A novel antibiotic class targeting the lipopolysaccharide transporter.

Carbapenem-resistant Acinetobacter baumannii (CRAB) has emerged as a major global pathogen with limited treatment options1. No new antibiotic chemical class with activity against A. baumannii has reached patients in over 50 years1. Here we report the identification and optimization of tethered macrocyclic peptide (MCP) antibiotics with potent antibacterial activity against CRAB. The mechanism of action of this molecule class involves blocking the transport of bacterial lipopolysaccharide from the inner membrane to its destination on the outer membrane, through inhibition of the LptB2FGC complex. A clinical candidate derived from the MCP class, zosurabalpin (RG6006), effectively treats highly drug-resistant contemporary isolates of CRAB both in vitro and in mouse models of infection, overcoming existing antibiotic resistance mechanisms. This chemical class represents a promising treatment paradigm for patients with invasive infections due to CRAB, for whom current treatment options are inadequate, and additionally identifies LptB2FGC as a tractable target for antimicrobial drug development.

Leveraging Cell Painting Images to Expand the Applicability Domain and Actively Improve Deep Learning Quantitative Structure-Activity Relationship Models.

The search for chemical hit material is a lengthy and increasingly expensive drug discovery process. To improve it, ligand-based quantitative structure-activity relationship models have been broadly applied to optimize primary and secondary compound properties. Although these models can be deployed as early as the stage of molecule design, they have a limited applicability domain─if the structures of interest differ substantially from the chemical space on which the model was trained, a reliable prediction will not be possible. Image-informed ligand-based models partly solve this shortcoming by focusing on the phenotype of a cell caused by small molecules, rather than on their structure. While this enables chemical diversity expansion, it limits the application to compounds physically available and imaged. Here, we employ an active learning approach to capitalize on both of these methods' strengths and boost the model performance of a mitochondrial toxicity assay (Glu/Gal). Specifically, we used a phenotypic Cell Painting screen to build a chemistry-independent model and adopted the results as the main factor in selecting compounds for experimental testing. With the additional Glu/Gal annotation for selected compounds we were able to dramatically improve the chemistry-informed ligand-based model with respect to the increased recognition of compounds from a 10% broader chemical space.

Pharmacological Enhancement of Adult Hippocampal Neurogenesis Improves Behavioral Pattern Separation in Young and Aged Mice.

BACKGROUND: Impairments in behavioral pattern separation (BPS)-the ability to distinguish between similar contexts or experiences-contribute to memory interference and overgeneralization seen in many neuropsychiatric conditions, including depression, anxiety, PTSD, dementia, and age-related cognitive decline. While BPS relies on the dentate gyrus and is sensitive to changes in adult hippocampal neurogenesis (AHN), its significance as a pharmacological target has not been tested. METHODS: In this study, we applied a human neural stem cell high-throughput screening cascade to identify compounds that increase human neurogenesis. One compound with a favorable profile, RO6871135, was then tested in BPS in mice. RESULTS: Chronic treatment with RO6871135, 7.5 mg/kg increased AHN and improved BPS in a fear discrimination task in both young and aged mice. RO6871135 treatment also lowered innate anxiety-like behavior, which was more apparent in mice exposed to chronic corticosterone. Ablation of AHN by hippocampal irradiation supported a neurogenesis-dependent mechanism for RO6871135-induced improvements in BPS. To identify possible mechanisms of action, in vitro and in vivo kinase inhibition and chemical proteomics assays were performed. These tests indicated that RO6871135 inhibited CDK8, CDK11, CaMK2a, CaMK2b, MAP2K6, and GSK3b. An analog compound also demonstrated high affinity for CDK8, CaMK2a, and GSK3b. CONCLUSIONS: These studies demonstrate a method for empirical identification and preclinical testing of novel neurogenic compounds that can improve BPS, and points to possible novel mechanisms that can be interrogated for the development of new therapies to improve specific endophenotypes such as impaired BPS.

A random motility assay based on image correlation spectroscopy.

We demonstrate the random motility (RAMOT) assay based on image correlation spectroscopy for the automated, label-free, high-throughput characterization of random cell migration. The approach is complementary to traditional migration assays, which determine only the collective net motility in a particular direction. The RAMOT assay is less demanding on image quality compared to single-cell tracking, does not require cell identification or trajectory reconstruction, and performs well on live-cell, time-lapse, phase contrast video microscopy of hundreds of cells in parallel. Effective diffusion coefficients derived from the RAMOT analysis are in quantitative agreement with Monte Carlo simulations and allowed for the detection of pharmacological effects on macrophage-like cells migrating on a planar collagen matrix. These results expand the application range of image correlation spectroscopy to multicellular systems and demonstrate a novel, to our knowledge, migration assay with little preparative effort.

Machine learning-powered antibiotics phenotypic drug discovery.

Identification of novel antibiotics remains a major challenge for drug discovery. The present study explores use of phenotypic readouts beyond classical antibacterial growth inhibition adopting a combined multiparametric high content screening and genomic approach. Deployment of the semi-automated bacterial phenotypic fingerprint (BPF) profiling platform in conjunction with a machine learning-powered dataset analysis, effectively allowed us to narrow down, compare and predict compound mode of action (MoA). The method identifies weak antibacterial hits allowing full exploitation of low potency hits frequently discovered by routine antibacterial screening. We demonstrate that BPF classification tool can be successfully used to guide chemical structure activity relationship optimization, enabling antibiotic development and that this approach can be fruitfully applied across species. The BPF classification tool could be potentially applied in primary screening, effectively enabling identification of novel antibacterial compound hits and differentiating their MoA, hence widening the known antibacterial chemical space of existing pharmaceutical compound libraries. More generally, beyond the specific objective of the present work, the proposed approach could be profitably applied to a broader range of diseases amenable to phenotypic drug discovery.

Molecular Phenotyping Combines Molecular Information, Biological Relevance, and Patient Data to Improve Productivity of Early Drug Discovery.

Today, novel therapeutics are identified in an environment which is intrinsically different from the clinical context in which they are ultimately evaluated. Using molecular phenotyping and an in vitro model of diabetic cardiomyopathy, we show that by quantifying pathway reporter gene expression, molecular phenotyping can cluster compounds based on pathway profiles and dissect associations between pathway activities and disease phenotypes simultaneously. Molecular phenotyping was applicable to compounds with a range of binding specificities and triaged false positives derived from high-content screening assays. The technique identified a class of calcium-signaling modulators that can reverse disease-regulated pathways and phenotypes, which was validated by structurally distinct compounds of relevant classes. Our results advocate for application of molecular phenotyping in early drug discovery, promoting biological relevance as a key selection criterion early in the drug development cascade.

mTORC1 Inhibition Corrects Neurodevelopmental and Synaptic Alterations in a Human Stem Cell Model of Tuberous Sclerosis.

Hyperfunction of the mTORC1 pathway has been associated with idiopathic and syndromic forms of autism spectrum disorder (ASD), including tuberous sclerosis, caused by loss of either TSC1 or TSC2. It remains largely unknown how developmental processes and biochemical signaling affected by mTORC1 dysregulation contribute to human neuronal dysfunction. Here, we have characterized multiple stages of neurogenesis and synapse formation in human neurons derived from TSC2-deleted pluripotent stem cells. Homozygous TSC2 deletion causes severe developmental abnormalities that recapitulate pathological hallmarks of cortical malformations in patients. Both TSC2(+/-) and TSC2(-/-) neurons display altered synaptic transmission paralleled by molecular changes in pathways associated with autism, suggesting the convergence of pathological mechanisms in ASD. Pharmacological inhibition of mTORC1 corrects developmental abnormalities and synaptic dysfunction during independent developmental stages. Our results uncouple stage-specific roles of mTORC1 in human neuronal development and contribute to a better understanding of the onset of neuronal pathophysiology in tuberous sclerosis.

White-to-brown metabolic conversion of human adipocytes by JAK inhibition.

The rising incidence of obesity and related disorders such as diabetes and heart disease has focused considerable attention on the discovery of new therapeutics. One promising approach has been to increase the number or activity of brown-like adipocytes in white adipose depots, as this has been shown to prevent diet-induced obesity and reduce the incidence and severity of type 2 diabetes. Thus, the conversion of fat-storing cells into metabolically active thermogenic cells has become an appealing therapeutic strategy to combat obesity. Here, we report a screening platform for the identification of small molecules capable of promoting a white-to-brown metabolic conversion in human adipocytes. We identified two inhibitors of Janus kinase (JAK) activity with no precedent in adipose tissue biology that stably confer brown-like metabolic activity to white adipocytes. Importantly, these metabolically converted adipocytes exhibit elevated UCP1 expression and increased mitochondrial activity. We further found that repression of interferon signalling and activation of hedgehog signalling in JAK-inactivated adipocytes contributes to the metabolic conversion observed in these cells. Our findings highlight a previously unknown role for the JAK-STAT pathway in the control of adipocyte function and establish a platform to identify compounds for the treatment of obesity.

Chemical conversion of human fibroblasts into functional Schwann cells.

Direct transdifferentiation of somatic cells is a promising approach to obtain patient-specific cells for numerous applications. However, conversion across germ-layer borders often requires ectopic gene expression with unpredictable side effects. Here, we present a gene-free approach that allows efficient conversion of human fibroblasts via a transient progenitor stage into Schwann cells, the major glial cell type of peripheral nerves. Using a multikinase inhibitor, we transdifferentiated fibroblasts into transient neural precursors that were subsequently further differentiated into Schwann cells. The resulting induced Schwann cells (iSCs) expressed numerous Schwann cell-specific proteins and displayed neurosupportive and myelination capacity in vitro. Thus, we established a strategy to obtain mature Schwann cells from human postnatal fibroblasts under chemically defined conditions without the introduction of ectopic genes.

Effect of GLP1R agonists taspoglutide and liraglutide on primary thyroid C-cells from rodent and man.

Glucagon-like peptide 1 (GLP1) analogs have been associated with an increased incidence of thyroid C-cell hyperplasia and tumors in rodents. This effect may be due to a GLP1 receptor (GLP1R)-dependent mechanism. As the expression of GLP1R is much lower in primates than in rodents, the described C-cell proliferative lesions may not be relevant to man. Here, we aimed to establish primary thyroid cell cultures of rat and human to evaluate the expression and function of GLP1R in C-cells. In our experiments, GLP1R expression was observed in primary rat C-cells (in situ hybridization) but was not detected in primary human C-cells (mRNA and protein levels). The functional response of the cultures to the stimulation with GLP1R agonists is an indirect measure of the presence of functional receptor. Liraglutide and taspoglutide elicited a modest increase in calcitonin release and in calcitonin expression in rat primary thyroid cultures. Contrarily, no functional response to GLP1R agonists was observed in human thyroid cultures, despite the presence of few calcitonin-positive C-cells. Thus, the lack of functional response of the human cultures adds to the weight of evidence indicating that healthy human C-cells have very low levels or completely lack GLP1R. In summary, our results support the hypothesis that the GLP1R agonist-induced C-cell responses in rodents may not be relevant to primates. In addition, the established cell culture method represents a useful tool to study the physiological and/or pathological roles of GLP1 and GLP1R agonists on normal, non-transformed primary C-cells from rats and man.

Topological analysis of the complex formed between neurokinin A and the NK2 tachykinin receptor.

Neurokinin A stimulates physiological responses in the peripheral and central nervous systems upon interacting primarily with the tachykinin NK2 receptor (NK2R). In this study, the structure of NKA bound to the NK2R is characterised by use of fluorescence resonance energy transfer. Four fluorescent NKA analogues with Texas red introduced at amino acid positions 1, 4, 7 and 10 were prepared. When bound to a NK2R carrying enhanced green fluorescent protein at the N-terminus, all peptides reduce green fluorescent protein fluorescence from 10% to 50% due to energy transfer. The derived donor-acceptor distances are 46, 55, 59 and 69 A for the fluorophore linked to positions 1-10, respectively. The monotonic increase in distance clearly indicates that the peptide adopts an extended structure when bound to its receptor. The present data are used, in combination with rhodopsin structure, fluorescence studies, photoaffinity labelling and site-directed mutagenesis data to design a computer model of the NKA-NK2R complex. We propose that the N-terminus of NKA is exposed and accessible to the extracellular medium. Subsequent amino acids of the NKA peptide become progressively more buried residues up to approximately one-third of the transmembrane-spanning domain.

Identification of the extracellular loop 2 as the point of interaction between the N terminus of the chemokine MIP-1alpha and its CCR1 receptor.

Macrophage inflammatory peptide-1alpha (MIP-1alpha)/CC-chemokine receptor ligand 3 is an 8-kDa peptide that induces chemotaxis of various lymphocytes to sites of inflammation through interaction with the G protein-coupled chemokine receptors CCR1 and CCR5. We recently described the preparation of a photoactivatable derivative of MIP-1alpha labeled with a benzophenone group at the extreme N-terminal end, which is a determinant for the agonist character of chemokines. Benzophenone-MIP-1alpha is a full agonist that specifically and covalently labels CCR1 and CCR5 receptors upon irradiation. In the present study, we use enzymatic and chemical cleavage methods on wild-type and mutated CCR1 receptors to show that the N terminus of the chemokine MIP-1alpha interacts in a specific manner with the second extracellular loop of the CCR1 receptor, within a segment comprising amino acids 178 to 194. This is the first report on the direct identification of a contact point between the N terminus of a chemokine and its membrane-bound receptor. The work shows that the part of chemokines that is endowed with agonist properties interacts with extracellular parts of the receptor rather than the transmembrane core of the protein.