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We synthesized and experimentally tested the passive permeability of more than thirty tetrapeptides mimicking the N-terminus of the pro-apoptotic protein Smac (Second mitochondria-derived activator of caspases). Each peptide bore one or two unnatural Hydrogen Bond Acceptor-bearing Amino Acid (HBA-AA) residues, such that intramolecular hydrogen bonding with proximal backbone amide N-H donors is feasible. Passive permeability of the synthetic peptides was determined using the parallel artificial membrane permeability assay (PAMPA). Experimental permeability values were found to span three orders of magnitude, providing useful empirical guidance for the design of more permeable Smac mimetics specifically, and peptidic ligands generally.

Ubiquitin specific protease 7 (USP7) regulates the protein stability of key cellular regulators in pathways ranging from apoptosis to neuronal development, making it a promising therapeutic target. Here we used an engineered, activated variant of the USP7 catalytic domain to perform structure-activity studies of electrophilic peptidomimetic inhibitors. Employing this USP7 variant, we found that inhibitors with a cyanopyrrolidine warhead unexpectedly promoted a β-elimination reaction of the initial covalent adducts, thereby converting the active-site cysteine residue to dehydroalanine. We determined that this phenomenon is specific for the USP7 catalytic cysteine and that structural features of the inhibitor and protein microenvironment impact elimination rates. Using comprehensive docking studies, we propose that the characteristic conformational dynamics of USP7 allow access to conformations that promote the ligand-induced elimination. Unlike in conventional reversible-covalent inhibition, the compounds described here irreversibly destroy a catalytic residue while simultaneously converting the inhibitor to a nonelectrophilic byproduct. Accordingly, this unexpected finding expands the scope of covalent inhibitor modalities and offers intriguing insights into enzyme-inhibitor dynamics.

Viruses are responsible for some of the most deadly human diseases, yet available vaccines and antivirals address only a fraction of the potential viral human pathogens. Here, we provide a methodology for managing human herpesvirus (HHV) infection by covalently inactivating the HHV maturational protease via a conserved, non-catalytic cysteine (C161). Using human cytomegalovirus protease (HCMV Pr) as a model, we screened a library of disulfides to identify molecules that tether to C161 and inhibit proteolysis, then elaborated hits into irreversible HCMV Pr inhibitors that exhibit broad-spectrum inhibition of other HHV Pr homologs. We further developed an optimized tool compound targeted toward HCMV Pr and used an integrative structural biology and biochemical approach to demonstrate inhibitor stabilization of HCMV Pr homodimerization, exploiting a conformational equilibrium to block proteolysis. Irreversible HCMV Pr inhibition disrupts HCMV infectivity in cells, providing proof of principle for targeting proteolysis via a non-catalytic cysteine to manage viral infection.

We describe the first systematic study of antimalarial 1,2,4-trioxolanes bearing a substitution pattern regioisomeric to that of arterolane. Conformational analysis suggested that trans-3″-substituted trioxolanes would exhibit Fe(II) reactivity and antiparasitic activity similar to that achieved with canonical cis-4″ substitution. The chiral 3″ analogues were prepared as single stereoisomers and evaluated alongside their 4″ congeners against cultured malaria parasites and in a murine malaria model. As predicted, the trans-3″ analogues exhibited in vitro antiplasmodial activity remarkably similar to that of their cis-4″ comparators. In contrast, efficacy in the Plasmodium berghei mouse model differed dramatically for some of the congeneric pairs. The best of the novel 3″ analogues (e.g., 12i) outperformed arterolane itself, producing cures in mice after a single oral exposure. Overall, this study suggests new avenues for modulating Fe(II) reactivity and the pharmacokinetic and pharmacodynamic properties of 1,2,4-trioxolane antimalarials.

Gram-negative pathogens expressing serine β-lactamases (SBLs) and metallo-β-lactamases (MBLs), especially those with carbapenemase activity, threaten the clinical utility of almost all β-lactam antibiotics. Here we describe the discovery of a heteroaryl phosphonate scaffold that exhibits non-covalent cross-class inhibition of representative carbapenemases, specifically the SBL KPC-2 and the MBLs NDM-1 and VIM-2. The most potent lead, compound 16, exhibited low nM to low μM inhibition of KPC-2, NDM-1, and VIM-2. Compound 16 potentiated imipenem efficacy against resistant clinical and laboratory bacterial strains expressing carbapenemases, while showing some cytotoxicity toward human HEK293T cells only at concentrations above 100 μg/mL. Complex structures with KPC-2, NDM-1, and VIM-2 demonstrate how these inhibitors achieve high binding affinity to both enzyme classes. These findings provide a structurally and mechanistically new scaffold for drug discovery targeting multidrug resistant Gram-negative pathogens, and more generally highlight the active site features of carbapenemases that can be leveraged for lead discovery.

Aberrant activation of caspase-6 (C6) in the absence of other hallmarks of apoptosis has been demonstrated in cells and tissues from patients with Huntington disease (HD) and animal models. C6 activity correlates with disease progression in patients with HD and the cleavage of mutant huntingtin (mHTT) protein is thought to strongly contribute to disease pathogenesis. Here we show that the mHTT1-586 fragment generated by C6 cleavage interacts with the zymogen form of the enzyme, stabilizing a conformation that contains an active site and is prone to full activation. This shift toward enhanced activity can be prevented by a small-molecule inhibitor that blocks the interaction between C6 and mHTT1-586. Molecular docking studies suggest that the inhibitor binds an allosteric site in the C6 zymogen. The interaction of mHTT1-586 with C6 may therefore promote a self-reinforcing, feedforward cycle of C6 zymogen activation and mHTT cleavage driving HD pathogenesis.

Here we describe a new approach for tumor targeting in which augmented concentrations of Fe(II) in cancer cells and/or the tumor microenvironment triggers drug release from an Fe(II)-reactive prodrug conjugate. The 1,2,4-trioxolane scaffold developed to enable this approach can in principle be applied to a broad range of cancer therapeutics and is illustrated here with Fe(II)-targeted forms of a microtubule toxin and a duocarmycin-class DNA-alkylating agent. We show that the intrinsic reactivity/toxicity of the duocarmycin analog is masked in the conjugated form and this greatly reduced toxicity in mice. This in turn permitted elevated dosing levels, leading to higher systemic exposure and a significantly improved response in tumor xenograft models. Overall our results suggest that Fe(II)-dependent drug delivery via trioxolane conjugates could have significant utility in expanding the therapeutic index of a range of clinical and preclinical stage cancer chemotherapeutics.

The integrated stress response comprises multiple signaling pathways for detecting and responding to cellular stress that converge at a single event-the phosphorylation of Ser51 on the α-subunit of eukaryotic translation initiation factor 2 (eIF2α). Phosphorylation of eIF2α (eIF2α-P) results in attenuation of global protein synthesis via the inhibitory effects of eIF2α-P on eIF2B, the guanine exchange factor (GEF) for eIF2. Herein we describe structure-activity relationship (SAR) studies of bis-O-arylglycolamides, first-in-class integrated stress response inhibitors (ISRIB). ISRIB analogues make cells insensitive to the effects of eIF2α-P by activating the GEF activity of eIF2B and allowing global protein synthesis to proceed with residual unphosphorylated eIF2α. The SAR studies described herein support the proposed pharmacology of ISRIB analogues as binding across a symmetrical protein-protein interface formed between protein subunits of the dimeric eIF2B heteropentamer.

Antibody-drug conjugates (ADCs) are antigen-targeted therapeutics that employ antibodies to deliver potent, cytotoxic effectors to cells with potentially high specificity. While promising clinical results have been achieved, significant pitfalls remain including internalization of ADCs in nontargeted cells expressing target antigen, which can limit therapeutic windows. Novel ADC linkers that are cleaved selectively in cancer cells but not in normal cells could minimize collateral damage caused by ADC uptake in nontargeted tissues. Here, we describe a prototypical ADC linker based on an Fe(II)-reactive 1,2,4-trioxolane scaffold (TRX) that by itself has demonstrated tumor-selective activity in preclinical cancer models. We prepared TRX-linked ADCs by site-selective conjugation to two sites in trastuzumab and compared their activity in Her2 positive and negative cells to ADC controls based on established linker chemistry. Our results confirm that the TRX moiety efficiently releases its payload following ADC uptake, affording picomolar potencies in antigen-positive cells. We also identified a destabilizing interaction between these initial TRX linkers and nearby antibody residues and suggest an approach to improve upon these prototypical designs.

Inhibition of the cysteine protease cruzain from Trypanosoma cruzi has been studied pre-clinically as a new chemotherapeutic approach to treat Chagas’ disease. Efficacious effects of vinylsulfone-based cruzain inhibitors in animal models support this therapeutic hypothesis. More recently, substrate–activity screening was used to identify nonpeptidic tetrafluorophenoxymethyl ketone inhibitors of cruzain that showed promising efficacy in animal models. Herein we report efforts to further optimize the in vitro potency and in vivo pharmacokinetic properties of this new class of cruzain inhibitors. Through modifications of the P1, P2 and/or P3 positions, new analogs have been identified with reduced lipophilicity, enhanced potency, and improved oral exposure and bioavailability.

Chagas disease affects 8 million people worldwide and remains a main cause of death due to heart failure in Latin America. The number of cases in the United States is now estimated to be 300,000, but there are currently no Food and Drug Administration (FDA)-approved drugs available for patients with Chagas disease. To fill this gap, we have established a public-private partnership between the University of California, San Francisco and the Genomics Institute of the Novartis Research Foundation (GNF) with the goal of delivering clinical candidates to treat Chagas disease. The discovery phase, based on the screening of more than 160,000 compounds from the GNF Academic Collaboration Library, led to the identification of new anti-Chagas scaffolds. Part of the screening campaign used and compared two screening methods, including a colorimetric-based assay using Trypanosoma cruzi expressing β-galactosidase and an image-based, high-content screening (HCS) assay using the CA-I/72 strain of T. cruzi. Comparing molecules tested in both assays, we found that ergosterol biosynthesis inhibitors had greater potency in the colorimetric assay than in the HCS assay. Both assays were used to inform structure-activity relationships for antiparasitic efficacy and pharmacokinetics. A new anti-T. cruzi scaffold derived from xanthine was identified, and we describe its development as lead series.

Summary Covalent inhibitors of K-Ras(G12C) have been reported that exclusively recognize the GDP state. Here, we utilize disulfide tethering of a non-natural cysteine (K-Ras(M72C)) to identify a new switch-II pocket (S-IIP) binding ligand (2C07) that engages the active GTP state. Co-crystal structures of 2C07 bound to H-Ras(M72C) reveal binding in a cryptic groove we term S-IIG. In the GppNHp state, 2C07 binding to a modified S-IIP pushes switch I away from the nucleotide, breaking the network of polar contacts essential for adopting the canonical GTP state. Biochemical studies show that 2C07 alters nucleotide preference and inhibits SOS binding and catalyzed nucleotide exchange. 2C07 was converted to irreversible covalent analogs, which target both nucleotide states, inhibit PI3K activation in vitro , and function as occupancy probes to detect reversible engagement in competition assays. Targeting both nucleotide states opens the possibility of inhibiting oncogenic mutants of Ras, which exist predominantly in the GTP state in cells.

Antimalarial agents artemisinin and arterolane act via initial reduction of a peroxide bond in a process likely mediated by ferrous iron sources in the parasite. Here, we report the synthesis and antiplasmodial activity of arterolane-like 1,2,4-trioxolanes specifically designed to release a tethered drug species within the malaria parasite. Compared with our earlier drug delivery scaffolds, these new arterolane-inspired systems are of significantly decreased molecular weight and possess superior metabolic stability. We describe an efficient, concise and scalable synthesis of the new systems, and demonstrate the use of the aminonucleoside antibiotic puromycin as a chemo/biomarker to validate successful drug release in live Plasmodium falciparum parasites. Together, the improved drug-like properties, more efficient synthesis, and proof of concept using puromycin, suggests these new molecules as improved vehicles for targeted drug delivery to the malaria parasite.

Creutzfeldt-Jakob disease (CJD) is a rare but invariably fatal neurodegenerative disease caused by misfolding of an endogenous protein into an alternative pathogenic conformation. The details of protein misfolding and aggregation are not well understood nor are the mechanism(s) by which the aggregated protein confers cellular toxicity. While there is as yet no clear consensus about how best to intervene therapeutically in CJD, prion infections can be propagated in cell culture and in experimental animals, affording both in vitro and in vivo models of disease. Here we review recent lead discovery efforts for CJD, with a focus on our own efforts to optimize 2-aminothiazole analogues for anti-prion potency in cells and for brain exposure in mice. The compounds that emerged from this effort were found to be efficacious in multiple animal models of prion disease even as they revealed new challenges for the field, including the emergence of resistant prion strains.

During prion diseases, a normally benign, host protein, denoted PrPC, undergoes alternative folding into the aberrant isoform, PrPSc. We used ELISA assays to identify and confirm hits in order to develop leads that reduce PrPSc in prion-infected dividing and stationary-phase mouse neuroblastoma (ScN2a-cl3) cells. We tested 52,830 diverse small molecules in dividing cells and 49,430 in stationary-phase cells. This led to 3,100 HTS and 970 single point confirmed (SPC) hits in dividing cells, 331 HTS and 55 confirmed SPC hits in stationary-phase cells as well as 36 confirmed SPC hits active in both. Fourteen chemical leads were identified from confirmed SPC hits in dividing cells and three in stationary-phase cells. From more than 682 compounds tested in concentration-effect relationships in dividing cells to determine potency (EC50), 102 had EC50 values between 1–10 µM and 50 had EC50 values of

Although they represent attractive therapeutic targets, caspases have so far proven recalcitrant to the development of drugs targeting the active site. Allosteric modulation of caspase activity is an alternate strategy that potentially avoids the need for anionic and electrophilic functionality present in most active-site inhibitors. Caspase-6 has been implicated in neurodegenerative disease, including Huntington's and Alzheimer's diseases. Herein we describe a fragment-based lead discovery effort focused on caspase-6 in its active and zymogen forms. Fragments were identified for procaspase-6 using surface plasmon resonance methods and subsequently shown by X-ray crystallography to bind a putative allosteric site at the dimer interface. A fragment-merging strategy was employed to produce nanomolar-affinity ligands that contact residues in the L2 loop at the dimer interface, significantly stabilizing procaspase-6. Because rearrangement of the L2 loop is required for caspase-6 activation, our results suggest a strategy for the allosteric control of caspase activation with drug-like small molecules.

K2P (KCNK) potassium channels generate "leak" potassium currents that strongly influence cellular excitability and contribute to pain, somatosensation, anesthesia, and mood. Despite their physiological importance, K2Ps lack specific pharmacology. Addressing this issue has been complicated by the challenges that the leak nature of K2P currents poses for electrophysiology-based high-throughput screening strategies. Here, we present a yeast-based high-throughput screening assay that avoids this problem. Using a simple growth-based functional readout, we screened a library of 106,281 small molecules and identified two new inhibitors and three new activators of the mammalian K2P channel K2P2.1 (KCNK2, TREK-1). By combining biophysical, structure-activity, and mechanistic analysis, we developed a dihydroacridine analogue, ML67-33, that acts as a low micromolar, selective activator of temperature- and mechano-sensitive K2P channels. Biophysical studies show that ML67-33 reversibly increases channel currents by activating the extracellular selectivity filter-based C-type gate that forms the core gating apparatus on which a variety of diverse modulatory inputs converge. The new K2P modulators presented here, together with the yeast-based assay, should enable both mechanistic and physiological studies of K2P activity and facilitate the discovery and development of other K2P small molecule modulators.

We evaluate experimentally and computationally the membrane permeability of matched sets of peptidic small molecules bearing natural or bioisosteric unnatural amino acids. We find that the intentional introduction of hydrogen bond acceptor-donor pairs in such molecules can improve membrane permeability while retaining or improving other favorable drug-like properties. We employ an all-atom force field based method to calculate changes in free energy associated with the transfer of the peptidic molecules from water to membrane. This computational method correctly predicts rank order experimental permeability trends within congeneric series and is much more predictive than calculations (e.g., clogP) that do not consider three-dimensional conformation.

The 1,2,4-trioxolanes are a new class of synthetic peroxidic antimalarials currently in human clinical trials. The well known reactivity of the 1,2,4-trioxolane ring towards inorganic ferrous iron and ferrous iron heme is proposed to play a role in the antimalarial action of this class of compounds. We have designed structurally relevant fluorescent chemical probes to study the sub-cellular localization of 1,2,4-trioxolanes in cultured Plasmodium falciparum parasites. Microscopy experiments revealed that a probe fluorescently labeled on the adamantane ring accumulated specifically in digestive vacuole-associated neutral lipid bodies within the parasite while an isosteric, but non-peroxidic congener did not. Probes fluorescently labeled on the cyclohexane ring showed no distinct localization pattern. In their sub-cellular localization and peroxidative effects, 1,2,4-trioxolane probes behave much like artemisinin-based probes studied previously. Our results are consistent with a role for adamantane-derived carbon-centered radicals in the antimalarial action of 1,2,4-trioxolanes, as hypothesized previously on the basis of chemical reactivity studies.

Artemisinin combination therapies (ACT) represent the current standard of care in the treatment of uncomplicated malaria. The widespread adoption of ACT has been motivated by a desire to minimize the emergence of drug resistance and to address the problem of recrudescence associated with artemisinin monotherapy.1–4 We set out to explore a single-molecule ‘fragmenting hybrid’ strategy in which an artemisinin-like peroxide is employed to deliver a partner drug, only upon activation by ferrous iron in the parasite. In principle, iron(II)-dependent drug delivery from a fragmenting hybrid could alleviate unwanted off-target bioactivities of the partner drug, which would be inactive in its hybrid form.

A docking screen identified reversible, non-covalent inhibitors (e.g. 1) of the parasite cysteine protease cruzain. Chemical optimization of 1 led to a series of oxadiazoles possessing interpretable SAR and potencies as much as 500-fold greater than 1. Detailed investigation of the SAR series subsequently revealed that many members of the oxadiazole class (and surprisingly also 1) act via divergent modes of inhibition – competitive or via colloidal aggregation – depending on the assay conditions employed.

A systematic study of P2 and P3 substitution in a series of vinyl sulfone cysteine protease inhibitors is described. The introduction of a methyl substituent in the P2 phenylalanine aryl ring had a favorable effect on protease inhibition and conferred modest selectivity for rhodesain over cruzain. Rhodesain selectivity could be enhanced further by combining these P2 modifications with certain P3 amide substituents.

Oxazolidinone analogs bearing substituted piperidine or azetidine C-rings are described. Analogs with a methyl group at the 3-position of the azetidine ring or the 4-position of the piperidine ring exhibited reduced mitochondrial protein synthesis inhibition while retaining good antibacterial potency.

Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics. Because no drug exists that halts or even slows any neurodegenerative disease, developing effective therapeutics for any prion disorder is urgent. We recently reported two compounds (IND24 and IND81) with the 2-aminothiazole (2-AMT) chemical scaffold that almost doubled the incubation times in scrapie prion-infected, wild-type (wt) FVB mice when given in a liquid diet. Remarkably, oral prophylactic treatment with IND24 beginning 14 days prior to intracerebral prion inoculation extended survival from ∼120 days to over 450 days. In addition to IND24, we evaluated the pharmacokinetics and efficacy of five additional 2-AMTs; one was not followed further because its brain penetration was poor. Of the remaining four new 2-AMTs, IND114338 doubled and IND125 tripled the incubation times of RML-inoculated wt and Tg4053 mice overexpressing wt mouse prion protein (PrP), respectively. Neuropathological examination of the brains from untreated controls showed a widespread deposition of self-propagating, β-sheet-rich "scrapie" isoform (PrPSc) prions accompanied by a profound astrocytic gliosis. In contrast, mice treated with 2-AMTs had lower levels of PrPScand associated astrocytic gliosis, with each compound resulting in a distinct pattern of deposition. Notably, IND125 prevented both PrPScaccumulation and astrocytic gliosis in the cerebrum. Progressive central nervous system dysfunction in the IND125-treated mice was presumably due to the PrPScthat accumulated in their brainstems. Disappointingly, none of the four new 2-AMTs prolonged the lives of mice expressing a chimeric human/mouse PrP transgene inoculated with Creutzfeldt-Jakob disease prions.

The oxazolidinones are a promising new class of synthetic antibacterial agents. Here, we review recent efforts directed at the discovery of new antibacterial compounds of this class. New structures and structure-activity relationships (SAR) are discussed in the context of earlier work in the field. Key issues of potency, spectrum, selectivity, in vivo efficacy, and pharmacokinetic profile of the new analogs are addressed.

The oxazolidinones are a new class of synthetic antibacterials effective against a broad range of pathogenic Gram-positive bacteria, including multi-drug-resistant strains. Linezolid is the first drug from this class to reach the market and has become an important new option for the treatment of serious infections, particularly those caused by methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enteroccocus faecium (VRE). In the search for novel oxazolidinones with improved potency and spectrum, we have prepared and evaluated the antibacterial properties of conformationally constrained analogues in which the morpholine ring of linezolid is replaced with various substituted azabicyclo[3.1.0]hexyl ring systems. Several classes of azabicyclic analogues were identified with activity comparable or superior to that of linezolid. These include analogues bearing hydroxyl, amino, amido, or carboxyl groups on the azabicyclic ring. The azabicyclic acid analogue 50 was 4 times more potent than linezolid against key Gram-positive and fastidious Gram-negative pathogens (S. aureus, Streptococcus pneumoniae, and E. faecalis MICs < or = 1 microg/mL; Haemophilus influenzae MIC = 4 microg/mL).

The ubiquitous AAA+ ATPase p97 functions as a dynamic molecular machine driving several cellular processes. It is essential in regulating protein homeostasis, and it represents a potential drug target for cancer, particularly when there is a greater reliance on the endoplasmic reticulum-associated protein degradation pathway and ubiquitin-proteasome pathway to degrade an overabundance of secreted proteins. Here, we report a case study for using fragment-based ligand design approaches against this large and dynamic hexamer, which has multiple potential binding sites for small molecules. A screen of a fragment library was conducted by surface plasmon resonance (SPR) and followed up by nuclear magnetic resonance (NMR), two complementary biophysical techniques. Virtual screening was also carried out to examine possible binding sites for the experimental hits and evaluate the potential utility of fragment docking for this target. Out of this effort, 13 fragments were discovered that showed reversible binding with affinities between 140 µM and 1 mM, binding stoichiometries of 1:1 or 2:1, and good ligand efficiencies. Structural data for fragment-protein interactions were obtained with residue-specific [U-(2)H] (13)CH3-methyl-labeling NMR strategies, and these data were compared to poses from docking. The combination of virtual screening, SPR, and NMR enabled us to find and validate a number of interesting fragment hits and allowed us to gain an understanding of the structural nature of fragment binding.

The production of β-lactamase is one of the primary resistance mechanisms used by Gram-negative bacterial pathogens to counter β-lactam antibiotics, such as penicillins, cephalosporins and carbapenems. There is an urgent need to develop novel β-lactamase inhibitors in response to ever evolving β-lactamases possessing an expanded spectrum of β-lactam hydrolyzing activity. Whereas traditional high-throughput screening has proven ineffective against serine β-lactamases, fragment-based approaches have been successfully employed to identify novel chemical matter, which in turn has revealed much about the specific molecular interactions possible in the active site of serine and metallo β-lactamases. In this review, we summarize recent progress in the field, particularly: the identification of novel inhibitor chemotypes through fragment-based screening; the use of fragment-protein structures to understand key features of binding hot spots and inform the design of improved leads; lessons learned and new prospects for β-lactamase inhibitor development using fragment-based approaches.

Purpose: Previous studies showed that lowering PrPC concomitantly reduced PrPSc in the brains of mice inoculated with prions. We aimed to develop assays that measure PrPC on the surface of human T98G glioblastoma and IMR32 neuroblastoma cells. Using these assays, we sought to identify chemical hits, confirmed hits, and scaffolds that potently lowered PrPC levels in human brains cells, without lethality, and that could achieve drug concentrations in the brain after oral or intraperitoneal dosing in mice. Methods: We utilized HTS ELISA assays to identify small molecules that lower PrPC levels by ≥30% on the cell surface of human glioblastoma (T98G) and neuroblastoma (IMR32) cells. Results: From 44,578 diverse chemical compounds tested, 138 hits were identified by single point confirmation (SPC) representing 7 chemical scaffolds in T98G cells, and 114 SPC hits representing 6 scaffolds found in IMR32 cells. When the confirmed SPC hits were combined with structurally related analogs, >300 compounds (representing 6 distinct chemical scaffolds) were tested for dose-response (EC50) in both cell lines, only studies in T98G cells identified compounds that reduced PrPC without killing the cells. EC50 values from 32 hits ranged from 65 nM to 4.1 μM. Twenty-eight were evaluated in vivo in pharmacokinetic studies after a single 10 mg/kg oral or intraperitoneal dose in mice. Our results showed brain concentrations as high as 16.2 μM, but only after intraperitoneal dosing. Conclusions: Our studies identified leads for future studies to determine which compounds might lower PrPC levels in rodent brain, and provide the basis of a therapeutic for fatal disorders caused by PrP prions. © 2014 Elsevier Ltd. All rights reserved.

The only small-molecule compound demonstrated to substantially extend survival in prion-infected mice is a biaryl hydrazone termed “Compd B” (4-pyridinecarboxaldehyde,2-[4-(5-oxazolyl)phenyl]hydrazone). However, the hydrazone moiety of Compd B results in toxic metabolites, making it a poor candidate for further drug development. We developed a pharmacophore model based on diverse antiprion compounds identified by high-throughput screening; based on this model, we generated biaryl amide analogs of Compd B. Medicinal chemistry optimization led to multiple compounds with increased potency, increased brain concentrations, and greater metabolic stability, indicating that they could be promising candidates for antiprion therapy. Replacing the pyridyl ring of Compd B with a phenyl group containing an electron-donating substituent increased potency, while adding an aryl group to the oxazole moiety increased metabolic stability. To test the efficacy of Compd B, we applied bioluminescence imaging (BLI), which was previously shown to detect prion disease onset in live mice earlier than clinical signs. In our studies, Compd B showed good efficacy in two lines of transgenic mice infected with the mouse-adapted Rocky Mountain Laboratory (RML) strain of prions, but not in transgenic mice infected with human prions. The BLI system successfully predicted the efficacies in all cases long before extension in survival could be observed. Our studies suggest that this BLI system has good potential to be applied in future antiprion drug efficacy studies.

Recently, we described the aminothiazole lead (4-biphenyl-4-ylthiazol-2-yl)-(6-methylpyridin-2-yl)-amine (1), which exhibits many desirable properties, including excellent stability in liver microsomes, oral bioavailability of ∼40%, and high exposure in the brains of mice. Despite its good pharmacokinetic properties, compound 1 exhibited only modest potency in mouse neuroblastoma cells overexpressing the disease-causing prion protein PrPSc. Accordingly, we sought to identify analogues of 1 with improved antiprion potency in ScN2a-cl3 cells while retaining similar or superior properties. Herein we report the discovery of improved lead compounds such as (6-methylpyridin-2-yl)-[4-(4-pyridin-3-yl-phenyl)thiazol-2-yl]amine and cyclopropanecarboxylic acid (4-biphenylthiazol-2-yl)amide, which exhibit brain exposure/EC50ratios at least tenfold greater than that of compound 1. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Purpose: To discover drugs lowering PrPScin prion-infected cultured neuronal cells that achieve high concentrations in brain to test in mouse models of prion disease and then treat people with these fatal diseases. Methods: We tested 2-AMT analogs for EC50and PK after a 40 mg/kg single dose and 40-210 mg/kg/day doses for 3 days. We calculated plasma and brain AUC, ratio of AUC/EC50after dosing. We reasoned that compounds with high AUC/EC50ratios should be good candidates going forward. Results: We evaluated 27 2-AMTs in single-dose and 10 in 3-day PK studies, of which IND24 and IND81 were selected for testing in mouse models of prion disease. They had high concentrations in brain after oral dosing. Absolute bioavailability ranged from 27-40%. AUC/EC50ratios after 3 days were >100 (total) and 48-113 (unbound). Stability in liver microsomes ranged from 30->60 min. Ring hydroxylated metabolites were observed in microsomes. Neither was a substrate for the MDR1 transporter. Conclusions: IND24 and IND81 are active in vitro and show high AUC/EC50ratios (total and unbound) in plasma and brain. These will be evaluated in mouse models of prion disease. © 2013 Springer Science+Business Media New York.

International audience; The emergence of CTX-M class A extended-spectrum beta-lactamases poses a serious health threat to the public. We have applied structure-based design to improve the potency of a novel noncovalent tetrazole-containing CTX-M inhibitor (K-i = 21 mu M) more than 200-fold via structural modifications targeting two binding hot spots, a hydrophobic shelf formed by Pro167 and a polar site anchored by Asp240. Functional groups contacting each binding hot spot independently in initial designs were later combined to produce analogues with submicromolar potencies, including 6-trifluoromethyl-3H-benzoimidazole-4-carboxylic acid [3-(1H-tetrazol-5-yl)-phenyl]-amide, which had a K-i value of 89 nM and reduced the MIC of cefotaxime by 64-fold in CTX-M-9 expressing Escherichia coli. The in vitro potency gains were accompanied by improvements in ligand efficiency (from 0.30 to 0.39) and LipE (from 1.37 to 3.86). These new analogues represent the first nM-affinity noncovalent inhibitors of a class A beta-lactamase. Their complex crystal structures provide valuable information about ligand binding for future inhibitor design.

The Sandler Center’s approach to target-based drug discovery for neglected tropical diseases is to focus on parasite targets that are homologous to human targets being actively investigated in the pharmaceutical industry. In this way we attempt to use both the know-how and actual chemical matter from other drug-development efforts to jump start the discovery process for neglected tropical diseases. Our approach is akin to drug repurposing, except that we seek to repurpose leads rather than drugs. Medicinal chemistry can then be applied to optimize the leads specifically for the desired antiparasitic indication.

2-Aminothiazoles are a new class of small molecules with antiprion activity in prion-infected neuroblastoma cell lines (J. Virol. 2010, 84, 3408). We report here structure-activity studies undertaken to improve the potency and physiochemical properties of 2-aminothiazoles, with a particular emphasis on achieving and sustaining high drug concentrations in the brain. The results of this effort include the generation of informative structure-activity relationships (SAR) and the identification of lead compounds that are orally absorbed and achieve high brain concentrations in animals. The new aminothiazole analogue (5-methylpyridin-2-yl)-[4-(3-phenylisoxazol-5-yl)-thiazol-2-yl]-amine (27), for example, exhibited an EC(50) of 0.94 μM in prion-infected neuroblastoma cells (ScN2a-cl3) and reached a concentration of ∼25 μM in the brains of mice following three days of oral administration in a rodent liquid diet. The studies described herein suggest 2-aminothiazoles as promising new leads in the search for effective therapeutics for prion diseases.

We describe here the identification of non-peptidic vinylsulfones that inhibit parasite cysteine proteases in vitro and inhibit the growth of Trypanosoma brucei brucei parasites in culture. A high resolution (1.75 A) co-crystal structure of 8a bound to cruzain reveals how the non-peptidic P2/P3 moiety in such analogs bind the S2 and S3 subsites of the protease, effectively recapitulating important binding interactions present in more traditional peptide-based protease inhibitors and natural substrates.

R. Jeffrey Neitz, Steven Chen, Frantisek Supek, Vince Yeh, Danielle Kellar, Jiri Gut, Clifford Bryant, Alejandra Gallardo-Godoy, Valentina Molteni, Steven L. Roach, Arnab K. Chatterjee, Stephanie Robertson, Adam R. Renslo, Michelle Arkin, Richard Glynne, James McKerrow, and Jair L. Siqueira-Neto. Lead Identification to Clinical Candidate Selection: Drugs for Chagas Disease J. Biomol. Screen. 2015, 20, 101-111. (Original doi:10.1177/1087057114553103) In the January 2015 issue of the Journal of Biomolecular Screening, the names of two authors were not published with this article, Shilpi Khare and Monique Stinson. Their names should have appeared after Steven L. Roach, and the affiliation for both of the missing authors is the Genomics Institute of the Novartis Research Foundation (GNF), San Diego, CA, USA.

A new series of antimicrobial oxazolidinones bearing unsaturated heterocyclic C-rings is described. Dihydrothiopyran derivatives were prepared from the saturated tetrahydrothiopyran sulfoxides via a Pummerer-rearrangement/elimination sequence. Two new synthetic approaches to the dihydrothiazine ring system were explored, the first involving a novel trifluoroacetylative-detrifluoroacetylative Pummerer-type reaction sequence and the second involving direct dehydrogenation of tetrahydrothiopyran S,S-dioxide intermediates. Final analogs such as 4 and 13 represent oxidized congeners of recent pre-clinical and clinical oxazolidinones.

A new class of oxazolidinone antibacterials incorporating oxygen-, nitrogen-, or sulfur-containing heterobicyclic C-rings is described. The in vitro potency and in vivo efficacy of these conformationally constrained oxazolidinone analogs are discussed.