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Pharmacological administration of fibroblast growth factor 21 (FGF21) improves metabolic profile in preclinical species and humans. FGF21 exerts its metabolic effects through formation of beta-klotho (KLB)/FGF receptor 1c FGFR1c complex and subsequent signaling. Data from various in vitro systems demonstrate the intact C- and N-terminus of FGF21 is required for binding with KLB, and interaction with FGFR1c, respectively. However the relative roles of the termini for in vivo pharmacological effects are unclear. Here we report PF-05231023, a long-acting FGF21 analogue which is unique in that the half-life and subcutaneous (s.c.) bioavailability of the intact C-terminus are significantly different from those of the intact N-terminus (2 vs. 22 hr for half-life and 4~7 vs. ~50% SC bioavailability). Therefore, this molecule serves as a valuable tool to evaluate the relative roles of intact C-terminus vs. N-terminus in in vivo pharmacology studies in preclinical species. We determined the effects of PF-05231023 administration on body weight (BW) loss and glucose reduction during an oral glucose tolerance test (OGTT) following SC and intravenous (i.v.) administration in diet-induced obese (DIO) and leptin-deficient obese (ob/ob) mice, respectively. Our data show that the intact N-terminus of FGF21 in PF-05231023 appears to be sufficient to drive glucose lowering during OGTT and sustain BW loss in DIOs. Further, PK/PD modeling suggests that while the intact FGF21 C-terminus is not strictly required for glucose lowering during OGTT in ob/ob mice or for BW reduction in DIO mice, the higher potency conferred by intact C-terminus contributes to a rapid initiation of pharmacodynamic effects immediately following dosing. These results provide additional insight into the strategy of developing stabilized versions of FGF21 analogs to harness the full spectrum of its metabolic benefits.

GDF15 is a distant TGF-β family member that induces anorexia and weight loss. Due to its function, GDF15 has attracted attention as a potential therapeutic for the treatment of obesity and its associated metabolic diseases. However, the pharmacokinetic and physicochemical properties of GDF15 present several challenges for its development as a therapeutic, including a short half-life, high aggregation propensity, and protease susceptibility in serum. Here, we report the design, characterization and optimization of GDF15 in an Fc-fusion protein format with improved therapeutic properties. Using a structure-based engineering approach, we combined knob-into-hole Fc technology and N-linked glycosylation site mutagenesis for half-life extension, improved solubility and protease resistance. In addition, we identified a set of mutations at the receptor binding site of GDF15 that show increased GFRAL binding affinity and led to significant half-life extension. We also identified a single point mutation that increases p-ERK signaling activity and results in improved weight loss efficacy in vivo. Taken together, our findings allowed us to develop GDF15 in a new therapeutic format that demonstrates better efficacy and potential for improved manufacturability.

This paper shows how noisy gene expression causes a bacterial cell to slowly lose track of time, as measured by its circadian clock. The theoretical framework thus introduced breaks the ground for the analysis of noise in other out-of-equilibrium living systems. Recent advances in measuring gene expression at the single-cell level have highlighted the stochastic nature of messenger RNA and protein synthesis 1 , 2 , 3 . Stochastic gene expression creates a source of variability in the abundance of cellular components, even among isogenic cells exposed to an identical environment. Recent integrated experimental and modelling studies 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 have shed light on the molecular sources of this variability. However, many of these studies focus on systems that have reached a steady state and therefore do not address a large class of dynamic phenomena including oscillatory gene expression. Here we develop a general protocol for analysing and predicting stochastic gene expression in systems that never reach steady states. We use this framework to analyse experimentally stochastic expression of genes driven by the Synechococcus elongatus circadian clock. We find that, although the average expression at two points in the circadian cycle separated by 12 hours is identical, the variability at these two time points can be different. We show that this is a general feature of out-of-steady-state systems. We demonstrate how intrinsic noise sources, owing to random births and deaths of mRNAs and proteins, or extrinsic noise sources, which introduce fluctuations in rate constants, affect the cell-to-cell variability. To distinguish experimentally between these sources, we measured how the correlation between expression fluctuations of two identical genes is modulated during the circadian cycle. This quantitative framework is generally applicable to any out-of-steady-state system and will be necessary for understanding the fidelity of dynamic cellular systems.

Characterization of cell surface target expression is critical for the development of therapeutics that bind membrane proteins or use changes in target abundance as efficacy biomarkers. Target level is often analyzed with flow-cytometry by converting signal intensity of fluorophore-conjugated target-specific monoclonal antibodies (mAbs) into an Ab binding capacity (ABC) number, as determined at binding equilibrium using a calibrator. Here, we examined the feasibility of simultaneous estimation of ABC as well as mAb binding and dissociation rates in cell populations of interest, by mathematical modeling of experimentally observed time-dependent changes in the mAb-target complex formation. First, using three different target-specific mAbs, conjugated to a fluorophore in an equimolar ratio, we demonstrated that by modeling mAb binding kinetics, simultaneous quantification of ABC and apparent affinity (Kd) by flow cytometry was feasible, and the estimates were similar to calibration-based benchmark measurements with these mAbs. Next, by comparing different fluorophore formats of the same mAbs, we found that while kinetic data fitting with employed model can estimate the ABC and apparent affinity for mAbs, regardless of fluorophore type, the results can be sensitive to the model’s assumptions of fluorophore to Ab conjugation ratio. Collectively, our findings indicated that the described experimental and modeling approaches can be applied for the concomitant analysis of membrane target level and Ab on-cell affinity, circumventing the need for complex multiple experiments, expediting data delivery for drug discovery. Graphical Abstract

Actin polymerization provides a powerful propulsion force for numerous types of cell motility. Although tremendous progress has been made in identifying the biochemical components necessary for actin-based motility, the precise biophysical mechanisms of force generation remain unclear. To probe the polymerization forces quantitatively, we introduce an experimental system in which lipid vesicles coated with the Listeria monocytogenes virulence factor ActA are propelled by actin polymerization. The polymerization forces cause significant deformations of the vesicle. We have used these deformations to obtain a spatially resolved measure of the forces exerted on the membrane using a model based on the competition between osmotic pressure and membrane stretching. Our results indicate that actin exerts retractile or propulsive forces depending on the local membrane curvature and that the membrane is strongly bound to the actin gel. These results are consistent with the observed dynamics. After a slow elongation of the vesicle from a spherical shape, the strong bonds between the actin gel and the membrane rupture if the retractile forces exceed a critical value, leading to a rapid release of the vesicle's trailing edge.

Osteopontin is a secreted glycophosphoprotein that is highly implicated in many physiological and pathological processes such as biomineralization, cell-mediated immunity, inflammation, fibrosis, cell survival, tumorigenesis and metastasis. Antibodies against osteopontin have been actively pursued as potential therapeutics for various diseases by pharmaceutical companies and academic laboratories. Many studies have demonstrated the efficacy of osteopontin inhibition in a variety of preclinical models of diseases such as rheumatoid arthritis, cancer, nonalcoholic steatohepatitis, but clinical utility has not yet been demonstrated. To evaluate the feasibility of osteopontin neutralization with antibodies in a clinical setting, we measured its physiological turnover rate in humans, a sensitive parameter required for mechanistic pharmacokinetic and pharmacodynamic (PK/PD) modeling of biotherapeutics. Results from a stable isotope-labelled amino acid pulse-chase study in healthy human subjects followed by mass spectrometry showed that osteopontin undergoes very rapid turnover. PK/PD modeling and simulation of different theoretical scenarios reveal that achieving sufficient target coverage using antibodies can be very challenging mostly due to osteopontin’s fast turnover, as well as its relatively high plasma concentrations in human. Therapeutic antibodies against osteopontin would need to be engineered to have much extended PK than conventional antibodies, and be administered at high doses and with short dosing intervals.

Pharmacological administration of FGF21 analogues has shown robust body weight reduction and lipid profile improvement in both dysmetabolic animal models and metabolic disease patients. Here we report the design, optimization, and characterization of a long acting glyco-variant of FGF21. Using a combination of N-glycan engineering for enhanced protease resistance and improved solubility, Fc fusion for further half-life extension, and a single point mutation for improving manufacturability in Chinese Hamster Ovary cells, we created a novel FGF21 analogue, Fc-FGF21[R19V][N171] or PF-06645849, with substantially improved solubility and stability profile that is compatible with subcutaneous (SC) administration. In particular, it showed a low systemic clearance (0.243 mL/hr/kg) and long terminal half-life (~200 hours for intact protein) in cynomolgus monkeys that approaches those of monoclonal antibodies. Furthermore, the superior PK properties translated into robust improvement in glucose tolerance and the effects lasted 14 days post single SC dose in ob/ob mice. PF-06645849 also caused greater body weight loss in DIO mice at lower and less frequent SC doses, compared to previous FGF21 analogue PF-05231023. In summary, the overall PK/PD and pharmaceutical profile of PF-06645849 offers great potential for development as weekly to twice-monthly SC administered therapeutic for chronic treatment of metabolic diseases.

Pharmacological administration of fibroblast growth factor 21 (FGF21) improves metabolic profile in preclinical species and humans. FGF21 exerts its metabolic effects through formation of beta-klotho (KLB)/FGF receptor 1c FGFR1c complex and subsequent signaling. Data from various in vitro systems demonstrate the intact C- and N-terminus of FGF21 is required for binding with KLB, and interaction with FGFR1c, respectively. However the relative roles of the termini for in vivo pharmacological effects are unclear. Here we report PF-05231023, a long-acting FGF21 analogue which is unique in that the half-life and subcutaneous (SC) bioavailability of the intact C-terminus are significantly different from those of the intact N-terminus (2 vs. 22 hr for half-life and 4~7 vs. ~50% SC bioavailability). Therefore, this molecule serves as a valuable tool to evaluate the relative roles of intact C-terminus vs. N-terminus in in vivo pharmacology studies in preclinical species. We determined the effects of PF-05231023 administration on body weight (BW) loss and glucose reduction during an oral glucose tolerance test (OGTT) following SC and intravenous (IV) administration in diet-induced obese (DIO) and leptin-deficient obese (ob/ob) mice, respectively. Our data show that the intact N-terminus of FGF21 in PF-05231023 appears to be sufficient to drive glucose lowering during OGTT and sustain BW loss in DIOs. Further, PK/PD modeling suggests that while the intact FGF21 C-terminus is not strictly required for glucose lowering during OGTT in ob/ob mice or for BW reduction in DIO mice, the higher potency conferred by intact C-terminus contributes to a rapid initiation of pharmacodynamic effects immediately following dosing. These results provide additional insight into the strategy of developing stabilized versions of FGF21 analogs to harness the full spectrum of its metabolic benefits.

Background The success of anti-TNF biologics for the treatment of rheumatoid arthritis has highlighted the importance of understanding the intracellular pathways that regulate TNF production in the quest for an orally-available small molecule inhibitor. p38 is known to strongly regulate TNF production via MK2. The failure of several p38 inhibitors in the clinic suggests the importance of other downstream pathways in normal cell function. Recent work has described a 'substrate-selective' p38 inhibitor that is able to preferentially block the activity of p38 against one substrate (MK2) versus another (ATF2). Using a combined experimental and computational approach, we have examined this mechanism in greater detail for two p38 substrates, MK2 and ATF2. Results We found that in a dual (MK2 and ATF2) substrate assay, MK2-p38 interaction reduced the activity of p38 against ATF2. We further constructed a detailed kinetic mechanistic model of p38 phosphorylation in the presence of multiple substrates and successfully predicted the performance of classical and so-called 'substrate-selective' p38 inhibitors in the dual substrate assay. Importantly, it was found that excess MK2 results in a stoichiometric effect in which the formation of p38-MK2-inhibitor complex prevents the phosphorylation of ATF2, despite the preference of the compound for the p38-MK2 complex over the p38-ATF2 complex. MK2 and p38 protein expression levels were quantified in U937, Thp-1 and PBMCs and found that [MK2] > [p38]. Conclusion Our integrated mechanistic modeling and experimental validation provides an example of how systems biology approaches can be applied to drug discovery and provide a basis for decision-making with limited chemical matter. We find that, given our current understanding, it is unlikely that 'substrate-selective' inhibitors of p38 will work as originally intended when placed in the context of more complex cellular environments, largely due to a stoichiometric excess of MK2 relative to p38.