A chemical probe to modulate human GID4 Pro/N-degron interactions.

The C-terminal to LisH (CTLH) complex is a ubiquitin ligase complex that recognizes substrates with Pro/N-degrons via its substrate receptor Glucose-Induced Degradation 4 (GID4), but its function and substrates in humans remain unclear. Here, we report PFI-7, a potent, selective and cell-active chemical probe that antagonizes Pro/N-degron binding to human GID4. Use of PFI-7 in proximity-dependent biotinylation and quantitative proteomics enabled the identification of GID4 interactors and GID4-regulated proteins. GID4 interactors are enriched for nucleolar proteins, including the Pro/N-degron-containing RNA helicases DDX21 and DDX50. We also identified a distinct subset of proteins whose cellular levels are regulated by GID4 including HMGCS1, a Pro/N-degron-containing metabolic enzyme. These data reveal human GID4 Pro/N-degron targets regulated through a combination of degradative and nondegradative functions. Going forward, PFI-7 will be a valuable research tool for investigating CTLH complex biology and facilitating development of targeted protein degradation strategies that highjack CTLH E3 ligase activity.

Chemical tools for the Gid4 subunit of the human E3 ligase C-terminal to LisH (CTLH) degradation complex.

We have developed a novel chemical handle (PFI-E3H1) and a chemical probe (PFI-7) as ligands for the Gid4 subunit of the human E3 ligase CTLH degradation complex. Through an efficient initial hit-ID campaign, structure-based drug design (SBDD) and leveraging the sizeable Pfizer compound library, we identified a 500 nM ligand for this E3 ligase through file screening alone. Further exploration identified a vector that is tolerant to addition of a linker for future chimeric molecule design. The chemotype was subsequently optimized to sub-100 nM Gid4 binding affinity for a chemical probe. These novel tools, alongside the suitable negative control also identified, should enable the interrogation of this complex human E3 ligase macromolecular assembly.

Intramolecular Ring-Opening Decomposition of Aryl Azetidines.

The ring strain present in azetidines can lead to undesired stability issues. Herein, we described a series of N-substituted azetidines which undergo an acid-mediated intramolecular ring-opening decomposition via nucleophilic attack of a pendant amide group. Studies were conducted to understand the decomposition mechanism enabling the design of stable analogues.

PF-07059013: A Noncovalent Modulator of Hemoglobin for Treatment of Sickle Cell Disease.

Sickle cell disease (SCD) is a genetic disorder caused by a single point mutation (ß6 Glu → Val) on the ß-chain of adult hemoglobin (HbA) that results in sickled hemoglobin (HbS). In the deoxygenated state, polymerization of HbS leads to sickling of red blood cells (RBC). Several downstream consequences of polymerization and RBC sickling include vaso-occlusion, hemolytic anemia, and stroke. We report the design of a noncovalent modulator of HbS, clinical candidate PF-07059013 (23). The seminal hit molecule was discovered by virtual screening and confirmed through a series of biochemical and biophysical studies. After a significant optimization effort, we arrived at 23, a compound that specifically binds to Hb with nanomolar affinity and displays strong partitioning into RBCs. In a 2-week multiple dose study using Townes SCD mice, 23 showed a 37.8% (±9.0%) reduction in sickling compared to vehicle treated mice. 23 (PF-07059013) has advanced to phase 1 clinical trials.

Aminopyrazole Carboxamide Bruton's Tyrosine Kinase Inhibitors. Irreversible to Reversible Covalent Reactive Group Tuning.

Potent covalent inhibitors of Bruton's tyrosine kinase (BTK) based on an aminopyrazole carboxamide scaffold have been identified. Compared to acrylamide-based covalent reactive groups leading to irreversible protein adducts, cyanamide-based reversible-covalent inhibitors provided the highest combined BTK potency and EGFR selectivity. The cyanamide covalent mechanism with BTK was confirmed through enzyme kinetic, NMR, MS, and X-ray crystallographic studies. The lead cyanamide-based inhibitors demonstrated excellent kinome selectivity and rat pharmacokinetic properties.

A Small-Molecule Anti-secretagogue of PCSK9 Targets the 80S Ribosome to Inhibit PCSK9 Protein Translation.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that downregulates low-density lipoprotein (LDL) receptor (LDL-R) levels on the surface of hepatocytes, resulting in decreased clearance of LDL-cholesterol (LDL-C). Phenotypic screening of a small-molecule compound collection was used to identify an inhibitor of PCSK9 secretion, (R)-N-(isoquinolin-1-yl)-3-(4-methoxyphenyl)-N-(piperidin-3-yl)propanamide (R-IMPP), which was shown to stimulate uptake of LDL-C in hepatoma cells by increasing LDL-R levels, without altering levels of secreted transferrin. Systematic investigation of the mode of action revealed that R-IMPP did not decrease PCSK9 transcription or increase PCSK9 degradation, but instead caused transcript-dependent inhibition of PCSK9 translation. In support of this surprising mechanism of action, we found that R-IMPP was able to selectively bind to human, but not E. coli, ribosomes. This study opens a new avenue for the development of drugs that modulate the activity of target proteins by mechanisms involving inhibition of eukaryotic translation.

Solution-based indirect affinity selection mass spectrometry--a general tool for high-throughput screening of pharmaceutical compound libraries.

We show here that an automated solution-based affinity selection mass spectrometry (ASMS) system can be built exclusively from commercially available parts. The value of this technology lies in the throughput (~1 × 10(5) compounds/day) coupled with a low hit rate. The system, being a binding assay, requires little development time yielding a fast timeline between target availability and hit identification. In addition, the use of exact mass simplifies the hit identification. We demonstrate this system using carbonic anhydrase as the target and a library of 144,000 proprietary compounds.

Integrating intramolecular hydrogen bonding (IMHB) considerations in drug discovery using ΔlogP as a tool.

This study demonstrates that ΔlogP(oct-tol) (difference between logP(octanol) and logP(toluene)) describes compounds propensity to form intramolecular hydrogen bonds (IMHB) and may be considered a privileged molecular descriptor for use in drug discovery and for prediction of IMHB in drug candidates. We identified experimental protocols for acquiring reliable ΔlogP(oct-tol) values on a set of compounds representing IMHB motifs most prevalent in medicinal chemistry, mainly molecules capable of forming 6-, 7-member IMHB rings. Furthermore, computational ΔlogP(oct-tol) values obtained with COSMO-RS software provided a good estimate of experimental results and can be used prospectively to assess IMHB. The proposed interpretation method based on ΔlogP(oct-tol) data allowed categorization of the compounds into 2 groups: with high propensity to form IMHB and poor propensity or poor relevance of IMHB. The relative (1)H NMR chemical shift of an exchangeable proton was used to verify presence of IMHB and to validate the IMHB interpretation scheme.

Discovery and optimization of a novel spiropyrrolidine inhibitor of ß-secretase (BACE1) through fragment-based drug design.

The aspartyl protease ß-secretase, or BACE, has been demonstrated to be a key factor in the proteolytic formation of Aß-peptide, a major component of plaques in the brains of Alzheimer's disease (AD) patients, and inhibition of this enzyme has emerged as a major strategy for pharmacologic intervention in AD. An X-ray-based fragment screen of Pfizer's proprietary fragment collection has resulted in the identification of a novel BACE binder featuring spiropyrrolidine framework. Although exhibiting only weak inhibitory activity against the BACE enzyme, the small compound was verified by biophysical and NMR-based methods as a bona fide BACE inhibitor. Subsequent optimization of the lead compound, relying heavily on structure-based drug design and computational prediction of physiochemical properties, resulted in a nearly 1000-fold improvement in potency while maintaining ligand efficiency and properties predictive of good permeability and low P-gp liability.

Metabolism, pharmacokinetics, and excretion of the 5-hydroxytryptamine1b receptor antagonist elzasonan in humans.

The metabolism, pharmacokinetics, and excretion of a potent and selective 5-hydroxytryptamine(1B) receptor antagonist elzasonan have been studied in six healthy male human subjects after oral administration of a single 10-mg dose of [(14)C]elzasonan. Total recovery of the administered dose was 79% with approximately 58 and 21% of the administered radioactive dose excreted in feces and urine, respectively. The average t(1/2) for elzasonan was 31.5 h. Elzasonan was extensively metabolized, and excreta and plasma were analyzed using mass spectrometry and NMR spectroscopy to elucidate the structures of metabolites. The major component of drug-related material in the excreta was in the feces and was identified as 5-hydroxyelzasonan (M3), which accounted for approximately 34% of the administered dose. The major human circulating metabolite was identified as the novel cyclized indole metabolite (M6) and accounted for ∼65% of the total radioactivity. A mechanism for the formation of M6 is proposed. Furthermore, metabolism-dependent covalent binding of drug-related material was observed upon incubation of [(14)C]elzasonan with liver microsomes, and data suggest that an indole iminium ion is involved. Overall, the major metabolic pathways of elzasonan were due to aromatic hydroxylation(s) of the benzylidene moiety, N-oxidation at the piperazine ring, N-demethylation, indirect glucuronidation, and oxidation, ring closure, and subsequent rearrangement to form M6.

Species differences in the biotransformation of an alpha 4 beta 2 nicotinic acetylcholine receptor partial agonist: the effects of distinct glucuronide metabolites on overall compound disposition.

The metabolism and disposition of (1R,5S)-2,3,4,5-tetrahydro-7-(trifluoromethyl)-1,5-methano-1H-3-benzazepine (1), an alpha(4)beta(2) nicotinic acetylcholine receptor partial agonist, was investigated in Sprague-Dawley rats and cynomolgus monkeys receiving (1R,5S)-2,3,4,5-tetrahydro-7-(trifluoromethyl)-1,5-methano-1H-4[(14)C]-3- benzazepine hydrochloride ([(14)C]1) orally. Although both species chiefly (>or=62%) cleared 1 metabolically, species-specific dispositional profiles were observed for both 1 and total radioactivity. Radioactivity was excreted equally in the urine and feces of intact rats but largely (72%) in bile in bile duct-cannulated animals. In monkeys, radioactivity recoveries were 50-fold greater in urine than feces and minimal (<5%) in bile. Both species metabolized 1 similarly: four-electron oxidation to one of four amino acids or two lactams (minor) and glucuronide formation (major). In rats, the latter pathway predominantly formed an N-carbamoyl glucuronide (M6), exclusively present in bile (69% of dose), whereas in monkeys it afforded an N-O-glucuronide (M5), a minor biliary component (4%) but the major plasma (62%) and urinary (42%) entity. In rats, first-pass hepatic conversion of 1 to M6, which was confirmed in rat hepatocytes, and its biliary secretion resulted in the indirect enterohepatic cycling of 1 via M6 and manifested in double-humped plasma concentration-time curves and long t(1/2) for both 1 and total radioactivity. In monkeys, in which only M5 was formed, double-humped plasma concentration-time curves were absent, and moderate t(1/2) for both 1 and total radioactivity were observed. A seemingly subtle, yet critical, difference in the chemical structures of these two glucuronide metabolites considerably affected the overall disposition of 1 in rats versus monkeys.

Biotransformation of an alpha4beta2 nicotinic acetylcholine receptor partial agonist in sprague-dawley rats and the dispositional characterization of its N-carbamoyl glucuronide metabolite.

The metabolism and disposition of (1S,5R)-2,3,4,5-tetrahydro-7-(trifluoromethyl)-1,5-methano-1H-3-benzazepine (1), an alpha(4)beta(2) nicotinic acetylcholine receptor partial agonist, was determined in Sprague-Dawley rats after oral administration of [(14)C]1. In intact animals, mass balance was achieved within 48 h, with 5 times more radioactivity excreted in urine than in feces. Compound 1 underwent renal and metabolic clearance equally and exhibited a very long half-life attributable to a secondary peak occurring 8 h postdose in its serum concentration-time curve. In bile duct-cannulated (BDC) rats, mass balance was also achieved within 48 h with 73.7, 23.4, and 5.5% of the dose detected in bile, urine, and feces, respectively. Rats metabolized 1 by two primary routes: four-electron oxidation to either four amino acids or a lactam and formation of an N-carbamoyl glucuronide (M6), which was only detected in bile. The presence of M6 solely in bile and the double-humped serum concentration-time curve of 1 suggested the indirect enterohepatic cycling of 1 via M6 after oral administration. To explore this mechanistic hypothesis further, intravenous studies were conducted with 1 in both intact and BDC rats to determine the extent of 1 undergoing indirect enterohepatic cycling via M6. Compared with the pharmacokinetics in intact rats, total serum clearance was higher (1.7-fold) and volume of distribution was lower (1.6-fold) in BDC rats, resulting in a correspondingly shorter (2.5-fold) half-life, with 56% of administered 1 undergoing recirculation, an amount consistent with that (68% of dose) of M6 observed in bile from rats dosed orally with [(14)C]1.

Comparison of LC-NMR and conventional NMR for structure elucidation in drug metabolism studies.

Liquid chromatography-nuclear magnetic resonance (LC-NMR) has proven to be a useful technique for the structure elucidation of novel metabolites from pharmaceutical compounds. Proponents of LC-NMR tout the advantage of eliminating the step of a separate chromatographic isolation. However, the advantages of directly coupling NMR and HPLC instrumentation must be weighed against compromises in performance made to each technique to achieve a hyphenated system. While significant advances have been made in LC-NMR technology, a strong case can be made that HPLC purification of metabolites followed by conventional tube NMR is equally useful. It is relatively rare that one approach will be successful and the other not. The fundamental consideration is whether there is sufficient chromatographic expertise in the NMR laboratory to adequately design and execute appropriate experiments such that a pure chromatographic peak will be produced in the hyphenated system. Due to speed and sensitivity differences between NMR spectroscopy and mass spectrometry, liquid chromatography/mass spectrometry (LC/MS) continues to be the front-line approach for the structure elucidation of metabolites.

Metabolism and disposition of varenicline, a selective alpha4beta2 acetylcholine receptor partial agonist, in vivo and in vitro.

The metabolism and disposition of varenicline (7,8,9,10-tetrahydro-6,10-methano-6H-pyrazino[2,3-h][3]benzazepine), a partial agonist of the nicotinic acetylcholine receptor for the treatment of tobacco addiction, was examined in rats, mice, monkeys, and humans after oral administration of [14C]varenicline. In the circulation of all species, the majority of drug-related material was composed of unchanged varenicline. In all four species, drug-related material was primarily excreted in the urine. A large percentage was excreted as unchanged parent drug (90, 84, 75, and 81% of the dose in mouse, rat, monkey, and human, respectively). Metabolites observed in excreta arose via N-carbamoyl glucuronidation and oxidation. These metabolites were also observed in the circulation, in addition to metabolites that arose via N-formylation and formation of a novel hexose conjugate. Experiments were conducted using in vitro systems to gain an understanding of the enzymes involved in the formation of the N-carbamoylglucuronide metabolite in humans. N-Carbamoyl glucuronidation was catalyzed by UGT2B7 in human liver microsomes when incubations were conducted under a CO2 atmosphere. The straightforward dispositional profile of varenicline should simplify its use in the clinic as an aid in smoking cessation.

Biotransformation of a GABAA receptor partial agonist in sprague-dawley rats and cynomolgus monkeys: identification of two unique N-carbamoyl metabolites.

The absorption, metabolism, and excretion of N-[3-fluoro-4-[2-(propylamino)ethoxy]phenyl]-4,5,6,7-tetrahydro-4-oxo-1H-indole-3-carboxamide monomethanesulfonate (1), a GABAA receptor partial agonist potentially useful in treating generalized anxiety disorder, have been evaluated in both Sprague-Dawley rats and cynomolgus monkeys using [14C]1. In both species, mass balance was achieved within 48 h postdose, with the majority of drug-related material excreted within the feces; the clearance of 1 in each species had both metabolic and renal components. In addition to the metabolites produced by aliphatic hydroxylation and/or N-dealkylation of 1, two unique metabolites were detected: a putative carbamic acid (M7) in rat plasma and monkey bile, and an N-carbamoyl glucuronide (M8) in both rat and monkey bile. Metabolite M8 was structurally deciphered by liquid chromatographytandem mass spectrometry and NMR, and was readily generated in vitro upon incubation of [14C]1 with rat liver microsomes fortified with uridine 5'-diphosphoglucuronic acid trisodium salt and alamethicin under a CO2 atmosphere. Treatment of M8 with beta-glucuronidase afforded 1 directly. The presence of M8 in bile and its notable absence from other matrices suggests the enterohepatic cycling of 1 via M8. Although the structure of M7 was not elucidated unequivocally due to its inability to be formed in vitro and its minimal absolute quantities in limited biological matrices, data herein clearly support its structural rationalization. Furthermore, since M7 is the precursor of M8, detection of M8 is indirect evidence of its existence. It is proposed that M7 arises from an equilibrium between 1 and dissolved CO2-equivalents both in vivo and in vitro, similar to carbamino bonds observed in hemoglobin and certain amino acids, respectively.

Identification of a novel glutathione adduct of diclofenac, 4'-hydroxy-2'-glutathion-deschloro-diclofenac, upon incubation with human liver microsomes.

Clinical use of the nonsteroidal anti-inflammatory drug diclofenac (DF) is associated with an incidence of idiosyncratic hepatoxicity. The formation of reactive metabolites of DF in vivo has been proposed to be responsible for such toxicity. One type of reactive metabolite, a benzoquinone imine of DF formed through oxidation by cytochromes P450, can be trapped by glutathione in vitro in liver microsomes to form glutathione (GS) adducts. Three GS adducts from DF were reported in the literature, namely, 5-hydroxy (OH)-4-glutathione-DF, 4'-OH-3'-glutathione-DF and 5-OH-6-glutathione-DF, and they all have the same molecular weight of 616. Recently, we developed a sensitive and high throughput method for the detection of GS adducts from liver microsome incubation. This method uses a constant neutral loss scan of m/z 129, a "structure-characteristic" fragment for GS adduct, on an automated chip-based nanoelectrospray (Advion NanoMate 100) attached to a tandem mass spectrometer (Sciex API 3000). The analysis of GS adducts from human liver microsome incubation with DF by the NanoMate 100-API 3000 method unambiguously revealed a new adduct ion with m/z 583 (MH+), in addition to the known adduct peak with m/z 617 (MH+). This new adduct was further confirmed to be 4'-OH-2'-glutathion-deschloro-diclofenac by liquid chromatography (LC) tandem mass spectrometry (MS), LC/MS-NMR, and comparison to a synthetic standard.

Characterization of novel dihydrothienopyridinium and thienopyridinium metabolites of ticlopidine in vitro: role of peroxidases, cytochromes p450, and monoamine oxidases.

Ticlopidine is an agent that inhibits adenosine diphosphate-induced platelet aggregation. Metabolic studies with ticlopidine have indicated that the principal routes of metabolism are N-dealkylation, N-oxidation, and oxidation of the thiophene ring. However, ticlopidine shares some structural features that are similar to those of cyclic tertiary amines such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and tetrahydroisoquinolines, which are converted to neurotoxic pyridinium metabolites, via the iminium (dihydropyridinium) species. The current in vitro studies examined the potential of ticlopidine to undergo a similar conversion by cytochrome P450 (P450), peroxidases, and monoamine oxidase (MAO). The results from these studies have suggested that ticlopidine undergoes an overall 4-electron oxidation to the novel thienopyridinium metabolite (M6) via the intermediate 2-electron oxidation product, the thienodihydropyridinium metabolite (M5) by P450, horseradish peroxidase, and myeloperoxidase and, to a lesser extent, by MAO. The structures of these metabolites were characterized by liquid chromatography (LC)-tandem mass spectrometry and LC-NMR. Qualitative studies with baculovirus-expressed P450s revealed the involvement of P450 3A4 in this conversion. Interestingly, M5 was the primary metabolite in the peroxidase-mediated reactions and was quite stable to air oxidation or disproportionation. It was less electrophilic and did not form cyanide, glutathione, or N-acetylcysteine adducts. On the other hand, M6 was the major metabolite in P450-catalyzed oxidation of ticlopidine. The results from this study have revealed that in addition to metabolism of the thiophene ring of ticlopidine, the tetrahydropyridine moiety of the compound is susceptible to a 2-electron and a 4-electron oxidation like other cyclic tertiary amines.