Tryptanthrin Analogs Substoichiometrically Inhibit Seeded and Unseeded Tau4RD Aggregation.

Microtubule-associated protein tau is an intrinsically disordered protein (IDP) that forms characteristic fibrillar aggregates in several diseases, the most well-known of which is Alzheimer's disease (AD). Despite keen interest in disrupting or inhibiting tau aggregation to treat AD and related dementias, there are currently no FDA-approved tau-targeting drugs. This is due, in part, to the fact that tau and other IDPs do not exhibit a single well-defined conformation but instead populate a fluctuating conformational ensemble that precludes finding a stable "druggable" pocket. Despite this challenge, we previously reported the discovery of two novel families of tau ligands, including a class of aggregation inhibitors, identified through a protocol that combines molecular dynamics, structural analysis, and machine learning. Here we extend our exploration of tau druggability with the identification of tryptanthrin and its analogs as potent, substoichiometric aggregation inhibitors, with the best compounds showing potencies in the low nanomolar range even at a ~100-fold molar excess of tau4RD. Moreover, conservative changes in small molecule structure can have large impacts on inhibitory potency, demonstrating that similar structure-activity relationship (SAR) principles as used for traditional drug development also apply to tau and potentially to other IDPs.

Use of Cryopreserved Hepatocytes as Part of an Integrated Strategy to Characterize In Vivo Clearance for Peptide-Antibody Conjugate Inhibitors of Nav1.7 in Preclinical Species.

The identification of nonopioid alternatives to treat chronic pain has received a great deal of interest in recent years. Recently, the engineering of a series of Nav1.7 inhibitory peptide-antibody conjugates has been reported, and herein, the preclinical efforts to identify novel approaches to characterize the pharmacokinetic properties of the peptide conjugates are described. A cryopreserved plated mouse hepatocyte assay was designed to measure the depletion of the peptide-antibody conjugates from the media, with a correlation being observed between percentage remaining in the media and in vivo clearance (Pearson r = -0.5525). Physicochemical (charge and hydrophobicity), receptor-binding [neonatal Fc receptor (FcRn)], and in vivo pharmacokinetic data were generated and compared with the results from our in vitro hepatocyte assay, which was hypothesized to encompass all of the aforementioned properties. Correlations were observed among hydrophobicity; FcRn binding; depletion rates from the hepatocyte assay; and ultimately, in vivo clearance. Subsequent studies identified potential roles for the low-density lipoprotein and mannose/galactose receptors in the association of the Nav1.7 peptide conjugates with mouse hepatocytes, although in vivo studies suggested that FcRn was still the primary receptor involved in determining the pharmacokinetics of the peptide conjugates. Ultimately, the use of the cryopreserved hepatocyte assay along with FcRn binding and hydrophobic interaction chromatography provided an efficient and integrated approach to rapidly triage molecules for advancement while reducing the number of in vivo pharmacokinetic studies. SIGNIFICANCE STATEMENT: Although multiple in vitro and in silico tools are available in small-molecule drug discovery, pharmacokinetic characterization of protein therapeutics is still highly dependent upon the use of in vivo studies in preclinical species. The current work demonstrates the combined use of cryopreserved hepatocytes, hydrophobic interaction chromatography, and neonatal Fc receptor binding to characterize a series of Nav1.7 peptide-antibody conjugates prior to conducting in vivo studies, thus providing a means to rapidly evaluate novel protein therapeutic platforms while concomitantly reducing the number of in vivo studies conducted in preclinical species.

Engineering NaV1.7 Inhibitory JzTx-V Peptides with a Potency and Basicity Profile Suitable for Antibody Conjugation To Enhance Pharmacokinetics.

Drug discovery research on new pain targets with human genetic validation, including the voltage-gated sodium channel NaV1.7, is being pursued to address the unmet medical need with respect to chronic pain and the rising opioid epidemic. As part of early research efforts on this front, we have previously developed NaV1.7 inhibitory peptide-antibody conjugates with tarantula venom-derived GpTx-1 toxin peptides with an extended half-life (80 h) in rodents but only moderate in vitro activity (hNaV1.7 IC50 = 250 nM) and without in vivo activity. We identified the more potent peptide JzTx-V from our natural peptide collection and improved its selectivity against other sodium channel isoforms through positional analogueing. Here we report utilization of the JzTx-V scaffold in a peptide-antibody conjugate and architectural variations in the linker, peptide loading, and antibody attachment site. We found conjugates with 100-fold improved in vitro potency relative to those of complementary GpTx-1 analogues, but pharmacokinetic and bioimaging analyses of these JzTx-V conjugates revealed a shorter than expected plasma half-life in vivo with accumulation in the liver. In an attempt to increase circulatory serum levels, we sought the reduction of the net +6 charge of the JzTx-V scaffold while retaining a desirable NaV in vitro activity profile. The conjugate of a JzTx-V peptide analogue with a +2 formal charge maintained NaV1.7 potency with 18-fold improved plasma exposure in rodents. Balancing the loss of peptide and conjugate potency associated with the reduction of net charge necessary for improved target exposure resulted in a compound with moderate activity in a NaV1.7-dependent pharmacodynamic model but requires further optimization to identify a conjugate that can fully engage NaV1.7 in vivo.

Structure-guided Discovery of Dual-recognition Chemibodies.

Small molecules and antibodies each have advantages and limitations as therapeutics. Here, we present for the first time to our knowledge, the structure-guided design of "chemibodies" as small molecule-antibody hybrids that offer dual recognition of a single target by both a small molecule and an antibody, using DPP-IV enzyme as a proof of concept study. Biochemical characterization demonstrates that the chemibodies present superior DPP-IV inhibition compared to either small molecule or antibody component alone. We validated our design by successfully solving a co-crystal structure of a chemibody in complex with DPP-IV, confirming specific binding of the small molecule portion at the interior catalytic site and the Fab portion at the protein surface. The discovery of chemibodies presents considerable potential for novel therapeutics that harness the power of both small molecule and antibody modalities to achieve superior specificity, potency, and pharmacokinetic properties.

Engineering Antibody Reactivity for Efficient Derivatization to Generate NaV1.7 Inhibitory GpTx-1 Peptide-Antibody Conjugates.

The voltage-gated sodium channel NaV1.7 is a genetically validated pain target under investigation for the development of analgesics. A therapeutic with a less frequent dosing regimen would be of value for treating chronic pain; however functional NaV1.7 targeting antibodies are not known. In this report, we describe NaV1.7 inhibitory peptide-antibody conjugates as an alternate construct for potential prolonged channel blockade through chemical derivatization of engineered antibodies. We previously identified NaV1.7 inhibitory peptide GpTx-1 from tarantula venom and optimized its potency and selectivity. Tethering GpTx-1 peptides to antibodies bifunctionally couples FcRn-based antibody recycling attributes to the NaV1.7 targeting function of the peptide warhead. Herein, we conjugated a GpTx-1 peptide to specific engineered cysteines in a carrier anti-2,4-dinitrophenol monoclonal antibody using polyethylene glycol linkers. The reactivity of 13 potential cysteine conjugation sites in the antibody scaffold was tuned using a model alkylating agent. Subsequent reactions with the peptide identified cysteine locations with the highest conversion to desired conjugates, which blocked NaV1.7 currents in whole cell electrophysiology. Variations in attachment site, linker, and peptide loading established design parameters for potency optimization. Antibody conjugation led to in vivo half-life extension by 130-fold relative to a nonconjugated GpTx-1 peptide and differential biodistribution to nerve fibers in wild-type but not NaV1.7 knockout mice. This study describes the optimization and application of antibody derivatization technology to functionally inhibit NaV1.7 in engineered and neuronal cells.

The discovery and optimization of aminooxadiazoles as potent Pim kinase inhibitors.

High levels of Pim expression have been implicated in several hematopoietic and solid tumor cancers. These findings suggest that inhibition of Pim signaling by a small molecule Pim-1,2 inhibitor could provide patients with therapeutic benefit. Herein, we describe our progress towards this goal starting from the highly Pim-selective indole-thiadiazole compound (1), which was derived from a nonselective hit identified in a high throughput screening campaign. Optimization of this compound's potency and its pharmacokinetic properties resulted in the discovery of compound 29. Cyclopropane 29 was found to exhibit excellent enzymatic potency on the Pim-1 and Pim-2 isoforms (Ki values of 0.55nM and 0.28nM, respectively), and found to inhibit the phosphorylation of BAD in the Pim-overexpressing KMS-12 cell line (IC50=150nM). This compound had moderate clearance and bioavailability in rat (CL=2.42L/kg/h; %F=24) and exhibited a dose-dependent inhibition of p-BAD in KMS-12 tumor pharmacodynamic (PD) model with an EC50 value of 6.74µM (18µg/mL) when dosed at 10, 30, 100 and 200mg/kg po in mice.

Discovery of 5-(1H-indol-5-yl)-1,3,4-thiadiazol-2-amines as potent PIM inhibitors.

PIM kinases are a family of Ser/Thr kinases that are implicated in tumorigenesis. The discovery of a new class of PIM inhibitors, 5-(1H-indol-5-yl)-1,3,4-thiadiazol-2-amines, is discussed with optimized compounds showing excellent potency against all three PIM isoforms.

Discovery of 2-methylpyridine-based biaryl amides as γ-secretase modulators for the treatment of Alzheimer's disease.

γ-Secretase modulators (GSMs) are potentially disease-modifying treatments for Alzheimer's disease. They selectively lower pathogenic Aß42 levels by shifting the enzyme cleavage sites without inhibiting γ-secretase activity, possibly avoiding known adverse effects observed with complete inhibition of the enzyme complex. A cell-based HTS effort identified the sulfonamide 1 as a GSM lead. Lead optimization studies identified compound 25 with improved cell potency, PKDM properties, and it lowered Aß42 levels in the cerebrospinal fluid (CSF) of Sprague-Dawley rats following oral administration. Further optimization of 25 to improve cellular potency is described.

Rapid identification of a novel small molecule phosphodiesterase 10A (PDE10A) tracer.

A radiolabeled tracer for imaging therapeutic targets in the brain is a valuable tool for lead optimization in CNS drug discovery and for dose selection in clinical development. We report the rapid identification of a novel phosphodiesterase 10A (PDE10A) tracer candidate using a LC-MS/MS technology. This structurally distinct PDE10A tracer, AMG-7980 (5), has been shown to have good uptake in the striatum (1.2% ID/g tissue), high specificity (striatum/thalamus ratio of 10), and saturable binding in vivo. The PDE10A affinity (K(D)) and PDE10A target density (B(max)) were determined to be 0.94 nM and 2.3 pmol/mg protein, respectively, using [(3)H]5 on rat striatum homogenate. Autoradiography on rat brain sections indicated that the tracer signal was consistent with known PDE10A expression pattern. The specific binding of [(3)H]5 to rat brain was blocked by another structurally distinct, published PDE10A inhibitor, MP-10. Lastly, our tracer was used to measure in vivo PDE10A target occupancy of a PDE10A inhibitor in rats using LC-MS/MS technology.

Fragment based drug discovery: practical implementation based on ¹9F NMR spectroscopy.

Fragment based drug discovery (FBDD) is a widely used tool for discovering novel therapeutics. NMR is a powerful means for implementing FBDD, and several approaches have been proposed utilizing (1)H-(15)N heteronuclear single quantum coherence (HSQC) as well as one-dimensional (1)H and (19)F NMR to screen compound mixtures against a target of interest. While proton-based NMR methods of fragment screening (FBS) have been well documented and are widely used, the use of (19)F detection in FBS has been only recently introduced (Vulpetti et al. J. Am. Chem. Soc.2009, 131 (36), 12949-12959) with the aim of targeting "fluorophilic" sites in proteins. Here, we demonstrate a more general use of (19)F NMR-based fragment screening in several areas: as a key tool for rapid and sensitive detection of fragment hits, as a method for the rapid development of structure-activity relationship (SAR) on the hit-to-lead path using in-house libraries and/or commercially available compounds, and as a quick and efficient means of assessing target druggability.

From fragment screening to in vivo efficacy: optimization of a series of 2-aminoquinolines as potent inhibitors of beta-site amyloid precursor protein cleaving enzyme 1 (BACE1).

Using fragment-based screening of a focused fragment library, 2-aminoquinoline 1 was identified as an initial hit for BACE1. Further SAR development was supported by X-ray structures of BACE1 cocrystallized with various ligands and molecular modeling studies to expedite the discovery of potent compounds. These strategies enabled us to integrate the C-3 side chain on 2-aminoquinoline 1 extending deep into the P2' binding pocket of BACE1 and enhancing the ligand's potency. We were able to improve the BACE1 potency to subnanomolar range, over 10(6)-fold more potent than the initial hit (900 µM). Further elaboration of the physical properties of the lead compounds to those more consistent with good blood-brain barrier permeability led to inhibitors with greatly improved cellular activity and permeability. Compound 59 showed an IC(50) value of 11 nM on BACE1 and cellular activity of 80 nM. This compound was advanced into rat pharmacokinetic and pharmacodynamic studies and demonstrated significant reduction of Aß levels in cerebrospinal fluid (CSF).

Selective inhibitors of the mutant B-Raf pathway: discovery of a potent and orally bioavailable aminoisoquinoline.

The discovery and optimization of a novel series of aminoisoquinolines as potent, selective, and efficacious inhibitors of the mutant B-Raf pathway is presented. The N-linked pyridylpyrimidine benzamide 2 was identified as a potent, modestly selective inhibitor of the B-Raf enzyme. Replacement of the benzamide with an aminoisoquinoline core significantly improved kinase selectivity and imparted favorable pharmacokinetic properties, leading to the identification of 1 as a potent antitumor agent in xenograft models.

Novel vanilloid receptor-1 antagonists: 1. Conformationally restricted analogues of trans-cinnamides.

The vanilloid receptor-1 (VR1 or TRPV1) is a member of the transient receptor potential (TRP) family of ion channels and plays a role as an integrator of multiple pain-producing stimuli. From a high-throughput screening assay, measuring calcium uptake in TRPV1-expressing cells, we identified an N-aryl trans-cinnamide (AMG9810, compound 9) that acts as a potent TRPV1 antagonist. We have demonstrated the antihyperalgesic properties of 9 in vivo and have also reported the discovery of novel, orally bioavailable cinnamides derived from this lead. Herein, we expand our investigations and describe the synthesis and biological evaluation of a series of conformationally constrained analogues of the s-cis conformer of compound 9. These investigations resulted in the identification of 4-amino- and 4-oxopyrimidine cores as suitable isosteric replacements for the trans-acrylamide moiety. The best examples from this series, pyrimidines 79 and 74, were orally bioavailable and exhibited potent antagonism of both rat (IC50 = 4.5 and 0.6 nM, respectively) and human TRPV1 (IC50 = 7.4 and 3.7 nM, respectively). In addition, compound 74 was shown to be efficacious at blocking a TRPV1-mediated physiological response in vivo in the capsaicin-induced hypothermia model in rats; however, it was ineffective at preventing thermal hyperalgesia induced by complete Freund's adjuvant in rats.

Novel vanilloid receptor-1 antagonists: 2. Structure-activity relationships of 4-oxopyrimidines leading to the selection of a clinical candidate.

A series of novel 4-oxopyrimidine TRPV1 antagonists was evaluated in assays measuring the blockade of capsaicin or acid-induced influx of calcium into CHO cells expressing TRPV1. The investigation of the structure-activity relationships in the heterocyclic A-region revealed the optimum pharmacophoric elements required for activity in this series and resulted in the identification of subnanomolar TRPV1 antagonists. The most potent of these antagonists were thoroughly profiled in pharmacokinetic assays. Optimization of the heterocyclic A-region led to the design and synthesis of 23, a compound that potently blocked multiple modes of TRPV1 activation. Compound 23 was shown to be effective in a rodent "on-target" biochemical challenge model (capsaicin-induced flinch, ED50 = 0.33 mg/kg p.o.) and was antihyperalgesic in a model of inflammatory pain (CFA-induced thermal hyperalgesia, MED = 0.83 mg/kg, p.o.). Based on its in vivo efficacy and pharmacokinetic profile, compound 23 (N-{4-[6-(4-trifluoromethyl-phenyl)-pyrimidin-4-yloxy]-benzothiazol-2-yl}-acetamide; AMG 517) was selected for further evaluation in human clinical trials.

One-pot microwave assisted preparation of pyrazoloquinazolinone libraries.

The novel solution-phase synthesis of an array of biologically relevant pyrazoloquinazolinones in a simple microwave driven one pot procedure is revelaed. Transformations are carried out in good to excellent yield by condensation of alpha-cyano-ketones and 2-hydrazino-benzoic acids. Subsequent microwave irradiation affords pyrazoloquinazolinones with six points of potential diversification. The protocol described represents a very attractive solution phase procedure for the rapid generation of arrays of such functionalized cores, further demonstrating the growing importance of economic and enabling complexity generating chemistries in the lead discovery arena.

Rapid assembly of molecular diversity via exploitation of isocyanide-based multi-component reactions.

Molecular diversity is the variability of physical properties between molecules, viewed in terms of molecular shape, polarity/charge, lipophilicity, polarizability and flexibility. Due to their widespread medicinal properties, natural products were one of the original sources of molecular diversity; however, new developments in the search for novel pharmacological agents over the last decade have focused on the preparation of chemical libraries as the source of new leads for drug discovery. A plethora of personal synthesizers and new automation technologies have emerged to help fuel the lead discovery engines of drug discovery organizations. Multistep solid-phase syntheses of diverse libraries in excess of 10,000 products can now be prepared via split-and-mix techniques. Simultaneously, a multitude of more efficient, diversity- or target-oriented solution-phase chemical methodologies have appeared in the chemical literature, enabling the relatively facile construction of successful lead generation libraries with low full-time equivalent input and little capital expenditure. Isocyanide-related multi-component reactions hold a pre-eminent position in this regard, and are finding increasing applications in the discovery process of new drugs and agrochemicals. This review is the authors' personal assessment of advances in the field over the last two years (2002 to 2003), with little emphasis placed on highly mechanistic details.