Diarrhoeal disease caused by Cryptosporidium is a major cause of morbidity and mortality in young and malnourished children from low- and middle-income countries, with no vaccine or effective treatment. Here we describe the discovery of EDI048, a Cryptosporidium PI(4)K inhibitor, designed to be active at the infection site in the gastrointestinal tract and undergo rapid metabolism in the liver. By using mutational analysis and crystal structure, we show that EDI048 binds to highly conserved amino acid residues in the ATP-binding site. EDI048 is orally efficacious in an immunocompromised mouse model despite negligible circulating concentrations, thus demonstrating that gastrointestinal exposure is necessary and sufficient for efficacy. In neonatal calves, a clinical model of cryptosporidiosis, EDI048 treatment resulted in rapid resolution of diarrhoea and significant reduction in faecal oocyst shedding. Safety and pharmacological studies demonstrated predictable metabolism and low systemic exposure of EDI048, providing a substantial safety margin required for a paediatric indication. EDI048 is a promising clinical candidate for the treatment of life-threatening paediatric cryptosporidiosis. EDI048 is a gastrointestinal-targeted Cryptosporidium PI(4)K inhibitor that undergoes a predictable metabolism and limits systemic exposure without compromising its anti-parasitic activity.

Millions who live in Latin America and sub-Saharan Africa are at risk of trypanosomatid infections, which cause Chagas disease and human African trypanosomiasis (HAT). Improved HAT treatments are available, but Chagas disease therapies rely on two nitroheterocycles, which suffer from lengthy drug regimens and safety concerns that cause frequent treatment discontinuation. We performed phenotypic screening against trypanosomes and identified a class of cyanotriazoles (CTs) with potent trypanocidal activity both in vitro and in mouse models of Chagas disease and HAT. Cryo-electron microscopy approaches confirmed that CT compounds acted through selective, irreversible inhibition of trypanosomal topoisomerase II by stabilizing double-stranded DNA:enzyme cleavage complexes. These findings suggest a potential approach toward successful therapeutics for the treatment of Chagas disease.

Human African Trypanosomiasis (HAT) is a vector-borne disease caused by kinetoplastid parasites of the Trypanosoma genus. The disease proceeds in two stages, with a hemolymphatic blood stage and a meningo-encephalic brain stage. In the latter stage, the parasite causes irreversible damage to the brain leading to sleep cycle disruption and is fatal if untreated. An orally bioavailable treatment is highly desirable. In this study, we present a brain-penetrant, parasite-selective 20S proteasome inhibitor that was rapidly optimized from an HTS singleton hit to drug candidate compound 7 that showed cure in a stage II mouse efficacy model. Here, we describe hit expansion and lead optimization campaign guided by cryo-electron microscopy and an in silico model to predict the brain-to-plasma partition coefficient Kp as an important parameter to prioritize compounds for synthesis. The model combined with in vitro and in vivo experiments allowed us to advance compounds with favorable unbound brain-to-plasma ratios (Kp,uu) to cure a CNS disease such as HAT.

Artificial intelligence (AI) is becoming established in drug discovery. For example, many in the industry are applying machine learning approaches to target discovery or to optimize compound synthesis. While our organization is certainly applying these sorts of approaches, we propose an additional approach: using AI to augment human intelligence. We have been working on a series of recommendation systems that take advantage of our existing laboratory processes, both wet and computational, in order to provide inspiration to our chemists, suggest next steps in their work, and automate existing workflows. We will describe five such systems in various stages of deployment within the Novartis Institutes for BioMedical Research. While each of these systems addresses different stages of the discovery pipeline, all of them share three common features: a trigger that initiates the recommendation, an analysis that leverages our existing systems with AI, and the delivery of a recommendation. The goal of all of these systems is to inspire and accelerate the drug discovery process.

Targeting of cryptic binding sites represents an attractive but underexplored approach to modulating protein function with small molecules. Using the dimeric protease (Pr) from Kaposi's sarcoma-associated herpes virus (KSHV) as a model system, we sought to dissect a putative allosteric network linking a cryptic site at the dimerization interface to enzyme function. Five cryogenic X-ray structures were solved of the monomeric protease with allosteric inhibitors bound to the dimer interface site. Distinct coordinated movements captured by the allosteric inhibitors were also revealed as alternative states in room temperature X-ray data and comparative analyses of other dimeric herpesvirus proteases. A two-step mechanism was elucidated through detailed kinetic analyses and suggests an enzyme isomerization model of inhibition. Finally, a representative allosteric inhibitor from this class was shown to be efficacious in a cellular model of viral infectivity. These studies reveal a coordinated dynamic network of atomic communication linking cryptic binding site occupancy and allosteric inactivation of KHSV Pr that can be exploited to target other members of this clinically relevant family of enzymes.

Fragment-based drug discovery has shown promise as an approach for challenging targets such as protein-protein interfaces. We developed and applied an activity-based fragment screen against dimeric Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr) using an optimized fluorogenic substrate. Dose-response determination was performed as a confirmation screen, and NMR spectroscopy was used to map fragment inhibitor binding to KSHV Pr. Kinetic assays demonstrated that several initial hits also inhibit human cytomegalovirus protease (HCMV Pr). Binding of these hits to HCMV Pr was also confirmed by NMR spectroscopy. Despite the use of a target-agnostic fragment library, more than 80% of confirmed hits disrupted dimerization and bound to a previously reported pocket at the dimer interface of KSHV Pr, not to the active site. One class of fragments, an aminothiazole scaffold, was further explored using commercially available analogues. These compounds demonstrated greater than 100-fold improvement of inhibition. This study illustrates the power of fragment-based screening for these challenging enzymatic targets and provides an example of the potential druggability of pockets at protein-protein interfaces.

Herpesviruses rely on a homodimeric protease for viral capsid maturation. A small molecule, DD2, previously shown to disrupt dimerization of Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr) by trapping an inactive monomeric conformation and two analogues generated through carboxylate bioisosteric replacement (compounds 2 and 3) were shown to inhibit the associated proteases of all three human herpesvirus (HHV) subfamilies (α, β, and γ). Inhibition data reveal that compound 2 has potency comparable to or better than that of DD2 against the tested proteases. Nuclear magnetic resonance spectroscopy and a new application of the kinetic analysis developed by Zhang and Poorman [Zhang, Z. Y., Poorman, R. A., et al. (1991) J. Biol. Chem. 266, 15591–15594] show DD2, compound 2, and compound 3 inhibit HHV proteases by dimer disruption. All three compounds bind the dimer interface of other HHV proteases in a manner analogous to binding of DD2 to KSHV protease. The determination and analysis of cocrystal structures of both analogues with the KSHV Pr monomer verify and elaborate on the mode of binding for this chemical scaffold, explaining a newly observed critical structure–activity relationship. These results reveal a prototypical chemical scaffold for broad-spectrum allosteric inhibition of human herpesvirus proteases and an approach for the identification of small molecules that allosterically regulate protein activity by targeting protein–protein interactions.

We have investigated the folding reaction of a model beta-barrel membrane protein, OmpA, using vibrational and electronic spectroscopy. Our primary focus is to interrogate changes in hydration during insertion and folding of the protein into the bilayer. The results suggest that expulsion of water and residual denaturant from the bilayer and/or protein core may give rise to the long-time component in folding kinetics. This finding is discussed in the context of in vitro mechanisms for membrane protein folding.

ADVERTISEMENT RETURN TO ISSUEPREVReviewCurrent and Potential Treatments for Ubiquitous but Neglected Herpesvirus InfectionsJonathan E. Gable†‡, Timothy M. Acker†, and Charles S. Craik*†View Author Information† Department of Pharmaceutical Chemistry, University of California, San Francisco, 600 16th Street, San Francisco, California 94158-2280, United States‡ Graduate Group in Biophysics, University of California, San Francisco, 600 16th Street, San Francisco, California 94158-2280, United States*E-mail: [email protected]. Phone: 415-476-8146.Cite this: Chem. Rev. 2014, 114, 22, 11382–11412Publication Date (Web):October 2, 2014Publication History Received13 May 2014Published online2 October 2014Published inissue 26 November 2014https://doi.org/10.1021/cr500255eCopyright © 2014 American Chemical SocietyRIGHTS & PERMISSIONSACS AuthorChoiceArticle Views3796Altmetric-Citations16LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (7 MB) Get e-AlertsSUBJECTS:DNA replication,Genetics,Infectious diseases,Inhibitors,Peptides and proteins Get e-Alerts

The Large-neutral Amino Acid Transporter 1 (LAT-1)—a sodium-independent exchanger of amino acids, thyroid hormones, and prescription drugs—is highly expressed in the blood–brain barrier and various types of cancer. LAT-1 plays an important role in cancer development as well as in mediating drug and nutrient delivery across the blood–brain barrier, making it a key drug target. Here, we identify four LAT-1 ligands, including one chemically novel substrate, by comparative modeling, virtual screening, and experimental validation. These results may rationalize the enhanced brain permeability of two drugs, including the anticancer agent acivicin. Finally, two of our hits inhibited proliferation of a cancer cell line by distinct mechanisms, providing useful chemical tools to characterize the role of LAT-1 in cancer metabolism.

All members of the human herpesvirus protease (HEW Pr) family are active as weakly associating dimers but inactive as monomers. A small-molecule allosteric inhibitor of Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr) traps the enzyme in an inactive monomeric state where the C-terminal helices are unfolded and the hydrophobic dimer interface is exposed. NMR titration studies demonstrate that the inhibitor binds to KSHV Pr monomers with low micromolar affinity. A 2.0-angstrom-resolution X-ray crystal structure of a C-terminal truncated KSHV Pr-inhibitor complex locates the binding pocket at the dimer interface and displays significant conformational perturbations at the active site, 15 angstrom from the allosteric site. NMR and CD data suggest that the small molecule inhibits human cytomegalovirus protease via a similar mechanism. As all HHV Prs are functionally and structurally homologous, the inhibitor represents a class of compounds that may be developed into broad-spectrum therapeutics that allosterically regulate enzymatic activity by disrupting protein-protein interactions. (C) 2011 Elsevier Ltd. All rights reserved.

We have identified a small molecule (DD2) that inhibits an essential dimeric protease of the human Kaposi’s sarcoma-associated herpesvirus (KSHV Pr). Proteolytic activity of KSHV Pr is disrupted by dissociation of dimeric state. Circular dichroism, chemical shift perturbation mapping and hydrogen exchange experiments indicate that a significant loss of structure at the C-terminal helices occurs concurrently with the dimer to monomer transition [1]. Complementary methods including size exclusion chromatography and 2D NMR spectroscopy show that the monomeric protease is enriched in the presence of DD2, leading to loss of enzymatic activity. 1H – 15N HSQC titration studies mapped the inhibitor-binding site to the dimer interface and mutagenesis studies targeting this region confirmed this binding mode [2]. As currently there are no specific treatments for herpesviral infections and with increased incidents of drug resistance, there is a need for the identification of new human herpesvirus therapeutics. The crystal structure of a monomeric form of KSHV Pr in complex with the small molecule inhibitor bound at the dimer interface was solved at 2.0 Å resolution. The final Rfactor/Rfree is 20.5%/24.3%, respectively. The complex was crystallized in the I222 space group. Two monomers of the truncated KSHV Pr were observed in the asymmetric unit with one DD2 molecule bound to monomer A, and two DD2 molecules bound to monomer B. The crystal structure is consistent with a mechanism where inhibitor binding prevents dimerization through the conformational selection of an inactive monomeric intermediate. This represents the first crystallographic structure of an inhibited monomeric Human Herpesvirus Protease to date.

Aromatic amino acids play critical roles in the stability and function of membrane-associated proteins and peptides. Here, we apply the site-selective vibrational tool, UV resonance Raman spectroscopy, to probe changes in the structure and microenvironment of tryptophan residues in integral membrane proteins and membrane-associated antimicrobial peptides. Alterations in molecular interactions, such as hydrogen-bonding states, cation-pi interactions, and local polarity, of tryptophan residues accompany the association and folding of these membrane-bound proteins to synthetic lipid bilayers. These results reveal the diversity of molecular interactions that help guide the in vitro assembly of membrane proteins and peptides in vesicles, and provide molecular clues to the mechanisms of membrane protein folding and vesicle disruption.

α‐Defensins are synthesized as inactive precursors that must undergo proteolytic conversion to mature bactericidal peptides. Acidic amino acids near the proCrp4 N terminus mediate inhibition of proCrp4 as shown by selective deletion and charge neutralizing mutagenesis (J. Biol. Chem. 284: 6826–6831, 2009). To study the inhibitory mechanism, we have compared the proximity of those anionic residues to the Crp4 component of native and mutant proCrp4 molecules using Förster resonance energy transfer (FRET). To provide FRET probes and enable FRET conformational studies in native and charge neutralized proCrp4, single Crp4 Trp substitutions were introduced in the α‐defensin moiety at V58W, F66W, and at the C terminus, and a Cys residue was added to proCrp4 N terminus (C N ) for IAEDANS labeling. ProCrp variants had bactericidal activities comparable to the parent molecules and were processed correctly by MMP‐7. FRET efficiency and distances between the FRET pairs in native and charge‐neutralized proCrp4 were determined. The N and C‐terminal regions of inactive C N (V58W)‐proCrp4 are closest with a FRET pair distance of 11.5 Å. In the active, bactericidal C N ‐(DE/G‐V58W)‐proCrp4 mutant, this distance increased to 14.3 Å. Thus, mutations that relieve proCrp4 inhibition do so without markedly repositioning the relation of the proregion N terminus to the Crp4 component of the precursor. Supported by NIH grants DK044632 and AI059346..

Ultraviolet resonance Raman (UVRR) spectra of tryptophan compounds in various solvents and a model peptide are presented and reveal systematic changes that reflect solvent polarity, hydrogen bond strength, and cation-pi interaction. The commonly utilized UVRR spectral marker for environment polarity that has been based on off-resonance Raman data, the tryptophan Fermi doublet ratio I1360/I1340, exhibits different values in on- and off-resonance Raman spectra as well as for different tryptophan derivatives. Specifically, the UVRR Fermi doublet ratio for indole ranges from 0.3 in polar solvents to 0.8 in nonpolar solvents, whereas the respective values reported here and previously for off-resonance Raman spectra are 0.5-1.3. UVRR Fermi doublet ratios for the more biologically relevant molecule, N-acetyl tryptophan ethyl ester (NATEE), are in a smaller range of 1.1 (polar solvent) to 1.7 (nonpolar solvent) and correlate to the solvent polarity/polarization parameters pi* and ETN. As has been reported previously, several UVRR modes are also sensitive to the hydrogen bond strength of the indole N-H moiety. Here, we report a new unambiguous marker for H-bonding: the ratio of the W10 (approximately 1237 cm-1) intensity to that of the W9 (approximately 1254 cm-1) mode (RW10). This ratio is 0.7 for NATEE in the absence of hydrogen bond acceptors and increases to 3.1 in the presence of strong hydrogen bond acceptors, with a value of 2.3 in water. The W8 and W17 modes shift more than +10 and approximately -5 cm-1 upon increase in hydrogen bond strength; this range for W17 is smaller than that reported previously and reflects a more realistic range for proteins and peptides in solution. Finally, our data provide evidence for change in the W18 and W16 relative intensity in the presence of cation-pi interactions. These UVRR markers are utilized to interpret spectra of model membrane-bound systems tryptophan octyl ester and the peptide toxin melittin. These spectra reveal the importance of intra- and intermolecular hydrogen bonding and cation-pi interactions that likely influence the partitioning of membrane-associated biomolecules to lipid bilayers or self-associated soluble oligomers. The UVRR analysis presented here modifies and augments prior reports and provides an unambiguous set of spectral makers that can be applied to elucidate the molecular microenvironment and structure of a wide range of complex systems, including anchoring tryptophan residues in membrane proteins and peptides.