid residues as an alternative source of stabilization. This study presents experimental support to provide a new perspective on how early protein–RNA interactions evolved, where the lack of aromatic/basic residues may have been compensated by acidic residues plus metal ions.

Galectin-1 is a β-galactoside-binding lectin with manifold biological functions. A single tryptophan residue (W68) in its carbohydrate binding site plays a major role in ligand binding and is highly conserved among galectins. To fine tune galectin-1 specificity, we introduced several non-canonical tryptophan analogues at this position of human galectin-1 and analyzed the resulting variants using glycan microarrays. Two variants containing 7-azatryptophan and 7-fluorotryptophan showed a reduced affinity for 3'-sulfated oligosaccharides. Their interaction with different ligands was further analyzed by fluorescence polarization competition assay. Using molecular modeling we provide structural clues that the change in affinities comes from modulated interactions and solvation patterns. Thus, we show that the introduction of subtle atomic mutations in the ligand binding site of galectin-1 is an attractive approach for fine-tuning its interactions with different ligands.

Information on how insulin and insulin-like growth factors 1 and 2 (IGF-1 and -2) activate insulin receptors (IR-A and -B) and the IGF-1 receptor (IGF-1R) is crucial for understanding the difference in the biological activities of these peptide hormones. Cryo-EM studies have revealed that insulin uses its binding sites 1 and 2 to interact with IR-A and have identified several critical residues in binding site 2. However, mutagenesis studies suggest that Ile-A10, Ser-A12, Leu-A13, and Glu-A17 also belong to insulin's site 2. Here, to resolve this discrepancy, we mutated these insulin residues and the equivalent residues in IGFs. Our findings revealed that equivalent mutations in the hormones can result in differential biological effects and that these effects can be receptor-specific. We noted that the insulin positions A10 and A17 are important for its binding to IR-A and IR-B and IGF-1R and that A13 is important only for IR-A and IR-B binding. The IGF-1/IGF-2 positions 51/50 and 54/53 did not appear to play critical roles in receptor binding, but mutations at IGF-1 position 58 and IGF-2 position 57 affected the binding. We propose that IGF-1 Glu-58 interacts with IGF-1R Arg-704 and belongs to IGF-1 site 1, a finding supported by the NMR structure of the less active Asp-58-IGF-1 variant. Computational analyses indicated that the aforementioned mutations can affect internal insulin dynamics and inhibit adoption of a receptor-bound conformation, important for binding to receptor site 1. We provide a molecular model and alternative hypotheses for how the mutated insulin residues affect activity.

RNA-peptide/protein interactions have been of utmost importance to life since its earliest forms, reaching even before the last universal common ancestor (LUCA). However, the ancient molecular mechanisms behind this key biological interaction remain enigmatic because extant RNA-protein interactions rely heavily on positively charged and aromatic amino acids that were absent (or heavily under-represented) in the early pre-LUCA evolutionary period. Here, an RNA-binding variant of the ribosomal L11 C-terminal domain was selected from a ∼1010 library of partially randomized sequences, all composed of 10 prebiotically plausible canonical amino acids. The selected variant binds to the cognate RNA with a similar overall affinity although it is less structured in the unbound form than the wild-type protein domain. The variant complex association and dissociation are both slower than for the wild-type, implying different mechanistic processes involved. The profile of the wild-type and mutant complex stabilities along with MD simulations uncover qualitative differences in the interaction modes. In the absence of positively charged and aromatic residues, the mutant L11 domain uses bridging ion (K+/Mg2+) interactions between the RNA sugar-phosphate backbone and glutamic acid residues as an alternative source of stabilization. This study presents experimental support to provide a new perspective on how early protein-RNA interactions evolved, where the lack of aromatic/basic residues was compensated by acidic residues plus metal ions.

Transient and fuzzy intermolecular interactions are fundamental to many biological processes. Despite their importance, they are notoriously challenging to characterize. Effects induced by paramagnetic ligands in the NMR spectra of interacting biomolecules provide an opportunity to amplify subtle manifestations of weak intermolecular interactions observed for diamagnetic ligands. Here, we present an approach to characterizing dynamic interactions between a partially flexible dimeric protein, HIV-1 protease, and a metallacarborane-based ligand, a system for which data obtained by standard NMR approaches do not enable detailed structural interpretation. We show that for the case where the experimental data are significantly averaged to values close to zero the standard fitting of pseudocontact shifts cannot provide reliable structural information. We based our approach on generating a large ensemble of full atomic models, for which the experimental data can be predicted, ensemble averaged and finally compared to the experiment. We demonstrate that a combination of paramagnetic NMR experiments, quantum chemical calculations, and molecular dynamics simulations offers a route towards structural characterization of dynamic protein-ligand complexes.

Schistosomiasis, a parasitic disease caused by blood flukes of the genus Schistosoma, is a global health problem with over 200 million people infected. Treatment relies on just one drug, and new chemotherapies are needed. Schistosoma mansoni cathepsin B1 (SmCB1) is a critical peptidase for the digestion of host blood proteins and a validated drug target. We screened a library of peptidomimetic vinyl sulfones against SmCB1 and identified the most potent SmCB1 inhibitors reported to date that are active in the subnanomolar range with second order rate constants (k2nd) of ∼2 × 105 M-1 s-1. High resolution crystal structures of the two best inhibitors in complex with SmCB1 were determined. Quantum chemical calculations of their respective binding modes identified critical hot spot interactions in the S1' and S2 subsites. The most potent inhibitor targets the S1' subsite with an N-hydroxysulfonic amide moiety and displays favorable functional properties, including bioactivity against the pathogen, selectivity for SmCB1 over human cathepsin B, and reasonable metabolic stability. Our results provide structural insights for the rational design of next-generation SmCB1 inhibitors as potential drugs to treat schistosomiasis.

CYC116 is a selective Aurora kinase inhibitor that has been tested in a Phase I study in patients with advanced solid tumors. Although CYC116 has shown desirable preclinical efficacy, the potential for emergence of resistance has not been explored. We established several CYC116 resistant clones from isogenic HCT116 p53+/+ and HCT116 p53−/− cell line pairs. We also generated resistant clones towards ZM447439 (quinazoline derivative), a model Aurora inhibitor. The selected clones were 10-80 fold resistant to CYC116 and cross-resistant to other synthetic Aurora inhibitors including AZD1152, VX-680, and MLN8054. Resistant clones displayed multidrug resistant phenotypes, tested by using 13 major cytostatics. All clones were highly resistant to etoposide followed by other drugs. Interestingly, all CYC116 clones but not ZM447439 became polyploid. ZM447439, but not CYC116 induced three novel mutations in Aurora B. Leu152Ser significantly affected ZM447439 binding, but not CYC116. Gene expression studies revealed differential expression of more than 200 genes. Some of these genes expression profiles were also observed in CYC116 resistant primary tumors. Bcl-xL (BCL2L1) was found to be overexpressed in CYC116 clones and its knockdown resensitized the p53+/+ resistant clone to CYC116. Finally Bcl-xL overexpressing p53+/+ CYC116 clones were highly sensitive to navitoclax (ABT-263) compared to parent cells. The data shed light on the genetic basis for resistance to Aurora kinase inhibitors which could be used to predict clinical response, to select patients who might benefit from therapy and to suggest suitable drug combinations for a particular patient population.

Multi-orthogonal molecular scaffolds can be applied as core structures of bioactive compounds. Here, we prepared four tri-orthogonal scaffolds based on adamantane or proline skeletons. The scaffolds were used for the solid-phase synthesis of model insulin mimetics bearing two different peptides on the scaffolds. We found that adamantane-derived compounds bind to the insulin receptor more effectively (Kd value of 0.5 μM) than proline-derived compounds (Kd values of 15–38 μM) bearing the same peptides. Molecular dynamics simulations suggest that spacers between peptides and central scaffolds can provide greater flexibility that can contribute to increased binding affinity. Molecular modeling showed possible binding modes of mimetics to the insulin receptor. Our data show that the structure of the central scaffold and flexibility of attached peptides in this type of compound are important and that different scaffolds should be considered when designing peptide hormone mimetics.

We report on the discovery of norbornyl moiety as a novel structural motif for cyclin-dependent kinase 2 (CDK2) inhibitors which was identified by screening a carbocyclic nucleoside analogue library. Three micromolar hits were expanded by the use of medicinal chemistry methods into a series of 16 novel compounds. They had prevailingly micromolar activities against CDK2 and the best compound of the series attained IC50 of 190 nM. The binding modes were explored in molecular details by modeling and docking. Quantum mechanics-based scoring was used to rationalize the affinities. In conclusion, the discovered 9-hydroxymethylnorbornyl moiety was shown by joint experimental-theoretical efforts to be able to serve as a novel substituent for CDK2 inhibitors. This finding opens door to the exploration of chemical space towards more effective derivatives targeting this important class of protein kinases.

A chalcogen bond is a nonclassical noncovalent interaction which can stabilise small-molecule crystals as well as protein structures. Here, we systematically explore the stabilising potential of chalcogen bonding in protein-ligand complexes in the Protein Data Bank (PDB). We have found that a large fraction (23 %) of complexes with a S/Se-containing ligand feature close S/Se⋅⋅⋅O/N/S contacts. Eleven non-redundant representative potential S/Se⋅⋅⋅O chalcogen-bond motifs were selected and truncated to model systems and seven more model systems were prepared by S-to-Se substitution. These systems were then subjected to analysis by quantum chemical (QM) methods-electrostatic potential, geometry optimisation or interaction energy calculations, including solvent effects. The QM calculations indicate that chalcogen bonding does indeed play a dominant role in stabilising some of the interaction motifs studied. We thus advocate further exploration of chalcogen bonding with the aim of potential future use in structure-based drug design.

FLT3 tyrosine kinase is a potential drug target in acute myeloid leukemia (AML) because patients with FLT3-ITD mutations respond poorly to standard cytotoxic agents and there is a clear link between the disease and the oncogenic properties of FLT3. We present novel 2,6,9-trisubstituted purine derivatives with potent FLT3 inhibitory activity. The lead compound 7d displays nanomolar activity in biochemical assays and selectively blocks proliferation of AML cell lines harboring FLT3-ITD mutations, whereas other transformed and normal human cells are several orders of magnitude less sensitive. The MV4-11 cells treated with 7d suppressed the phosphorylation of FLT3 and its downstream signaling pathways, with subsequent G1 cell cycle arrest and apoptosis. Additionally, a single dose of 7d in mice with subcutaneous MV4-11 xenografts caused sustained inhibition of FLT3 and STAT5 phosphorylation over 48 h, in contrast to the shorter effect observed after administration of the reference FLT3 inhibitor quizartinib.

Accurate prediction of protein-ligand binding affinities is essential for hit-to-lead optimization and virtual screening. The reliability of scoring functions can be improved by including quantum effects. Here, we demonstrate the ranking power of the semiempirical quantum mechanics (SQM)/implicit solvent (COSMO) scoring function by using a challenging set of 10 inhibitors binding to carbonic anhydrase II through Zn

General and reliable description of structures and energetics in protein–ligand (PL) binding using the docking/scoring methodology has until now been elusive. We address this urgent deficiency of scoring functions (SFs) by the systematic development of corrected semiempirical quantum mechanical (SQM) methods, which correctly describe all types of noncovalent interactions and are fast enough to treat systems of thousands of atoms. Two most accurate SQM methods, PM6-D3H4X and SCC-DFTB3-D3H4X, are coupled with the conductor-like screening model (COSMO) implicit solvation model in so-called “SQM/COSMO” SFs and have shown unique recognition of native ligand poses in cognate docking in four challenging PL systems, including metalloprotein. Here, we apply the two SQM/COSMO SFs to 17 diverse PL complexes and compare their performance with four widely used classical SFs (Glide XP, AutoDock4, AutoDock Vina, and UCSF Dock). We observe superior performance of the SQM/COSMO SFs and identify challenging systems. This method, due to its generality, comparability across the chemical space, and lack of need for any system-specific parameters, gives promise of becoming, after comprehensive large-scale testing in the near future, a useful computational tool in structure-based drug design and serving as a reference method for the development of other SFs.

We have recently introduced the “SQM/COSMO” scoring function which combines a semiempirical quantum mechanical description of noncovalent interactions at the PM6-D3H4X level and the COSMO implicit model of solvation. This approach outperformed standard scoring functions but faced challenges with a metalloprotein featuring a Zn2+···S– interaction. Here, we invoke SCC-DFTB3-D3H4, a higher-level SQM method, and observe improved behavior for the metalloprotein and high-quality results for the other systems. This method holds promise for diverse protein–ligand complexes including metalloproteins.

Close B–H⋯π contacts have recently been observed in crystallographic structures of Ir–dithiolene–phosphine complexes containing boron hydride cluster. This finding was interpreted using quantum chemical calculations as a new type of electrostatically driven nonclassical hydrogen bonding. However, such an explanation contradicts the wealth of evidence for unique noncovalent interactions of boron hydrides. Moreover, care must be exercised when computational methods are used to interpret new phenomena. Therefore, here, we cautiously examine the B–H⋯π interaction by means of advanced quantum chemistry and disprove the claimed attractive electrostatic nature and rather define it as a nonspecific dispersion-driven contact. In summary, we present evidence that the crystallographically observed B–H⋯π contacts do not constitute a novel type of hydrogen bonding of boron hydride clusters.

Pathogenic micro-organisms utilize protein receptors in adhesion to host tissues, a process that in some cases relies on the interaction between lectin and human glycoconjugates. Oligosaccharide epitopes are recognized through their three-dimensional structure and their flexibility is a key issue in specificity. In this paper, we analyse by X-ray crystallography the structures of the lectin LecB from two strains of Pseudomonas aeruginosa in complex with Lewis x oligosaccharide present on cell surfaces of human tissues. An unusual conformation of the glycan was observed in all binding sites with a non-canonical syn orientation of the N-acetyl group of N-acetyl-glucosamine. A PDB-wide search revealed that such an orientation occurs only in 2% of protein/carbohydrate complexes. Theoretical chemistry calculations showed that the observed conformation is unstable in solution but stabilised by the lectin. A reliable description of LecB/Lewis x complex by force field-based methods had proven as especially challenging due to the special feature of the binding site, two closely apposed Ca2+ ions which induce strong charge delocalisation. By comparing various force-field parametrisations, we design general protocols which will be useful in near future for designing carbohydrate-based ligands (glycodrugs) against other calcium-dependent protein receptors.

Quantum mechanical (QM) methods have been gaining importance in structure-based drug design where a reliable description of protein-ligand interactions is of utmost significance. However, strategies i. e. QM/MM, fragmentation or semiempirical (SQM) methods had to be pursued to overcome the unfavorable scaling of QM methods. Various SQM-based approaches have significantly contributed to the accuracy of docking and improvement of lead compounds. Parametrizations of SQM and implicit solvent methods in our laboratory have been instrumental to obtain a reliable SQM-based scoring function. The experience gained in its application for activity ranking of ligands binding to tens of protein targets resulted in setting up a faster SQM/COSMO scoring approach, which outperforms standard scoring methods in native pose identification for two dozen protein targets with ten thousand poses. Recently, SQM/COSMO was effectively applied in a proof-of-concept study of enrichment in virtual screening. Due to its superior performance, feasibility and chemical generality, we propose the SQM/COSMO approach as an efficient tool in structure-based drug design.

Lens epithelium-derived growth factor/p75 (LEDGF/p75, or PSIP1) is a transcriptional coactivator that tethers other proteins to gene bodies. The chromatin tethering function of LEDGF/p75 is hijacked by HIV integrase to ensure viral integration at sites of active transcription. LEDGF/p75 is also important for the development of mixed-lineage leukemia (MLL), where it tethers the MLL1 fusion complex at aberrant MLL targets, inducing malignant transformation. However, little is known about how the LEDGF/p75 protein interaction network is regulated. Here, we obtained solution structures of the complete interfaces between the LEDGF/p75 integrase binding domain (IBD) and its cellular binding partners and validated another binding partner, Mediator subunit 1 (MED1). We reveal that structurally conserved IBD-binding motifs (IBMs) on known LEDGF/p75 binding partners can be regulated by phosphorylation, permitting switching between low- and high-affinity states. Finally, we show that elimination of IBM phosphorylation sites on MLL1 disrupts the oncogenic potential of primary MLL1-rearranged leukemic cells. Our results demonstrate that kinase-dependent phosphorylation of MLL1 represents a previously unknown oncogenic dependency that may be harnessed in the treatment of MLL-rearranged leukemia. ispartof: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA vol:115 issue:30 pages:E7053-E7062 ispartof: location:United States status: published

FLT3 tyrosine kinase is a potential drug target in acute myeloid leukemia (AML) because patients with FLT3-ITD mutations respond poorly to standard cytotoxic agents and there is a clear link between the disease and the oncogenic properties of FLT3. We present novel 2,6,9-trisubstituted purine derivatives with potent FLT3 inhibitory activity. The lead compound 7d displays nanomolar activity in biochemical assays and selectively blocks proliferation of AML cell lines harboring FLT3-ITD mutations, whereas other transformed and normal human cells are several orders of magnitude less sensitive. The MV4-11 cells treated with 7d suppressed the phosphorylation of FLT3 and its downstream signaling pathways, with subsequent G1 cell cycle arrest and apoptosis. Additionally, a single dose of 7d in mice with subcutaneous MV4-11 xenografts caused sustained inhibition of FLT3 and STAT5 phosphorylation over 48 h, in contrast to the shorter effect observed after administration of the reference FLT3 inhibitor quizartinib.

Inhibition of Bordetella pertussis adenylate cyclase toxin (ACT) and Bacillus anthracis edema factor (EF), key virulence factors with adenylate cyclase activity, represents a potential method for treating or preventing toxemia related to whooping cough and anthrax, respectively. Novel α-branched acyclic nucleoside phosphonates (ANPs) having a hemiaminal ether moiety were synthesized as potential inhibitors of bacterial adenylate cyclases. ANPs prepared as bisamidates were not cytotoxic, but did not exhibit any profound activity (IC50 >10 μm) toward ACT in J774A.1 macrophages. The apparent lack of activity of the bisamidates is speculated to be due to the inefficient formation of the biologically active species (ANPpp) in the cells. Conversely, two 5-haloanthraniloyl-substituted ANPs in the form of diphosphates were shown to be potent ACT and EF inhibitors with IC50 values ranging from 55 to 362 nm.

We describe the synthesis of a trifunctional scaffold constructed from a planar core of trimesic acid derivatized with three propargylamine moieties. The scaffold was attached to a solid-phase resin through the carboxylic group of a fluorinated alkyl spacer arm. The orthogonal protection of two of the alkyne groups with triethylsilyl and triisopropylsilyl moieties enabled modular and efficient derivatization of the scaffold with three different azides by using solid-phase synthesis on amphiphilic ChemMatrix resin. We showed that a fluorine label can be used to quantify the content of fluorine-containing compounds by 19F NMR spectroscopic analysis after cleavage from the resin. We have thus designed a versatile and convenient tool that could be useful for simple and rapid solid-phase syntheses of combinatorial libraries of the scaffold-based compounds, for example as new protein binders.

A systematic quantum mechanical study of σ-hole (chalcogen, pnicogen, and halogen) bonding in neutral experimentally known closo-heteroboranes is performed. Chalcogens and pnicogens are incorporated in the borane cage, whereas halogens are considered as exo-substituents of dicarbaboranes. The chalcogen and pnicogen atoms in the heteroborane cages have partial positive charge and thus more positive σ-holes. Consequently, these heteroboranes form very strong chalcogen and pnicogen bonds. Halogen atoms in dicarbaboranes also have a highly positive σ-hole, but only in the case of C-bonded halogen atoms. In such cases, the halogen bond of heteroboranes is also strong and comparable to halogen bonds in organic compounds with several electron-withdrawing groups being close to the halogen atom involved in the halogen bond.

We present comprehensive testing of solvent representation in quantum mechanics (QM)-based scoring of protein-ligand affinities. To this aim, we prepared 21 new inhibitors of cyclin-dependent kinase 2 (CDK2) with the pyrazolo[1,5-a]pyrimidine core, whose activities spanned three orders of magnitude. The crystal structure of a potent inhibitor bound to the active CDK2/cyclin A complex revealed that the biphenyl substituent at position 5 of the pyrazolo[1,5-a]pyrimidine scaffold was located in a previously unexplored pocket and that six water molecules resided in the active site. Using molecular dynamics, protein-ligand interactions and active-site water H-bond networks as well as thermodynamics were probed. Thereafter, all the inhibitors were scored by the QM approach utilizing the COSMO implicit solvent model. Such a standard treatment failed to produce a correlation with the experiment (R-2 = 0.49). However, the addition of the active-site waters resulted in significant improvement (R-2 = 0.68). The activities of the compounds could thus be interpreted by taking into account their specific noncovalent interactions with CDK2 and the active-site waters. In summary, using a combination of several experimental and theoretical approaches we demonstrate that the inclusion of explicit solvent effects enhance QM/COSMO scoring to produce a reliable structure activity relationship with physical insights. More generally, this approach is envisioned to contribute to increased accuracy of the computational design of novel inhibitors. (C) 2016 Elsevier Masson SAS. All rights reserved.

The weakly coordinating binary macropolyhedral anion closo,closo-[B

Rhomboid-family intramembrane proteases regulate important biological processes and have been associated with malaria, cancer, and Parkinson's disease. However, due to the lack of potent, selective, and pharmacologically compliant inhibitors, the wide therapeutic potential of rhomboids is currently untapped. Here, we bridge this gap by discovering that peptidyl α-ketoamides substituted at the ketoamide nitrogen by hydrophobic groups are potent rhomboid inhibitors active in the nanomolar range, surpassing the currently used rhomboid inhibitors by up to three orders of magnitude. Such peptidyl ketoamides show selectivity for rhomboids, leaving most human serine hydrolases unaffected. Crystal structures show that these compounds bind the active site of rhomboid covalently and in a substrate-like manner, and kinetic analysis reveals their reversible, slow-binding, non-competitive mechanism. Since ketoamides are clinically used pharmacophores, our findings uncover a straightforward modular way for the design of specific inhibitors of rhomboid proteases, which can be widely applicable in cell biology and drug discovery. ispartof: Cell Chemical Biology vol:24 issue:12 pages:1523- ispartof: location:United States status: published

The weakly coordinating binary macropolyhedral anion closo,closo-[BH] (B21; D symmetry) has been synthesized using a simplified strategy compared to that in the literature. While gas-phase complexes of B21 with β- and γ-cyclodextrin (CD) were detected using ESI FT-ICR spectrometric measurements, α-CD did not bind to the B21 guest. This spectroscopic evidence has been interpreted using quantum-chemical computations, showing that β- and γ-CD are able to interact with B21 due to their larger cavities, in contrast to the smaller α-CD. The hydridic B-H vectors of the B21 anion interact with K counterions and, via dihydrogen bonding, also with the partially positively charged hydrogens of the CD sugar units in the modeled β- and γ-CD complexes. In summary, it has been shown by combined spectrometric/computational analysis that macropolyhedral boron hydride anions with two counterions can form stable complexes with β- and γ-CD in the gas phase, offering a new perspective for the future investigation of this remarkable anion in the areas of supramolecular and medicinal chemistries.

The mechanisms of intramembrane proteases are incompletely understood due to the lack of structural data on substrate complexes. To gain insight into substrate binding by rhomboid proteases, we have synthesised a series of novel peptidyl-chloromethylketone (CMK) inhibitors and analysed their interactions with Escherichia coli rhomboid GlpG enzymologically and structurally. We show that peptidyl-CMKs derived from the natural rhomboid substrate TatA from bacterium Providencia stuartii bind GlpG in a substrate-like manner, and their co-crystal structures with GlpG reveal the S1 to S4 subsites of the protease. The S1 subsite is prominent and merges into the ‘water retention site’, suggesting intimate interplay between substrate binding, specificity and catalysis. Unexpectedly, the S4 subsite is plastically formed by residues of the L1 loop, an important but hitherto enigmatic feature of the rhomboid fold. We propose that the homologous region of members of the wider rhomboid-like protein superfamily may have similar substrate or client-protein binding function. Finally, using molecular dynamics, we generate a model of the Michaelis complex of the substrate bound in the active site of GlpG.

We report enzymologic, thermodynamic and structural analyses of a series of six clinically derived mutant HIV proteases (PR) resistant to darunavir. As many as 20 mutations in the resistant PRs decreased the binding affinity of darunavir by up to 13 000-fold, mostly because of a less favorable enthalpy of binding that was only partially compensated by the entropic contribution. X-ray structure analysis suggested that the drop in enthalpy of darunavir binding to resistant PR species was mostly the result of a decrease in the number of hydrogen bonds and a loosening of the fit between the inhibitor and the mutated enzymes. The favorable entropic contribution to darunavir binding to mutated PR variants correlated with a larger burial of the nonpolar solvent-accessible surface area upon inhibitor binding. We show that even very dramatic changes in the PR sequence leading to the loss of hydrogen bonds with the inhibitor could be partially compensated by the entropy contribution as a result of the burial of the larger nonpolar surface area of the mutated HIV PRs. Database Atomic coordinates and structure factors for the crystal structures PRwt–DRV and PRDRV2–DRV complex have been deposited in the Protein Data Bank under accession codes 4LL3 and 3TTP, respectively. Structured digital abstract • PR and PR bind by x-ray crystallography (View interaction)

The quantum mechanics (QM)-based scoring function that we previously developed for the description of noncovalent binding in protein-ligand complexes has been modified and extended to treat covalent binding of inhibitory ligands. The enhancements are (i) the description of the covalent bond breakage and formation using hybrid QM/semiempirical QM (QM/SQM) restrained optimizations and (ii) the addition of the new ΔG(cov)' term to the noncovalent score, describing the "free" energy difference between the covalent and noncovalent complexes. This enhanced QM-based scoring function is applied to a series of 20 vinyl sulfone-based inhibitory compounds inactivating the cysteine peptidase cathepsin B1 of the Schistosoma mansoni parasite (SmCB1). The available X-ray structure of the SmCB1 in complex with a potent vinyl sulfone inhibitor K11017 is used as a template to build the other covalently bound complexes and to model the derived noncovalent complexes. We present the correlation of the covalent score and its constituents with the experimental binding data. Four outliers are identified. They contain bulky R1' substituents structurally divergent from the template, which might induce larger protein rearrangements than could be accurately modeled. In summary, we propose a new computational approach and an optimal protocol for the rapid evaluation and prospective design of covalent inhibitors with a conserved binding mode.

In this paper, we studied a designed series of aldose reductase (AR) inhibitors. The series was derived from a known AR binder, which had previously been shown to form a halogen bond between its bromine atom and the oxygen atom of the Thr-113 side chain of AR. In the series, the strength of the halogen bond was modulated by two factors, namely bromine-iodine substitution and the fluorination of the aromatic ring in several positions. The role of the single halogen bond in AR-ligand binding was elucidated by advanced binding free energy calculations involving the semiempirical quantum chemical Hamiltonian. The results were complemented with ultrahigh-resolution X-ray crystallography and IC50 measurements. All of the AR inhibitors studied were shown by X-ray crystallography to bind in an identical manner. Further, it was demonstrated that it was possible to decrease the IC50 value by about 1 order of magnitude by tuning the strength of the halogen bond by a monoatomic substitution. The calculations revealed that the protein-ligand interaction energy increased upon the substitution of iodine for bromine or upon the addition of electron-withdrawing fluorine atoms to the ring. However, the effect on the binding affinity was found to be more complex due to the change of the solvation/desolvation properties within the ligand series. The study shows that it is possible to modulate the strength of a halogen bond in a protein-ligand complex as was designed based on the previous studies of low-molecular-weight complexes.