Selective targeting of Plasmodium falciparum Hsp90 disrupts the 26S proteasome.

The molecular chaperone heat shock protein 90 (Hsp90) has an essential but largely undefined role in maintaining proteostasis in Plasmodium falciparum, the most lethal malaria parasite. Herein, we identify BX-2819 and XL888 as potent P. falciparum (Pf)Hsp90 inhibitors. Derivatization of XL888's scaffold led to the development of Tropane 1, as a PfHsp90-selective binder with nanomolar affinity. Hsp90 inhibitors exhibit anti-Plasmodium activity against the liver, asexual blood, and early gametocyte life stages. Thermal proteome profiling was implemented to assess PfHsp90-dependent proteome stability, and the proteasome-the main site of cellular protein recycling-was enriched among proteins with perturbed stability upon PfHsp90 inhibition. Subsequent biochemical and cellular studies suggest that PfHsp90 directly promotes proteasome hydrolysis by chaperoning the active 26S complex. These findings expand our knowledge of the PfHsp90-dependent proteome and protein quality control mechanisms in these pathogenic parasites, as well as further characterize this chaperone as a potential antimalarial drug target.

Shotgun Kinetic Target-Guided Synthesis Approach Enables the Discovery of Small-Molecule Inhibitors against Pathogenic Free-Living Amoeba Glucokinases.

Pathogenic free-living amoebae (pFLA) can cause life-threatening central nervous system (CNS) infections and warrant the investigation of new chemical agents to combat the rise of infection from these pathogens. Naegleria fowleri glucokinase (NfGlck), a key metabolic enzyme involved in generating glucose-6-phosphate, was previously identified as a potential target due to its limited sequence similarity with human Glck (HsGlck). Herein, we used our previously demonstrated multifragment kinetic target-guided synthesis (KTGS) screening strategy to identify inhibitors against pFLA glucokinases. Unlike the majority of previous KTGS reports, our current study implements a "shotgun" approach, where fragments were not biased by predetermined binding potentials. The study resulted in the identification of 12 inhibitors against 3 pFLA glucokinase enzymes─NfGlck, Balamuthia mandrillaris Glck (BmGlck), and Acanthamoeba castellanii Glck (AcGlck). This work demonstrates the utility of KTGS to identify small-molecule binders for biological targets where resolved X-ray crystal structures are not readily accessible.

Benzothiazole Substitution Analogs of Rhodacyanine Hsp70 Inhibitors Modulate Tau Accumulation.

The accumulation and aggregation of the microtubule-associated protein tau (tau) into intracellular neuronal tangles are a hallmark of a range of progressive neurodegenerative tauopathies, including Alzheimer's disease (AD), frontotemporal dementia, Pick's disease, and progressive supranuclear palsy. The aberrant phosphorylation of tau is associated with tau aggregates in AD. Members of the heat shock protein 70 kDa (Hsp70) family of chaperones bind directly to tau and modulate tau clearance and aggregation. Small molecules that inhibit the Hsp70 family of chaperones have been shown to reduce the accumulation of tau, including phosphorylated tau. Here, eight analogs of the rhodacyanine inhibitor, JG-98, were synthesized and evaluated. Like JG-98, many of the compounds inhibited ATPase activity of the cytosolic heat shock cognate 70 protein (Hsc70) and reduced total, aggregated, and phosphorylated tau accumulation in cultured cells. Three compounds, representing divergent clogP values, were evaluated for in vivo blood-brain barrier penetration and tau reduction in an ex vivo brain slice model. AL69, the compound with the lowest clogP and the lowest membrane retention in a parallel artificial membrane permeability assay (PAMPA), reduced phosphorylated tau accumulation. Our results suggest that benzothiazole substitutions of JG-98 that increase hydrophilicity may increase the efficacy of these Hsp70 inhibitors to reduce phosphorylated tau.

Alkyne modified purines for assessment of activation of Plasmodium vivax hypnozoites and growth of pre-erythrocytic and erythrocytic stages in Plasmodium spp.

Malaria is a major global health problem which predominantly afflicts developing countries. Although many antimalarial therapies are currently available, the protozoan parasite causing this disease, Plasmodium spp., continues to evade eradication efforts. One biological phenomenon hampering eradication efforts is the parasite's ability to arrest development, transform into a drug-insensitive form, and then resume growth post-therapy. Currently, the mechanisms by which the parasite enters arrested development, or dormancy, and later recrudesces or reactivates to continue development, are unknown and the malaria field lacks techniques to study these elusive mechanisms. Since Plasmodium spp. salvage purines for DNA synthesis, we hypothesised that alkyne-containing purine nucleosides could be used to develop a DNA synthesis marker which could be used to investigate mechanisms behind dormancy. Using copper-catalysed click chemistry methods, we observe incorporation of alkyne modified adenosine, inosine, and hypoxanthine in actively replicating asexual blood stages of Plasmodium falciparum and incorporation of modified adenosine in actively replicating liver stage schizonts of Plasmodium vivax. Notably, these modified purines were not incorporated in dormant liver stage hypnozoites, suggesting this marker could be used as a tool to differentiate replicating and non-replicating liver forms and, more broadly, as a tool for advancing our understanding of Plasmodium dormancy mechanisms.

Naegleria fowleri: Protein structures to facilitate drug discovery for the deadly, pathogenic free-living amoeba.

Naegleria fowleri is a pathogenic, thermophilic, free-living amoeba which causes primary amebic meningoencephalitis (PAM). Penetrating the olfactory mucosa, the brain-eating amoeba travels along the olfactory nerves, burrowing through the cribriform plate to its destination: the brain's frontal lobes. The amoeba thrives in warm, freshwater environments, with peak infection rates in the summer months and has a mortality rate of approximately 97%. A major contributor to the pathogen's high mortality is the lack of sensitivity of N. fowleri to current drug therapies, even in the face of combination-drug therapy. To enable rational drug discovery and design efforts we have pursued protein production and crystallography-based structure determination efforts for likely drug targets from N. fowleri. The genes were selected if they had homology to drug targets listed in Drug Bank or were nominated by primary investigators engaged in N. fowleri research. In 2017, 178 N. fowleri protein targets were queued to the Seattle Structural Genomics Center of Infectious Disease (SSGCID) pipeline, and to date 89 soluble recombinant proteins and 19 unique target structures have been produced. Many of the new protein structures are potential drug targets and contain structural differences compared to their human homologs, which could allow for the development of pathogen-specific inhibitors. Five of the structures were analyzed in more detail, and four of five show promise that selective inhibitors of the active site could be found. The 19 solved crystal structures build a foundation for future work in combating this devastating disease by encouraging further investigation to stimulate drug discovery for this neglected pathogen.

Identification and Characterization of Novel Bronchodilator Agonists Acting at Human Airway Smooth Muscle Cell TAS2R5.

Bitter taste receptors (TAS2Rs) are recognized as being expressed on multiple cell types and organs, including human airway smooth muscle (HASM) cells, where agonists promote significant relaxation to constrictive stimuli. Thus, the HASM TAS2Rs have been targeted as novel bronchodilators for the treatment of asthma and other obstructive lung diseases. The TAS2R5 subtype, a dominant receptor on HASM, has few known agonists, all with reported low potency and efficacy. We screened multiple compounds by measuring [Ca2+]i release in HASM (a consequence of receptor-G protein coupling) to establish structure-activity relationships and arrive at a potent agonist for TAS2R5. HASM physiological studies using magnetic twisting cytometry confirmed the relaxation effects of lead compounds. 1,10-Phenanthroline-5,6-dione had the greatest potency (EC50 ≈ 120 nM), amounting to a >1000-fold improvement over the other compounds, and displayed maximal efficacy. These studies revealed critical structural requirements for favorable potencies and efficacies for a potential first-in-class bronchodilator targeting TAS2R5 of the airway.

Characterization of Arylalkylamine N-Acyltransferase from Tribolium castaneum: An Investigation into a Potential Next-Generation Insecticide Target.

The growing issue of insecticide resistance has meant the identification of novel insecticide targets has never been more important. Arylalkylamine N-acyltransferases (AANATs) have been suggested as a potential new target. These promiscuous enzymes are involved in the N-acylation of biogenic amines to form N-acylamides. In insects, this process is a key step in melanism, hardening of the cuticle, removal of biogenic amines, and in the biosynthesis of fatty acid amides. The unique nature of each AANAT isoform characterized indicates each organism accommodates an assembly of discrete AANATs relatively exclusive to that organism. This implies a high potential for selectivity in insecticide design, while also maintaining polypharmacology. Presented here is a thorough kinetic and structural analysis of AANAT found in one of the most common secondary pests of all plant commodities in the world, Tribolium castaneum. The enzyme, named TcAANAT0, catalyzes the formation of short-chain N-acylarylalkylamines, with short-chain acyl-CoAs (C2-C10), benzoyl-CoA, and succinyl-CoA functioning in the role of acyl donor. Recombinant TcAANAT0 was expressed and purified from E. coli and was used to investigate the kinetic and chemical mechanism of catalysis. The kinetic mechanism is an ordered sequential mechanism with the acyl-CoA binding first. pH-rate profiles and site-directed mutagenesis studies identified amino acids critical to catalysis, providing insights about the chemical mechanism of TcAANAT0. A crystal structure was obtained for TcAANAT0 bound to acetyl-CoA, revealing valuable information about its active site. This combination of kinetic analysis and crystallography alongside mutagenesis and sequence analysis shines light on some approaches possible for targeting TcAANAT0 and other AANATs for novel insecticide design.

Targeting the FKBP51/GR/Hsp90 Complex to Identify Functionally Relevant Treatments for Depression and PTSD.

Genetic and epigenetic alterations in FK506-binding protein 5 ( FKBP5) have been associated with increased risk for psychiatric disorders, including post-traumatic stress disorder (PTSD). Some of these common variants can increase the expression of FKBP5, the gene that encodes FKBP51. Excess FKBP51 promotes hypothalamic-pituitary-adrenal (HPA) axis dysregulation through altered glucocorticoid receptor (GR) signaling. Thus, we hypothesized that GR activity could be restored by perturbing FKBP51. Here, we screened 1280 pharmacologically active compounds and identified three compounds that rescued FKBP51-mediated suppression of GR activity without directly activating GR. One of the three compounds, benztropine mesylate, disrupted the association of FKBP51 with the GR/Hsp90 complex in vitro. Moreover, we show that removal of FKBP51 from this complex by benztropine restored GR localization in ex vivo brain slices and primary neurons from mice. In conclusion, we have identified a novel disruptor of the FKBP51/GR/Hsp90 complex. Targeting this complex may be a viable approach to developing treatments for disorders related to aberrant FKBP51 expression.

Enantioselective Total Synthesis of Cannabinoids-A Route for Analogue Development.

A practical synthetic approach to Δ9-tetrahydrocannabinol (1) and cannabidiol (2) that provides scalable access to these natural products and should enable the generation of novel synthetic analogues is reported.

Binding-based proteomic profiling and the fatty acid amides.

Fatty acid amides represent a diverse and underappreciated family of lipids found in vertebrates and invertebrates. The most recognized, most studied, and best understood members of the fatty acid amide family are N-arachidonoylethanolamine (anandamide) and oleamide. Over 70 other fatty acid amides have been identified from biological systems and these non-anandamide and non-oleamide fatty acid amides are not well understood: their cellular functions, transport, biosynthesis, and degradation are, at best, partially elucidated. Most of the fatty acid amides are "orphan" ligands for "orphan" or unknown receptors. Interest in the fatty acid amides will wane without a more complete understanding of their function in vivo and most of these lipids will be mentioned in a few sentences in reviews on ananamide and/or olemide. In this commentary, we suggest that one strategy to dramatically increase our understanding of any member of the fatty acid amide family is the design, synthesis, and proper use of binding-based profiling probes (BBPPs) based on the structure of a specific fatty acid amide. A BBPP is an analog of a fatty acid amide that enables the controlled covalent attachment of the probe to a fatty acid amide-binding protein and, also, possesses a chemical moiety that will allow the purification and/or detection of the BBPP-labeled proteins. The identification of the proteins that specifically bind a fatty acid amide will foster a better understanding of the function, transport, and metabolism of a fatty acid amide.

Structural and Mechanistic Analysis of Drosophila melanogaster Agmatine N-Acetyltransferase, an Enzyme that Catalyzes the Formation of N-Acetylagmatine.

Agmatine N-acetyltransferase (AgmNAT) catalyzes the formation of N-acetylagmatine from acetyl-CoA and agmatine. Herein, we provide evidence that Drosophila melanogaster AgmNAT (CG15766) catalyzes the formation of N-acetylagmatine using an ordered sequential mechanism; acetyl-CoA binds prior to agmatine to generate an AgmNAT•acetyl-CoA•agmatine ternary complex prior to catalysis. Additionally, we solved a crystal structure for the apo form of AgmNAT with an atomic resolution of 2.3 Å, which points towards specific amino acids that may function in catalysis or active site formation. Using the crystal structure, primary sequence alignment, pH-activity profiles, and site-directed mutagenesis, we evaluated a series of active site amino acids in order to assign their functional roles in AgmNAT. More specifically, pH-activity profiles identified at least one catalytically important, ionizable group with an apparent pKa of ~7.5, which corresponds to the general base in catalysis, Glu-34. Moreover, these data led to a proposed chemical mechanism, which is consistent with the structure and our biochemical analysis of AgmNAT.

Synthesis and Activity of a New Series of Antileishmanial Agents.

We have determined that tetrahydroindazoles such as 1 show potent activity against Leishmania donovani, the causative agent of leishmaniasis. While the Hsp90 activity and anticancer properties of 1 have previously been explored, we present here our efforts to optimize their activity against L. donovani via the synthesis of novel analogues designed to probe the hydrophobic pocket of the protozoan Hsp90 orthologue, specifically through the auspices of functionalization of an amine embedded into the scaffold.

Identification of a Hit Series of Antileishmanial Compounds through the Use of Mixture-Based Libraries.

From a screening campaign that included mixture-based libraries containing more than 6 million compounds, a lead series of bis-cyclic guanidines was identified as the most promising. Lead optimization resulted in the identification of potent (IC50 < 500 nM) and selective compounds within this series as well as potent and selective monoguanidines.

Synthesis of the phorboxazoles-potent, architecturally novel marine natural products.

The phorboxazoles have attracted the attention of synthetic chemists around the globe due to their potent biological activity and novel structure. A review of the recent synthetic efforts as well as new phorboxazole analogs is discussed.

Inactivation of peptidylglycine α-hydroxylating monooxygenase by cinnamic acid analogs.

Peptidylglycine α-amidating monooxygenase (PAM) is a bifunctional enzyme that catalyzes the final reaction in the maturation of α-amidated peptide hormones. Peptidylglycine α-hydroxylating monooxygenase (PHM) is the PAM domain responsible for the copper-, ascorbate- and O2-dependent hydroxylation of a glycine-extended peptide. Peptidylamidoglycolate lyase is the PAM domain responsible for the Zn(II)-dependent dealkylation of the α-hydroxyglycine-containing precursor to the final α-amidated peptide. We report herein that cinnamic acid and cinnamic acid analogs are inhibitors or inactivators of PHM. The inactivation chemistry exhibited by the cinnamates exhibits all the attributes of a suicide-substrate. However, we find no evidence for the formation of an irreversible linkage between cinnamate and PHM in the inactivated enzyme. Our data support the reversible formation of a Michael adduct between an active site nucleophile and cinnamate that leads to inactive enzyme. Our data are of significance given that cinnamates are found in foods, perfumes, cosmetics and pharmaceuticals.

Discovery of a novel class of highly potent, selective, ATP-competitive, and orally bioavailable inhibitors of the mammalian target of rapamycin (mTOR).

A series of novel, highly potent, selective, and ATP-competitive mammalian target of rapamycin (mTOR) inhibitors based on a benzoxazepine scaffold have been identified. Lead optimization resulted in the discovery of inhibitors with low nanomolar activity and greater than 1000-fold selectivity over the closely related PI3K kinases. Compound 28 (XL388) inhibited cellular phosphorylation of mTOR complex 1 (p-p70S6K, pS6, and p-4E-BP1) and mTOR complex 2 (pAKT (S473)) substrates. Furthermore, this compound displayed good pharmacokinetics and oral exposure in multiple species with moderate bioavailability. Oral administration of compound 28 to athymic nude mice implanted with human tumor xenografts afforded significant and dose-dependent antitumor activity.

SAR and in vivo evaluation of 4-aryl-2-aminoalkylpyrimidines as potent and selective Janus kinase 2 (JAK2) inhibitors.

We report the discovery of a series of 4-aryl-2-aminoalkylpyrimidine derivatives as potent and selective JAK2 inhibitors. High throughput screening of our in-house compound library led to the identification of hit 1, from which optimization resulted in the discovery of highly potent and selective JAK2 inhibitors. Advanced lead 10d demonstrated a significant dose-dependent pharmacodynamic and antitumor effect in a mouse xenograft model. Based upon the desirable profile of 10d (XL019) it was advanced into clinical trials.

Discovery of a novel series of potent and orally bioavailable phosphoinositide 3-kinase γ inhibitors.

The phosphoinositide 3-kinases (PI3Ks) have been linked to an extraordinarily diversified group of cellular functions making these enzymes compelling targets for the treatment of disease. A large body of evidence has linked PI3Kγ to the modulation of autoimmune and inflammatory processes making it an intriguing target for drug discovery. Our high-throughput screening (HTS) campaign revealed two hits that were nominated for further optimization studies. The in vitro activity of the first HTS hit, designated as the sulfonylpiperazine scaffold, was optimized utilizing structure-based design. However, nonoptimal pharmacokinetic properties precluded this series from further studies. An overlay of the X-ray structures of the sulfonylpiperazine scaffold and the second HTS hit within their complexes with PI3Kγ revealed a high degree of overlap. This feature was utilized to design a series of hybrid analogues including advanced leads such as 31 with desirable potency, selectivity, and oral bioavailability.

Discovery of a novel class of potent and orally bioavailable sphingosine 1-phosphate receptor 1 antagonists.

A series of subtype selective sphingosine 1-phosphate receptor 1 (S1P(1)) antagonists are disclosed. Our high-throughput screening campaign revealed hit 1 for which an increase in potency and mouse oral exposure was achieved with minor modifications to the chemical scaffold. In vivo efficacy revealed that at high doses compounds 12 and 15 inhibited tumor growth. Further optimization of our lead series led to the discovery of proline derivatives 37 (XL541) and 38 which had similar efficacy as our first generation analogues at significantly lower doses. Analogue 37 displayed excellent pharmacokinetics and oral exposure in multiple species.

Enantioselective total synthesis of the antitumor macrolide rhizoxin D.

The convergent, highly enantioselective synthesis of rhizoxin D, a natural product possessing potent antitumor and antifungal bioactivity, is described. The C(1)-C(9) fragment of the molecule was synthesized utilizing a threefold pseudosymmetric intermediate ultimately derived from gamma-butyrolactone. The central core of rhizoxin D was prepared via a chiral resolution/asymmetric aldol protocol. Several methods for the generation of the polyene fragment were explored, and the side-chain was ultimately prepared from serine in six steps. The unification of the left and right wings of the molecule was achieved using a one-step olefination protocol, and the macrocyclization was carried out using a Horner-Emmons olefination at the C(2)-C(3) olefin.