The use of peptides in medicine is limited by low membrane permeability, metabolic instability, high clearance, and negligible oral bioavailability. The prediction of oral bioavailability of drugs ...relies on physicochemical properties that favor passive permeability and oxidative metabolic stability, but these may not be useful for peptides. Here we investigate effects of heterocyclic constraints, intramolecular hydrogen bonds, and side chains on the oral bioavailability of cyclic heptapeptides. NMR‐derived structures, amide H–D exchange rates, and temperature‐dependent chemical shifts showed that the combination of rigidification, stronger hydrogen bonds, and solvent shielding by branched side chains enhances the oral bioavailability of cyclic heptapeptides in rats without the need for N‐methylation.
The oral absorption and bioavailability of a cyclic heptapeptide in rats was improved by combining heterocyclic constraints, intramolecular hydrogen bonds, and branched amino acid side chains to shield polar backbone atoms. These improvements were solely guided by amide H–D exchange rates, NMR‐derived structures, and solvent‐exposed polar surfaces.
Peptide agonists of the glucagon-like peptide-1 receptor (GLP-1R) have revolutionized diabetes therapy, but their use has been limited because they require injection. Herein, we describe the ...discovery of the orally bioavailable, small-molecule, GLP-1R agonist PF-06882961 (danuglipron). A sensitized high-throughput screen was used to identify 5-fluoropyrimidine-based GLP-1R agonists that were optimized to promote endogenous GLP-1R signaling with nanomolar potency. Incorporation of a carboxylic acid moiety provided considerable GLP-1R potency gains with improved off-target pharmacology and reduced metabolic clearance, ultimately resulting in the identification of danuglipron. Danuglipron increased insulin levels in primates but not rodents, which was explained by receptor mutagensis studies and a cryogenic electron microscope structure that revealed a binding pocket requiring a primate-specific tryptophan 33 residue. Oral administration of danuglipron to healthy humans produced dose-proportional increases in systemic exposure (NCT03309241). This opens an opportunity for oral small-molecule therapies that target the well-validated GLP-1R for metabolic health.
Ever since eukaryotes subsumed the bacterial ancestor of mitochondria, the nuclear and mitochondrial genomes have had to closely coordinate their activities, as each encode different subunits of the ...oxidative phosphorylation (OXPHOS) system. Mitochondrial dysfunction is a hallmark of aging, but its causes are debated. We show that, during aging, there is a specific loss of mitochondrial, but not nuclear, encoded OXPHOS subunits. We trace the cause to an alternate PGC-1α/β-independent pathway of nuclear-mitochondrial communication that is induced by a decline in nuclear NAD+ and the accumulation of HIF-1α under normoxic conditions, with parallels to Warburg reprogramming. Deleting SIRT1 accelerates this process, whereas raising NAD+ levels in old mice restores mitochondrial function to that of a young mouse in a SIRT1-dependent manner. Thus, a pseudohypoxic state that disrupts PGC-1α/β-independent nuclear-mitochondrial communication contributes to the decline in mitochondrial function with age, a process that is apparently reversible.
Display omitted
•A specific decline in mitochondrially encoded genes occurs during aging in muscle•Nuclear NAD+ levels regulate mitochondrial homeostasis independently of PGC-1α/β•Declining NAD+ during aging causes pseudohypoxia, which disrupts OXPHOS function•Raising nuclear NAD+ in old mice reverses pseudohypoxia and metabolic dysfunction
Aging disrupts an NAD+-dependent nuclear-mitochondrial communication pathway, causing a decline in the mitochondrially encoded oxidative phosphorylation components relative to nuclear-encoded components. Raising NAD+ levels in old mice alleviates the pseudohypoxic conditions that disrupt the communication pathway, reversing this mitochondrial defect.
Resveratrol induces mitochondrial biogenesis and protects against metabolic decline, but whether SIRT1 mediates these benefits is the subject of debate. To circumvent the developmental defects of ...germline SIRT1 knockouts, we have developed an inducible system that permits whole-body deletion of SIRT1 in adult mice. Mice treated with a moderate dose of resveratrol showed increased mitochondrial biogenesis and function, AMPK activation, and increased NAD+ levels in skeletal muscle, whereas SIRT1 knockouts displayed none of these benefits. A mouse overexpressing SIRT1 mimicked these effects. A high dose of resveratrol activated AMPK in a SIRT1-independent manner, demonstrating that resveratrol dosage is a critical factor. Importantly, at both doses of resveratrol no improvements in mitochondrial function were observed in animals lacking SIRT1. Together these data indicate that SIRT1 plays an essential role in the ability of moderate doses of resveratrol to stimulate AMPK and improve mitochondrial function both in vitro and in vivo.
Display omitted
► Resveratrol's ability to improve mitochondrial function requires SIRT1 in vivo ► Moderate doses of resveratrol activate AMPK and raise NAD+ in a SIRT1-dependent manner ► Activation of AMPK in the absence of SIRT1 does not improve mitochondrial function ► Overexpression of SIRT1 mimics resveratrol's effects on AMPK and mitochondria
Human immunodeficiency virus (HIV)-1 is able to replicate in primary human macrophages without stimulating innate immunity despite reverse transcription of genomic RNA into double-stranded DNA, an ...activity that might be expected to trigger innate pattern recognition receptors. We reasoned that if correctly orchestrated HIV-1 uncoating and nuclear entry is important for evasion of innate sensors then manipulation of specific interactions between HIV-1 capsid and host factors that putatively regulate these processes should trigger pattern recognition receptors and stimulate type 1 interferon (IFN) secretion. Here we show that HIV-1 capsid mutants N74D and P90A, which are impaired for interaction with cofactors cleavage and polyadenylation specificity factor subunit 6 (CPSF6) and cyclophilins (Nup358 and CypA), respectively, cannot replicate in primary human monocyte-derived macrophages because they trigger innate sensors leading to nuclear translocation of NF-κB and IRF3, the production of soluble type 1 IFN and induction of an antiviral state. Depletion of CPSF6 with short hairpin RNA expression allows wild-type virus to trigger innate sensors and IFN production. In each case, suppressed replication is rescued by IFN-receptor blockade, demonstrating a role for IFN in restriction. IFN production is dependent on viral reverse transcription but not integration, indicating that a viral reverse transcription product comprises the HIV-1 pathogen-associated molecular pattern. Finally, we show that we can pharmacologically induce wild-type HIV-1 infection to stimulate IFN secretion and an antiviral state using a non-immunosuppressive cyclosporine analogue. We conclude that HIV-1 has evolved to use CPSF6 and cyclophilins to cloak its replication, allowing evasion of innate immune sensors and induction of a cell-autonomous innate immune response in primary human macrophages.
SARS-CoV-2-specific memory T cells will likely prove critical for long-term immune protection against COVID-19. Here, we systematically mapped the functional and phenotypic landscape of ...SARS-CoV-2-specific T cell responses in unexposed individuals, exposed family members, and individuals with acute or convalescent COVID-19. Acute-phase SARS-CoV-2-specific T cells displayed a highly activated cytotoxic phenotype that correlated with various clinical markers of disease severity, whereas convalescent-phase SARS-CoV-2-specific T cells were polyfunctional and displayed a stem-like memory phenotype. Importantly, SARS-CoV-2-specific T cells were detectable in antibody-seronegative exposed family members and convalescent individuals with a history of asymptomatic and mild COVID-19. Our collective dataset shows that SARS-CoV-2 elicits broadly directed and functionally replete memory T cell responses, suggesting that natural exposure or infection may prevent recurrent episodes of severe COVID-19.
Display omitted
•Acute-phase SARS-CoV-2-specific T cells display an activated cytotoxic phenotype•Convalescent-phase SARS-CoV-2-specific T cells generate broad responses•Polyfunctional SARS-CoV-2-specific T cells also occur in seronegative individuals
Sekine et al. provide a functional and phenotypic map of SARS-CoV-2-specific T cells across the full spectrum of exposure, infection, and COVID-19 severity. They observe that SARS-CoV-2 elicits broadly directed and functionally replete memory T cells that may protect against recurrent episodes of severe COVID-19.
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.
To assess 5-year Descemet's stripping endothelial keratoplasty (DSEK) graft survival and endothelial cell loss in the surviving grafts.
Retrospective, interventional case series.
One hundred ...sixty-five eyes of 149 patients treated with primary DSEK.
Donor corneal-scleral rims were dissected manually or with a microkeratome and were cut with a trephine. The graft was folded endothelial side inward and was inserted with forceps through a 5-mm incision. The cumulative probability of secondary graft failure was calculated using Kaplan-Meier survival analysis and the log-rank test. Endothelial cell density (ECD) was determined from baseline preoperative donor and 1-year, 3-year, and 5-year postoperative central endothelial images.
Graft survival and ECD at 5 years.
The median recipient age was 71 years (range, 22-90 years) and 62% were female. Eighteen eyes (11%) were treated for pseudophakic or aphakic corneal edema and 147 eyes (89%) were treated for Fuchs' dystrophy. The cumulative 5-year survival rate was significantly lower in pseudophakic or aphakic corneal edema eyes (76%) versus Fuchs' eyes (95%; P = 0.0087). In particular, the 5-year survival rate was reduced significantly in eyes with prior glaucoma shunt or trabeculectomy surgery vs. those without (40% vs. 95%; P<0.0001). The causes of secondary graft failure were endothelial decompensation in 6 eyes (3.6%) and unsatisfactory corrected distance acuity (20/60 to 20/100) in 4 eyes (2.4%). No grafts experienced traumatic wound rupture or failed as a result of ocular surface complications. The median 5-year endothelial cell loss was 53% (range, 7.5%-89%). The 5-year graft ECD was correlated weakly with the baseline donor ECD (r = 0.22 and P = 0.04) and was not significantly correlated with recipient gender (P = 0.075), age (P = 0.85), or diagnosis (P = 0.78).
The 5-year graft survival rates for DSEK were similar to those reported for penetrating keratoplasty in the multicenter Cornea Donor Study (95% vs. 93% for Fuchs' dystrophy and 76% vs. 73% for pseudophakic or aphakic corneal edema). Furthermore, the 5-year endothelial cell loss after DSEK compared favorably with that measured after penetrating keratoplasty in the Cornea Donor Study (53% vs. 70%).
Significance Peptides are valuable leads for drug development, offering advantages over other molecular classes. Specifically, they can bind potently and selectively to drug targets, including ...protein–protein interactions that are too challenging for small-molecule therapeutics. However, peptides are poor drugs because of their low in vivo stability and poor oral bioavailability. We propose a strategy for improving the oral bioavailability of peptides by identifying appropriate amides for chemical modification using temperature coefficients measured by NMR. The modified peptides have improved solvation properties, making them more membrane permeable. This approach for identifying sites for modification is a rapid method for guiding peptide drug design.
Enhancing the oral bioavailability of peptide drug leads is a major challenge in drug design. As such, methods to address this challenge are highly sought after by the pharmaceutical industry. Here, we propose a strategy to identify appropriate amides for N -methylation using temperature coefficients measured by NMR to identify exposed amides in cyclic peptides. N -methylation effectively caps these amides, modifying the overall solvation properties of the peptides and making them more membrane permeable. The approach for identifying sites for N -methylation is a rapid alternative to the elucidation of 3D structures of peptide drug leads, which has been a commonly used structure-guided approach in the past. Five leucine-rich peptide scaffolds are reported with selectively designed N -methylated derivatives. In vitro membrane permeability was assessed by parallel artificial membrane permeability assay and Caco-2 assay. The most promising N -methylated peptide was then tested in vivo. Here we report a novel peptide (15), which displayed an oral bioavailability of 33% in a rat model, thus validating the design approach. We show that this approach can also be used to explain the notable increase in oral bioavailability of a somatostatin analog.
The capacity of the immune system to adapt to rapidly evolving viruses is a primary feature of effective immunity, yet its molecular basis is unclear. Here, we investigated protective HIV-1-specific ...CD8+ T cell responses directed against the immunodominant p24 Gag-derived epitope KK10 (KRWIILGLNK263-272) presented by human leukocyte antigen (HLA)-B∗2705. We found that cross-reactive CD8+ T cell clonotypes were mobilized to counter the rapid emergence of HIV-1 variants that can directly affect T cell receptor (TCR) recognition. These newly recruited clonotypes expressed TCRs that engaged wild-type and mutant KK10 antigens with similar affinities and almost identical docking modes, thereby accounting for their antiviral efficacy in HLA-B∗2705+ individuals. A protective CD8+ T cell repertoire therefore encompasses the capacity to control TCR-accessible mutations, ultimately driving the development of more complex viral escape variants that disrupt antigen presentation.
► New T cell clonotypes are recruited to counter TCR-accessible HIV mutations ► These T cells express TCRs that crossrecognize wild-type and mutant epitopes ► Crossreactive CD8+ T cells underpin anti-HIV efficacy in HLA-B∗27+ patients ► T cell efficacy ultimately drives escape mutations that impact antigen presentation
PDF
