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Quantitatively determining in vivo achievable drug concentrations in targeted organs of animal models and subsequent target engagement confirmation is a challenge to drug discovery and translation due to lack of bioassay technologies that can discriminate drug binding with different mechanisms. We have developed a multiplexed and high-throughput method to quantify drug distribution in tissues by integrating high content screening (HCS) with U-Net based deep learning (DL) image analysis models. This technology combination allowed direct visualization and quantification of biologics drug binding in targeted tissues with cellular resolution, thus enabling biologists to objectively determine drug binding kinetics.

The Allotrope Foundation (AF) is a group of pharmaceutical, device vendor, and software companies that develops and releases technologies [the Allotrope Data Format (ADF), the Allotrope Foundation Ontology (AFO), and the Allotrope Data Models (ADM)] to simplify the exchange of electronic data. We present here the first comprehensive history of the AF, its structure, a list of members and partners, and an introduction to the technologies. Finally, we provide current insights into the adoption and development of the technologies by summarizing the Fall 2020 Allotrope Connect virtual conference. This overview provides an easy access to the AF and highlights opportunities for collaboration.

Malaria is a devastating infection caused by protozoa of the genus Plasmodium. Drug resistance is widespread, no new chemical class of antimalarials has been introduced into clinical practice since 1996 and there is a recent rise of parasite strains with reduced sensitivity to the newest drugs. We screened nearly 2 million compounds in GlaxoSmithKline's chemical library for inhibitors of P. falciparum, of which 13,533 were confirmed to inhibit parasite growth by at least 80% at 2 microM concentration. More than 8,000 also showed potent activity against the multidrug resistant strain Dd2. Most (82%) compounds originate from internal company projects and are new to the malaria community. Analyses using historic assay data suggest several novel mechanisms of antimalarial action, such as inhibition of protein kinases and host-pathogen interaction related targets. Chemical structures and associated data are hereby made public to encourage additional drug lead identification efforts and further research into this disease.

Two new series of potent and selective dual EGFR/ErbB-2 kinase inhibitors derived from novel thienopyrimidine cores have been identified. Isomeric thienopyrimidine cores were evaluated as isosteres for a 4-anilinoquinazoline core and several analogs containing the thieno[3,2-d]pyrimidine core showed anti-proliferative activity with IC(50) values less than 1 microM against human tumor cells in vitro.

Numerous biochemical and structural studies have shown that the conformation of the estrogen receptor alpha (ERalpha) can be influenced by ligand binding. In turn, the conformational state of ERalpha affects the ability of the receptor to interact with a wide variety of protein accessory factors. To globally investigate ligand-based cofactor recruitment activities of ERalpha, we have applied a flow cytometric multiplexed binding assay to determine the simultaneous binding of ERalpha to over 50 different peptides derived from both known cofactor proteins and random peptide phage display. Using over 400 ERalpha-binding compounds, we have observed that the multiplexed in vitro peptide-binding profiles are distinct for a number of compounds and that these profiles can predict the effect that ERalpha ligands have on various cellular activities. These cell-based activities include transcriptional regulation at an estrogen response element, MCF-7 cell proliferation, and Ishikawa endometrial cell stimulation. The majority of the compound-induced diversity in the peptide profiling assay is provided by the unique phage display peptides. Importantly, some of these peptides show a sequence relationship with the corepressor motif, suggesting that peptides identified via phage display might represent natural binding partners of ERalpha. These in vitro:cellular correlations may in part explain tissue-specific activities of ERalpha-modulating compounds.

Tuberculosis (TB) remains a leading cause of infectious disease in the world today and therapies developed over the last forty years are becoming increasingly ineffective against resistant strains of Mycobacterium tuberculosis. In an effort to explore new mechanisms for drug development, we have investigated the enzymes of the diaminopimelate biosynthetic pathway as potential targets. Specifically, dihydrodipicolinate reductase, the essential gene product of dapB, was screened for novel inhibitors. Inhibitors were identified both by a molecular modeling approach which utilized the available crystal structure of the enzyme with an inhibitor bound at the active site as well as by more conventional screening strategies. The resulting compounds contain a number of structural motifs and were all found to be competitive with respect to the DHDP substrate. The Ki values for the inhibitors range from 10 to 90 μM. The molecular modeling approach was very effective in identifying novel inhibitors of the enzyme. These compounds were obtained at a higher frequency based on the number of compounds analyzed than those inhibitors discovered via conventional screening. However, conventional screening proved beneficial in identifying compounds with greater structural diversity.

Resistance to carbapenem antibiotics in gram-negative bacteria is due, in part, to expression of a wide spectrum metallo-beta-lactamase, which renders the drug inactive. Biphenyl tetrazoles containing 3-n-butyl-1-phenylpyrazole-5-carboxylates or the corresponding 5-ethyl esters were found to inhibit metallo-beta-lactamases as well as renal dehydropeptidase I to a lesser extent.

NMR studies of the hydroperoxide form of cobalt deglycoBleomycin A2 (HOO−CodGBLM) and its complex with oligonucleotide d(CCAGGCCTGG) (1, C is the site of cleavage) are presented in an effort to establish deglycoBLM as a prototype for studies with BLM analogues, synthesized without the sugar moieties (Boger et al. Bioorg. Med. Chem. 1995, 3, 1281−1295). The structure determination of free HOO−CodGBLM has been hampered by the lack of NOE or ROE information. By direct comparison of the chemical shifts and coupling constants of HOO−CodGBLM with its glycosylated parent HOO−CoBLM, the former is shown to share many global structural features with the latter, including the nature of axial ligands (a point recently disputed by Caceres-Cortes et al. Eur. J. Biochem. 1997, 244, 818−828), the screw sense of metal-coordinating ligands, and the folding of the peptide linker region. The structure of HOO−CodGBLM bound to 1 is reported based on molecular modeling using NMR constraints (39 intermolecular and 44 intramolecu...

A model for the structure of the aquated form of cobalt bleomycin (H2O-CoBLM or CoBLM A2 brown), free and bound to d(CCAGGCCTGG)2 (1) is reported based on molecular modeling using the constraints obtained from 2D NMR studies. CoBLM A2 brown has a chiral organization of its ligands, including the axial primary amine of β-aminoalanine, identical to the hydroperoxide form of cobalt BLM (HOO-CoBLM or CoBLM A2 green). H2O-CoBLM forms a 1:1 complex with 1 with a Kd of 2 × 10-6 M which is in slow exchange on the NMR time scale. The complex exhibits 44 intermolecular NOEs and 56 intramolecular NOEs within H2O-CoBLM itself. Molecular modeling reveals that H2O-CoBLM's mode of binding, basis for sequence specificity, and chemical specificity are almost identical to that previously reported for HOO-CoBLM (Wu, W.; Vanderwall, D. E.; Turner, C. J.; Kozarich, J. W.; Stubbe, J. J. Am. Chem. Soc. 1996, 118, 1281−1294). The bithiazole tail is inserted from the minor groove 3‘ to C6 and the terminal thiazolium ring is well ...

The structure of homogeneous Co·Bleomycin (CoBLM) A2 green (the hydroperoxide form of CoBLM) has been determined using 2D NMR methods and molecular dynamics calculations. Previous studies of Xu et al. (Xu, R. X.; Nettesheim, D.; Otvos, J. D.; Petering, D. H. Biochemistry 1994, 33, 907−916) reported several possible structures for CoBLM A2 green compatible with their NMR data acquired on a mixture of CoBLM A2 green and A2 brown forms. The availability of the pure CoBLM A2 green, which is stable for months at neutral pH, has allowed the complete assignments of the 1H and 13C chemical shifts, observation of 55 intramolecular NOEs, and determination of 15 coupling constants allowing the definition of dihedral angles. These results are a prerequisite to determining its structure with duplex DNA of a defined sequence (Wu, W.; Vanderwall, D. E.; Turner, C. J.; Kozarich, J. W.; Stubbe, J. J. Am. Chem. Soc. 1996, 118, 1281−1294). Two screw sense isomers each containing two possible axial ligands (the primary amine...

The solution structure of Co·Bleomycin (CoBLM) A2 green (the hydroperoxide form of CoBLM) complexed with the self-complementary oligonucleotide d(CCAGGCCTGG) with a cleavage site at C6 has been determined by 2D NMR spectroscopic methods and molecular dynamics calculations. Intermolecular NOEs (60 between CoBLM A2 green and DNA) and intramolecular NOEs (61 within CoBLM A2 green) have defined the position and orientation of CoBLM A2 green with respect to its single binding site in the duplex. CoBLM A2 green is a stable analog of the activated BLM, the Fe3+ hydroperoxide (Sam, J. W.; Tang, X.-J.; Peisach, J. J. Am. Chem. Soc. 1994, 116, 5250−5256). These studies have provided the first structural insight into the mode of binding of the bithiazole tail of CoBLM A2 green to DNA, the basis for specificity of its cleavage at pyrimidines (Py) in d(G-Py) sequences, and the orientation of its terminal oxygen of the hydroperoxide relative to the 4‘ carbon hydrogen bond being cleaved in the DNA. The bithiazole tail i...

Drug toxicity is a major cause of late-stage product attrition. During lead identification and optimization phases little information is typically available about which molecules might have safety concerns. A system was built linking chemistry, preclinical and human safety information, enabling scientists to lever safety knowledge across multiple disciplines. The system consists of a data warehouse with chemical structures and chemical and biological properties for ∼80000 compounds and tools to access and analyze clinical data, toxicology, in vitro pharmacology and drug metabolism data. Tapping into this safety knowledge enables rapid clinically focused risk assessments of drug candidates. Use of this strategy adds value to the drug discovery process at GSK via efficient triage of compounds based on their potential for toxicity.

Analysis of the x-ray crystal structure of mono-substituted acetylenic thienopyrimidine 6 complexed with the ErbB family enzyme ErbB-4 revealed a covalent bond between the terminal carbon of the acetylene moiety and the sulfhydryl group of Cys-803 at the solvent interface. The identification of this covalent adduct suggested that acetylenic thienopyrimidine 6 and related analogs might also be capable of forming an analogous covalent adduct with EGFR, which has a conserved cysteine (797) near the ATP binding pocket. To test this hypothesis, we treated a truncated, catalytically competent form of EGFR (678–1020) with a structurally related propargylic amine ( 8 ). An investigation of the resulting complex by mass spectrometry revealed the formation of a covalent complex of thienopyrimidine 8 with Cys-797 of EGFR. This finding enabled us to readily assess the irreversibility of various inhibitors and also facilitated a structure–activity relationship understanding of the covalent modifying potential and biological activity of a series of acetylenic thienopyrimidine compounds with potent antitumor activity. Several ErbB family enzyme and cell potent 6-ethynyl thienopyrimidine kinase inhibitors were found to form covalent adducts with EGFR.

A series of 6-alkoxy-4-anilinoquinazoline compounds was prepared and evaluated for in vitro inhibition of the erbB2 and EGFR kinase activity. The IC(50) values of the best compounds were below 0.10 uM. Further, several of these compounds inhibit the growth of erbB2 and EGFR over-expressing tumor cell lines at concentrations below 1 uM.

Phosphodiesterase catalyzes the hydrolysis of the intracellular second messenger 3′,5′-cyclic AMP (cAMP) into the corresponding 5′-nucleotide. Phosphodiesterase 4 (PDE4), the major cAMP-specific PDE in inflammatory and immune cells, is an attractive target for the treatment of asthma and COPD. We have determined crystal structures of the catalytic domain of PDE4B complexed with AMP (2.0 A), 8-Br-AMP (2.13 A) and the potent inhibitor rolipram (2.0 A). All the ligands bind in the same hydrophobic pocket and can interact directly with the active site metal ions. The identity of these metal ions was examined using X-ray anomalous difference data. The structure of the AMP complex confirms the location of the catalytic site and allowed us to speculate about the detailed mechanism of catalysis. The high-resolution structures provided the experimental insight into the nucleotide selectivity of phosphodiesterase. 8-Br-AMP binds in the syn conformation to the enzyme and demonstrates an alternative nucleotide-binding mode. Rolipram occupies much of the AMP-binding site and forms two hydrogen bonds with Gln443 similar to the nucleotides.

The bleomycins (BLMs) are natural products that in the presence of iron and oxygen bind to and cause single-strand and double-strand cleavage of DNA. The mode(s) of binding of the FeBLMs that leads to sequence-specific cleavage at pyrimidines 3' to guanines and chemical-specific cleavage at the C-4' H of the deoxyribose of the pyrimidine has remained controversial. 2D NMR studies using the hydroperoxide of CoBLM (HOO-CoBLM) have demonstrated that its bithiazole tail binds by partial intercalation to duplex DNA. Studies with ZnBLM demonstrate that the bithiazole tail binds in the minor groove. Phleomycins (PLMs) are BLM analogs in which the penultimate thiazolium ring of the bithiazole tail is reduced. The disruption of planarity of this ring and the similarities between FePLM- and FeBLM-mediated DNA cleavage have led Hecht and co-workers to conclude that a partial intercalative mode of binding is not feasible. The interaction of HOO-CoPLM with d(CCAGGCCTGG)2 has therefore been investigated. Binding studies indicate a single site with a K(d) of 16 micro M, 100-fold greater than HOO-CoBLM for the same site. 2D NMR methods and molecular modeling using NMR-derived restraints have led to a structural model of HOO-CoPLM complexed to d(CCAGGCCTGG)2. The model reveals a partial intercalative mode of binding and the basis for sequence specificity of binding and chemical specificity of cleavage. The importance of the bithiazoles and the partial intercalative mode of binding in the double-strand cleavage of DNA is discussed.

Cyclic nucleotides are second messengers that are essential in vision, muscle contraction, neurotransmission, exocytosis, cell growth, and differentiation. These molecules are degraded by a family of enzymes known as phosphodiesterases, which serve a critical function by regulating the intracellular concentration of cyclic nucleotides. We have determined the three-dimensional structure of the catalytic domain of phosphodiesterase 4B2B to 1.77 angstrom resolution. The active site has been identified and contains a cluster of two metal atoms. The structure suggests the mechanism of action and basis for specificity and will provide a framework for structure-assisted drug design for members of the phosphodiesterase family.

Background: High level resistance to carbapenem antibiotics in gram negative bacteria such as Bacteroides fragilis is caused, in part, by expression of a wide-spectrum metallo-β-lactamase that hydrolyzes the drug to an inactive form. Co-administration of metallo-β-lactamase inhibitors to resistant bacteria is expected to restore the antibacterial activity of carbapenems. Results: Biphenyl tetrazoles (BPTs) are a structural class of potent competitive inhibitors of metallo-β-lactamase identified through screening and predicted using molecular modeling of the enzyme structure. The X-ray crystal structure of the enzyme bound to the BPT L-159,061 shows that the tetrazole moiety of the inhibitor interacts directly with one of the two zinc atoms in the active site, replacing a metal-bound water molecule. Inhibition of metallo-β-lactamase by BPTs in vitro correlates well with antibiotic sensitization of resistant B. fragilis . Conclusions: BPT inhibitors can sensitize a resistant B. fragilis clinical isolate expressing metallo-β-lactamase to the antibiotics imipenem or penicillin G but not to rifampicin.

Background : The bleomycins (BLMs) are a family of natural products used clinically as antitumor agents. In the presence of their required cofactors, iron and oxygen, BLMs bind to and mediate single-stranded and double-stranded DNA cleavage. Recently, two dimensional nuclear magnetic resonance (2D NMR) spectroscopic studies and molecular modeling have provided a picture of how the hydroperoxide form of cobalt BLM A2 (HOO-CoBLM), an analog of ‘activated' iron BLM (HOO-FeBLM), binds to a d(GpC) motif and of the basis for both sequence specificity and chemical specificity of DNA cleavage. Results : The solution structure of HOO-CoBLM bound to d(CCAGTACTGG) containing a'hot spot' for double-stranded DNA cleavage at T 5 and T 15 is reported using constraints from 2D NMR spectroscopy. The mode of binding and basis for sequence specificity and chemical specificity of cleavage is almost identical to that of a d(GpC) motif. This structure has allowed formulation of a structural model for how a single molecule of FeBLM can mediate a double-stranded DNA cleavage event without dissociation from the DNA. Conclusions : The structural similarity of HOO-CoBLM bound to d(GpT) in d(CCAGTACTGG) compared to a d(GpC) motif suggests a general paradigm for the binding of HOO-CoBLM to DNA and, by analogy, for the binding of the biological significant entity 100-FeBLM.

Bleomycin is a metal- and oxygen-dependent DNA cleaver. The chemistry of DNA damage has been proposed to involve rate-limiting abstraction of the 4'-hydrogen. A DNA fragment has been prepared that contains [4'-2H]thymidine residues of high isotopic content. Primary kinetic isotope effects have been directly observed at individual thymidine residues with DNA sequencing technology.

Background Retrieving pertinent information from biological scientific literature requires cutting-edge text mining methods which may be able to recognize the meaning of the very ambiguous names of biological entities. Aliases of a gene share a common vocabulary in their respective collections of PubMed abstracts. This may be true even when these aliases are not associated with the same subset of documents. This gene-specific vocabulary defines a unique fingerprint that can be used to disclose ambiguous aliases. The present work describes an original method for automatically assessing the ambiguity levels of gene aliases in large gene terminologies based exclusively in the content of their associated literature. The method can deal with the two major problems restricting the usage of current text mining tools: 1) different names associated with the same gene; and 2) one name associated with multiple genes, or even with non-gene entities. Important, this method does not require training examples. Results Aliases were considered “ambiguous” when their Jaccard distance to the respective official gene symbol was equal or greater than the smallest distance between the official gene symbol and one of the three internal controls (randomly picked unrelated official gene symbols). Otherwise, they were assigned the status of “synonyms”. We evaluated the coherence of the results by comparing the frequencies of the official gene symbols in the text corpora retrieved with their respective “synonyms” or “ambiguous” aliases. Official gene symbols were mentioned in the abstract collections of 42 % (70/165) of their respective synonyms. No official gene symbol occurred in the abstract collections of any of their respective ambiguous aliases. In overall, querying PubMed with official gene symbols and “synonym” aliases allowed a 3.6-fold increase in the number of unique documents retrieved. Conclusions These results confirm that this method is able to distinguish between synonyms and ambiguous gene aliases based exclusively on their vocabulary fingerprint. The approach we describe could be used to enhance the retrieval of relevant literature related to a gene.