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.

Optimizing the Benefit/Risk of Acetyl-CoA Carboxylase Inhibitors through Liver Targeting.

Preclinical and clinical data suggest that acetyl-CoA carboxylase (ACC) inhibitors have the potential to rebalance disordered lipid metabolism, leading to improvements in nonalcoholic steatohepatitis (NASH). Consistent with these observations, first-in-human clinical trials with our ACC inhibitor PF-05175157 led to robust reduction of de novo lipogenesis (DNL), albeit with concomitant reductions in platelet count, which were attributed to the inhibition of fatty acid synthesis within bone marrow. Herein, we describe the design, synthesis, and evaluation of carboxylic acid-based ACC inhibitors with organic anion transporting polypeptide (OATP) substrate properties, which facilitated selective distribution of the compounds at the therapeutic site of action (liver) relative to the periphery. These efforts led to the discovery of clinical candidate PF-05221304 (12), which selectively inhibits liver DNL in animals, while demonstrating considerable safety margins against platelet reduction in a nonhuman primate model.

Permeability of Cyclic Peptide Macrocycles and Cyclotides and Their Potential as Therapeutics.

Macrocycles have emerged as a viable approach for the modulation of tough targets in drug discovery. In this Innovations article we discuss recent progress toward the design of cell permeable and orally bioavailable peptide macrocycles and cyclotides and provide a perspective for their potential as therapeutics. We highlight design concepts that may be broadly relevant to drug discovery efforts beyond the rule of five.

Structural basis for selective stalling of human ribosome nascent chain complexes by a drug-like molecule.

The drug-like molecule PF-06446846 (PF846) binds the human ribosome and selectively blocks the translation of a small number of proteins by an unknown mechanism. In structures of PF846-stalled human ribosome nascent chain complexes, PF846 binds in the ribosome exit tunnel in a eukaryotic-specific pocket formed by 28S ribosomal RNA, and alters the path of the nascent polypeptide chain. PF846 arrests the translating ribosome in the rotated state of translocation, in which the peptidyl-transfer RNA 3'-CCA end is improperly docked in the peptidyl transferase center. Selections of messenger RNAs from mRNA libraries using translation extracts reveal that PF846 can stall translation elongation, arrest termination or even enhance translation, depending on nascent chain sequence context. These results illuminate how a small molecule selectively targets translation by the human ribosome, and provides a foundation for developing small molecules that modulate the production of proteins of therapeutic interest.

Small Molecule Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Inhibitors: Hit to Lead Optimization of Systemic Agents.

The optimization of a new class of small molecule PCSK9 mRNA translation inhibitors is described. The potency, physicochemical properties, and off-target pharmacology associated with the hit compound (1) were improved by changes to two regions of the molecule. The last step in the synthesis of the congested amide center was enabled by three different routes. Subtle structural changes yielded significant changes in pharmacology and off-target margins. These efforts led to the identification of 7l and 7n with overall profiles suitable for in vivo evaluation. In a 14-day toxicology study, 7l demonstrated an improved safety profile vs lead 7f. We hypothesize that the improved safety profile is related to diminished binding of 7l to nontranslating ribosomes and an apparent improvement in transcript selectivity due to the lower strength of 7l stalling of off-target proteins.

Discovery of Potent and Selective Periphery-Restricted Quinazoline Inhibitors of the Cyclic Nucleotide Phosphodiesterase PDE1.

We disclose the discovery and X-ray cocrystal data of potent, selective quinazoline inhibitors of PDE1. Inhibitor ( S)-3 readily attains free plasma concentrations above PDE1 IC50 values and has restricted brain access. The racemic compound 3 inhibits >75% of PDE hydrolytic activity in soluble samples of human myocardium, consistent with heightened PDE1 activity in this tissue. These compounds represent promising new tools to probe the value of PDE1 inhibition in the treatment of cardiovascular disease.

Donated chemical probes for open science.

Potent, selective and broadly characterized small molecule modulators of protein function (chemical probes) are powerful research reagents. The pharmaceutical industry has generated many high-quality chemical probes and several of these have been made available to academia. However, probe-associated data and control compounds, such as inactive structurally related molecules and their associated data, are generally not accessible. The lack of data and guidance makes it difficult for researchers to decide which chemical tools to choose. Several pharmaceutical companies (AbbVie, Bayer, Boehringer Ingelheim, Janssen, MSD, Pfizer, and Takeda) have therefore entered into a pre-competitive collaboration to make available a large number of innovative high-quality probes, including all probe-associated data, control compounds and recommendations on use (https://openscienceprobes.sgc-frankfurt.de/). Here we describe the chemical tools and target-related knowledge that have been made available, and encourage others to join the project.

Correction: Selective stalling of human translation through small-molecule engagement of the ribosome nascent chain.

[This corrects the article DOI: 10.1371/journal.pbio.2001882.].

Receptor-Mediated Delivery of CRISPR-Cas9 Endonuclease for Cell-Type-Specific Gene Editing.

CRISPR-Cas RNA-guided endonucleases hold great promise for disrupting or correcting genomic sequences through site-specific DNA cleavage and repair. However, the lack of methods for cell- and tissue-selective delivery currently limits both research and clinical uses of these enzymes. We report the design and in vitro evaluation of S. pyogenes Cas9 proteins harboring asialoglycoprotein receptor ligands (ASGPrL). In particular, we demonstrate that the resulting ribonucleoproteins (Cas9-ASGPrL RNP) can be engineered to be preferentially internalized into cells expressing the corresponding receptor on their surface. Uptake of such fluorescently labeled proteins in liver-derived cell lines HEPG2 (ASGPr+) and SKHEP (control; diminished ASGPr) was studied by live cell imaging and demonstrates increased accumulation of Cas9-ASGPrL RNP in HEPG2 cells as a result of effective ASGPr-mediated endocytosis. When uptake occurred in the presence of a peptide with endosomolytic properties, we observed receptor-facilitated and cell-type specific gene editing that did not rely on electroporation or the use of transfection reagents. Overall, these in vitro results validate the receptor-mediated delivery of genome-editing enzymes as an approach for cell-selective gene editing and provide a framework for future potential applications to hepatoselective gene editing in vivo.

Liver-Targeted Small-Molecule Inhibitors of Proprotein Convertase Subtilisin/Kexin Type 9 Synthesis.

Targeting of the human ribosome is an unprecedented therapeutic modality with a genome-wide selectivity challenge. A liver-targeted drug candidate is described that inhibits ribosomal synthesis of PCSK9, a lipid regulator considered undruggable by small molecules. Key to the concept was the identification of pharmacologically active zwitterions designed to be retained in the liver. Oral delivery of the poorly permeable zwitterions was achieved by prodrugs susceptible to cleavage by carboxylesterase 1. The synthesis of select tetrazole prodrugs was crucial. A cell-free in vitro translation assay containing human cell lysate and purified target mRNA fused to a reporter was used to identify active zwitterions. In vivo PCSK9 lowering by oral dosing of the candidate prodrug and quantification of the drug fraction delivered to the liver utilizing an oral positron emission tomography 18 F-isotopologue validated our liver-targeting approach.

Discovery of Potent and Orally Bioavailable Macrocyclic Peptide-Peptoid Hybrid CXCR7 Modulators.

The chemokine receptor CXCR7 is an attractive target for a variety of diseases. While several small-molecule modulators of CXCR7 have been reported, peptidic macrocycles may provide advantages in terms of potency, selectivity, and reduced off-target activity. We produced a series of peptidic macrocycles that incorporate an N-linked peptoid functionality where the peptoid group enabled us to explore side-chain diversity well beyond that of natural amino acids. At the same time, theoretical calculations and experimental assays were used to track and reduce the polarity while closely monitoring the physicochemical properties. This strategy led to the discovery of macrocyclic peptide-peptoid hybrids with high CXCR7 binding affinities (Ki < 100 nM) and measurable passive permeability (Papp > 5 × 10-6 cm/s). Moreover, bioactive peptide 25 (Ki = 9 nM) achieved oral bioavailability of 18% in rats, which was commensurate with the observed plasma clearance values upon intravenous administration.

Comparative pharmacokinetic profile of cyclosporine (CsA) with a decapeptide and a linear analogue.

The synthesis and in vivo pharmacokinetic profile of an analogue of cyclosporine is disclosed. An acyclic congener was also profiled in in vitro assays to compare cell permeability. The compounds possess similar calculated and measured molecular descriptors however have different behaviors in an RRCK assay to assess cell permeability.

Selective stalling of human translation through small-molecule engagement of the ribosome nascent chain.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a key role in regulating the levels of plasma low-density lipoprotein cholesterol (LDL-C). Here, we demonstrate that the compound PF-06446846 inhibits translation of PCSK9 by inducing the ribosome to stall around codon 34, mediated by the sequence of the nascent chain within the exit tunnel. We further show that PF-06446846 reduces plasma PCSK9 and total cholesterol levels in rats following oral dosing. Using ribosome profiling, we demonstrate that PF-06446846 is highly selective for the inhibition of PCSK9 translation. The mechanism of action employed by PF-06446846 reveals a previously unexpected tunability of the human ribosome that allows small molecules to specifically block translation of individual transcripts.

Efficient Liver Targeting by Polyvalent Display of a Compact Ligand for the Asialoglycoprotein Receptor.

A compact and stable bicyclic bridged ketal was developed as a ligand for the asialoglycoprotein receptor (ASGPR). This compound showed excellent ligand efficiency, and the molecular details of binding were revealed by the first X-ray crystal structures of ligand-bound ASGPR. This analogue was used to make potent di- and trivalent binders of ASGPR. Extensive characterization of the function of these compounds showed rapid ASGPR-dependent cellular uptake in vitro and high levels of liver/plasma selectivity in vivo. Assessment of the biodistribution in rodents of a prototypical Alexa647-labeled trivalent conjugate showed selective hepatocyte targeting with no detectable distribution in nonparenchymal cells. This molecule also exhibited increased ASGPR-directed hepatocellular uptake and prolonged retention compared to a similar GalNAc derived trimer conjugate. Selective release in the liver of a passively permeable small-molecule cargo was achieved by retro-Diels-Alder cleavage of an oxanorbornadiene linkage, presumably upon encountering intracellular thiol. Therefore, the multicomponent construct described here represents a highly efficient delivery vehicle to hepatocytes.

Nonclassical Size Dependence of Permeation Defines Bounds for Passive Adsorption of Large Drug Molecules.

Macrocyclic peptides are considered large enough to inhibit "undruggable" targets, but the design of passively cell-permeable molecules in this space remains a challenge due to the poorly understood role of molecular size on passive membrane permeability. Using split-pool combinatorial synthesis, we constructed a library of cyclic, per-N-methlyated peptides spanning a wide range of calculated lipohilicities (0 < AlogP < 8) and molecular weights (∼800 Da < MW < ∼1200 Da). Analysis by the parallel artificial membrane permeability assay revealed a steep drop-off in apparent passive permeability with increasing size in stark disagreement with current permeation models. This observation, corroborated by a set of natural products, helps define criteria for achieving permeability in larger molecular size regimes and suggests an operational cutoff, beyond which passive permeability is constrained by a sharply increasing penalty on membrane permeation.

Helixconstraints and amino acid substitution in GLP-1 increase cAMP and insulin secretion but not beta-arrestin 2 signaling.

Glucagon-like peptide (GLP-1) is an endogenous hormone that induces insulin secretion from pancreatic islets and modified forms are used to treat diabetes mellitus type 2. Understanding how GLP-1 interacts with its receptor (GLP-1R) can potentially lead to more effective drugs. Modeling and NMR studies of the N-terminus of GLP-1 suggest a ß-turn between residues Glu9-Phe12 and a kinked alpha helix between Val16-Gly37. N-terminal turn constraints attenuated binding affinity and activity (compounds 1-8). Lys-Asp (i, i+4) crosslinks in the middle and at the C-terminus increased alpha helicity and cAMP stimulation without much effect on binding affinity or beta-arrestin 2 recruitment (compounds 9-18). Strategic positioning of helix-inducing constraints and amino acid substitutions (Tyr16, Ala22) increased peptide helicity and produced ten-fold higher cAMP potency (compounds 19-28) over GLP-1(7-37)-NH2. The most potent cAMP activator (compound 23) was also the most potent inducer of insulin secretion.

Chiral Sulfoxide-Induced Single Turn Peptide α-Helicity.

Inducing α-helicity through side-chain cross-linking is a strategy that has been pursued to improve peptide conformational rigidity and bio-availability. Here we describe the preparation of small peptides tethered to chiral sulfoxide-containing macrocyclic rings. Furthermore, a study of structure-activity relationships (SARs) disclosed properties with respect to ring size, sulfur position, oxidation state, and stereochemistry that show a propensity to induce α-helicity. Supporting data include circular dichroism spectroscopy (CD), NMR spectroscopy, and a single crystal X-ray structure for one such stabilized peptide. Finally, theoretical studies are presented to elucidate the effect of chiral sulfoxides in inducing backbone α-helicity.

Truncated Glucagon-like Peptide-1 and Exendin-4 α-Conotoxin pl14a Peptide Chimeras Maintain Potency and α-Helicity and Reveal Interactions Vital for cAMP Signaling in Vitro.

Glucagon-like peptide-1 (GLP-1) signaling through the glucagon-like peptide 1 receptor (GLP-1R) is a key regulator of normal glucose metabolism, and exogenous GLP-1R agonist therapy is a promising avenue for the treatment of type 2 diabetes mellitus. To date, the development of therapeutic GLP-1R agonists has focused on producing drugs with an extended serum half-life. This has been achieved by engineering synthetic analogs of GLP-1 or the more stable exogenous GLP-1R agonist exendin-4 (Ex-4). These synthetic peptide hormones share the overall structure of GLP-1 and Ex-4, with a C-terminal helical segment and a flexible N-terminal tail. Although numerous studies have investigated the molecular determinants underpinning GLP-1 and Ex-4 binding and signaling through the GLP-1R, these have primarily focused on the length and composition of the N-terminal tail or on how to modulate the helicity of the full-length peptides. Here, we investigate the effect of C-terminal truncation in GLP-1 and Ex-4 on the cAMP pathway. To ensure helical C-terminal regions in the truncated peptides, we produced a series of chimeric peptides combining the N-terminal portion of GLP-1 or Ex-4 and the C-terminal segment of the helix-promoting peptide α-conotoxin pl14a. The helicity and structures of the chimeric peptides were confirmed using circular dichroism and NMR, respectively. We found no direct correlation between the fractional helicity and potency in signaling via the cAMP pathway. Rather, the most important feature for efficient receptor binding and signaling was the C-terminal helical segment (residues 22-27) directing the binding of Phe(22) into a hydrophobic pocket on the GLP-1R.

Cyclic alpha-conotoxin peptidomimetic chimeras as potent GLP-1R agonists.

Type 2 diabetes mellitus (T2DM) results from compromised pancreatic ß-cell function, reduced insulin production, and lowered insulin sensitivity in target organs resulting in hyperglycemia. The GLP-1 hormone has two biologically active forms, GLP-1-(7-37) and GLP-1-(7-36)amide, which are equipotent at the glucagon-like peptide-1 receptor (GLP-1R). These peptides are central both to normal glucose metabolism and dysregulation in T2DM. Several structurally modified GLP-1 analogues are now approved drugs, and a number of other analogues are in clinical trials. None of these compounds is orally bioavailable and all require parenteral delivery. Recently, a number of smaller peptidomimetics containing 11-12 natural and unnatural amino acids have been identified that have similar insulin regulating profiles as GLP-1. The α-conotoxins are a class of disulfide rich peptide venoms isolated from cone snails, and are known for their highly constrained structures and resistance to enzymatic degradation. In this study, we examined whether 11-residue peptidomimetics incorporated into α-conotoxin scaffolds, forming monocyclic or bicyclic compounds constrained by disulfide bonds and/or backbone cyclization, could activate the GLP-1 receptor (GLP-1R). Several compounds showed potent (nanomolar) agonist activity at GLP-1R, as evaluated via cAMP signaling. In addition, HPLC retention times and in silico calculations suggested that mono- and bicyclic compounds had more favorable n-octanol/water partition coefficients according to the virtual partition coefficient model (vLogP), while maintaining a smaller radius of gyration compared to corresponding uncyclized peptidomimetics. Our findings suggest that cyclic peptidomimetics provide a potential avenue for future design of potent, compact ligands targeting GLP-1R and possessing improved physicochemical properties.

Peptide to Peptoid Substitutions Increase Cell Permeability in Cyclic Hexapeptides.

The effect of peptide-to-peptoid substitutions on the passive membrane permeability of an N-methylated cyclic hexapeptide is examined. In general, substitutions maintained permeability but increased conformational heterogeneity. Diversification with nonproteinogenic side chains increased permeability up to 3-fold. Additionally, the conformational impact of peptoid substitutions within a ß-turn are explored. Based on these results, the strategic incorporation of peptoid residues into cyclic peptides can maintain or improve cell permeability, while increasing access to diverse side-chain functionality.