Machine-Learning-Guided Peptide Drug Discovery: Development of GLP-1 Receptor Agonists with Improved Drug Properties.

Peptide-based drug discovery has surged with the development of peptide hormone-derived analogs for the treatment of diabetes and obesity. Machine learning (ML)-enabled quantitative structure-activity relationship (QSAR) approaches have shown great promise in small molecule drug discovery but have been less successful in peptide drug discovery due to limited data availability. We have developed a peptide drug discovery platform called streaMLine, enabling rigorous design, synthesis, screening, and ML-driven analysis of large peptide libraries. Using streaMLine, this study systematically explored secretin as a peptide backbone to generate potent, selective, and long-acting GLP-1R agonists with improved physicochemical properties. We synthesized and screened a total of 2688 peptides and applied ML-guided QSAR to identify multiple options for designing stable and potent GLP-1R agonists. One candidate, GUB021794, was profiled in vivo (S.C., 10 nmol/kg QD) and showed potent body weight loss in diet-induced obese mice and a half-life compatible with once-weekly dosing.

Comprehensive Peptide Cyclization Examination Yields Optimized APP Scaffolds with Improved Affinity toward Mint2.

Peptides targeting disease-relevant protein-protein interactions are an attractive class of therapeutics covering the otherwise undruggable space between small molecules and therapeutic proteins. However, peptides generally suffer from poor metabolic stability and low membrane permeability. Hence, peptide cyclization has become a valuable approach to develop linear peptide motifs into metabolically stable and potentially cell-permeable cyclic leads. Furthermore, cyclization of side chains, also known as "stapling", can stabilize particular secondary peptide structures. Here, we demonstrate that a comprehensive examination of cyclization strategies in terms of position, chemistry, and length is a prerequisite for the selection of optimal cyclic peptide scaffolds. Our systematic approach identifies cyclic APP dodecamer peptides targeting the phosphotyrosine binding domain of Mint2 with substantially improved affinity. We show that especially all-hydrocarbon stapling provides improved metabolic stability, a significantly stabilized secondary structure and membrane permeability.

Danish teleophthalmology platform reduces optometry referrals into the national eye care system.

OBJECTIVE: The purpose of this study was to evaluate the stratification of follow-up and referral pathways after implementation of a systematic cloud-based electronic-referral teleophthalmological service for optometry-initiated ocular posterior segment disease referrals to the Danish national eye care system. METHODS AND ANALYSIS: A retrospective cohort study was conducted in the period from 1 August 2018 to 31 July 2019. Patients with suspected ocular posterior segment disease reviewed by the telemedical ophthalmology service were included. The service stratified patients into the categories: no need for follow-up, follow-up by optometrist, follow-up by the telemedical service and referral to the national Danish eye care service. RESULTS: From a pool of 386 361 customers, 9938 patients were enrolled into this study. 19.5% of all patients were referred to the Danish national eye care system, while 80.5% of the patients in the telemedical service were not, in the period from 1 August 2018 to 31 July 2019. 14.4% of the optometrist referrals did not need any follow-up, while a majority of 66.1% needed some follow-up either by the optometrist themselves or within the telemedical service. CONCLUSION: Optometrist posterior segment disease referrals can be considerably reduced with a risk stratified approach and optimal use of technology. New models can improve and streamline the healthcare system.

Orchestration of signaling by structural disorder in class 1 cytokine receptors.

BACKGROUND: Class 1 cytokine receptors (C1CRs) are single-pass transmembrane proteins responsible for transmitting signals between the outside and the inside of cells. Remarkably, they orchestrate key biological processes such as proliferation, differentiation, immunity and growth through long disordered intracellular domains (ICDs), but without having intrinsic kinase activity. Despite these key roles, their characteristics remain rudimentarily understood. METHODS: The current paper asks the question of why disorder has evolved to govern signaling of C1CRs by reviewing the literature in combination with new sequence and biophysical analyses of chain properties across the family. RESULTS: We uncover that the C1CR-ICDs are fully disordered and brimming with SLiMs. Many of these short linear motifs (SLiMs) are overlapping, jointly signifying a complex regulation of interactions, including network rewiring by isoforms. The C1CR-ICDs have unique properties that distinguish them from most IDPs and we forward the perception that the C1CR-ICDs are far from simple strings with constitutively bound kinases. Rather, they carry both organizational and operational features left uncovered within their disorder, including mechanisms and complexities of regulatory functions. CONCLUSIONS: Critically, the understanding of the fascinating ability of these long, completely disordered chains to orchestrate complex cellular signaling pathways is still in its infancy, and we urge a perceptional shift away from the current simplistic view towards uncovering their full functionalities and potential. Video abstract.

Analyzing Biomolecular Ensembles.

Several techniques are available to generate conformational ensembles of proteins and other biomolecules either experimentally or computationally. These methods produce a large amount of data that need to be analyzed to identify structure-dynamics-function relationship. In this chapter, we will cover different tools to unveil the information hidden in conformational ensemble data and to guide toward the rationalization of the data. We included routinely used approaches such as dimensionality reduction, as well as new methods inspired by high-order statistics and graph theory.

Molecular dynamics-guided discovery of an ago-allosteric modulator for GPR40/FFAR1.

The long-chain fatty acid receptor FFAR1/GPR40 binds agonists in both an interhelical site between the extracellular segments of transmembrane helix (TM)-III and TM-IV and a lipid-exposed groove between the intracellular segments of these helices. Molecular dynamics simulations of FFAR1 with agonist removed demonstrated a major rearrangement of the polar and charged anchor point residues for the carboxylic acid moiety of the agonist in the interhelical site, which was associated with closure of a neighboring, solvent-exposed pocket between the extracellular poles of TM-I, TM-II, and TM-VII. A synthetic compound designed to bind in this pocket, and thereby prevent its closure, was identified through structure-based virtual screening and shown to function both as an agonist and as an allosteric modulator of receptor activation. This discovery of an allosteric agonist for a previously unexploited, dynamic pocket in FFAR1 demonstrates both the power of including molecular dynamics in the drug discovery process and that this specific, clinically proven, but difficult, antidiabetes target can be addressed by chemotypes different from existing ligands.

HUWE1 E3 ligase promotes PINK1/PARKIN-independent mitophagy by regulating AMBRA1 activation via IKKα.

The selective removal of undesired or damaged mitochondria by autophagy, known as mitophagy, is crucial for cellular homoeostasis, and prevents tumour diffusion, neurodegeneration and ageing. The pro-autophagic molecule AMBRA1 (autophagy/beclin-1 regulator-1) has been defined as a novel regulator of mitophagy in both PINK1/PARKIN-dependent and -independent systems. Here, we identified the E3 ubiquitin ligase HUWE1 as a key inducing factor in AMBRA1-mediated mitophagy, a process that takes place independently of the main mitophagy receptors. Furthermore, we show that mitophagy function of AMBRA1 is post-translationally controlled, upon HUWE1 activity, by a positive phosphorylation on its serine 1014. This modification is mediated by the IKKα kinase and induces structural changes in AMBRA1, thus promoting its interaction with LC3/GABARAP (mATG8) proteins and its mitophagic activity. Altogether, these results demonstrate that AMBRA1 regulates mitophagy through a novel pathway, in which HUWE1 and IKKα are key factors, shedding new lights on the regulation of mitochondrial quality control and homoeostasis in mammalian cells.

An Efficient Method for Estimating the Hydrodynamic Radius of Disordered Protein Conformations.

Intrinsically disordered proteins play important roles throughout biology, yet our understanding of the relationship between their sequences, structural properties, and functions remains incomplete. The dynamic nature of these proteins, however, makes them difficult to characterize structurally. Many disordered proteins can attain both compact and expanded conformations, and the level of expansion may be regulated and important for function. Experimentally, the level of compaction and shape is often determined either by small-angle x-ray scattering experiments or pulsed-field-gradient NMR diffusion measurements, which provide ensemble-averaged estimates of the radius of gyration and hydrodynamic radius, respectively. Often, these experiments are interpreted using molecular simulations or are used to validate them. We here provide, to our knowledge, a new and efficient method to calculate the hydrodynamic radius of a disordered protein chain from a model of its structural ensemble. In particular, starting from basic concepts in polymer physics, we derive a relationship between the radius of gyration of a structure and its hydrodynamic ratio, which in turn can be used, for example, to compare a simulated ensemble of conformations to NMR diffusion measurements. The relationship may also be valuable when using NMR diffusion measurements to restrain molecular simulations.

The Mutational Landscape of the Oncogenic MZF1 SCAN Domain in Cancer.

SCAN domains in zinc-finger transcription factors are crucial mediators of protein-protein interactions. Up to 240 SCAN-domain encoding genes have been identified throughout the human genome. These include cancer-related genes, such as the myeloid zinc finger 1 (MZF1), an oncogenic transcription factor involved in the progression of many solid cancers. The mechanisms by which SCAN homo- and heterodimers assemble and how they alter the transcriptional activity of zinc-finger transcription factors in cancer and other diseases remain to be investigated. Here, we provide the first description of the conformational ensemble of the MZF1 SCAN domain cross-validated against NMR experimental data, which are probes of structure and dynamics on different timescales. We investigated the protein-protein interaction network of MZF1 and how it is perturbed in different cancer types by the analyses of high-throughput proteomics and RNASeq data. Collectively, we integrated many computational approaches, ranging from simple empirical energy functions to all-atom microsecond molecular dynamics simulations and network analyses to unravel the effects of cancer-related substitutions in relation to MZF1 structure and interactions.

A single mutation Gln142Lys doubles the catalytic activity of VPR, a cold adapted subtilisin-like serine proteinase.

Structural comparisons of the cold adapted subtilase VPR and its thermophilic homologue, aqualysin I (AQUI) indicated the presence of additional salt bridges in the latter. Few of those appear to contribute significantly to thermal stability of AQUI. This includes a putative salt bridge between residues Lys142 and Glu172 as its deletion did not have any significant effect on its stability or activity (Jónsdóttir et al. (2014)). Insertion of this putative salt bridge into the structure of VPR, in a double mutant (VPRΔC_Q142K/S172E), however was detrimental to the stability of the enzyme. Incorporation of either the Q142K or S172E mutations into VPR, were found to significantly affect the catalytic properties of the enzyme. The single mutation Q142K was highly effective, as it increased the kcat and kcat/Km more than twofold. When the Q142K mutation was inserted into a thermostabilized, but a low activity mutant of VPR (VPRΔC_N3P/I5P), the activity increased about tenfold in terms of kcat and kcat/Km, while retaining the stability of the mutant. Molecular dynamics simulations of the single mutants were carried out to provide structural rationale for these experimental observations. Based on root mean square fluctuation (RMSF) profiles, the two mutants were more flexible in certain regions of the structure and the Q142K mutant had the highest overall flexibility of the three enzymes. The results suggest that weakening of specific H-bonds resulting from the mutations may be propagated over some distance giving rise to higher flexibility in the active site regions of the enzyme, causing higher catalytic activity in the mutants.

A Residue Quartet in the Extracellular Domain of the Prolactin Receptor Selectively Controls Mitogen-activated Protein Kinase Signaling.

Cytokine receptors elicit several signaling pathways, but it is poorly understood how they select and discriminate between them. We have scrutinized the prolactin receptor as an archetype model of homodimeric cytokine receptors to address the role of the extracellular membrane proximal domain in signal transfer and pathway selection. Structure-guided manipulation of residues involved in the receptor dimerization interface identified one residue (position 170) that in cell-based assays profoundly altered pathway selectivity and species-specific bio-characteristics. Subsequent in vitro spectroscopic and nuclear magnetic resonance analyses revealed that this residue was part of a residue quartet responsible for specific local structural changes underlying these effects. This included alteration of a novel aromatic T-stack within the membrane proximal domain, which promoted selective signaling affecting primarily the MAPK (ERK1/2) pathway. Importantly, activation of the MAPK pathway correlated with in vitro stabilities of ternary ligand·receptor complexes, suggesting a threshold mean lifetime of the complex necessary to achieve maximal activation. No such dependence was observed for STAT5 signaling. Thus, this study establishes a residue quartet in the extracellular membrane proximal domain of homodimeric cytokine receptors as a key regulator of intracellular signaling discrimination.

Residue 146 regulates prolactin receptor folding, basal activity and ligand-responsiveness: potential implications in breast tumorigenesis.

PRLR(I146L) is the first identified gain-of-function variant of the prolactin receptor (PRLR) that was proposed to be associated with benign breast tumorigenesis. Structural investigations suggested this hydrophobic core position in the extracellular D2 domain to be linked to receptor dimerization. Here, we used a mutational approach to address how the conservative I-to-L substitution induced constitutive activity. Using cell-based assays of different I146-PRLR variants in combination with spectroscopic/nuclear magnetic resonance analyses we found that chemical manipulation of position 146 profoundly altered folding, PRL-responsiveness, and ligand-independent activity of the receptor in a mutation-specific manner. Together, these data further add to the critical role of position 146, showing it to also be crucial to structural integrity thereby imposing on the biological PRLR properties. When stably introduced in MCF-7 (luminal) and MDA-MB231 (mesenchymal) breast cancer cells, the most potent of the PRL-insensitive mutants (PRLR(I146D)) had minimal impact on cell proliferation and cell differentiation status.