Morphological profiling for drug discovery in the era of deep learning.

Morphological profiling is a valuable tool in phenotypic drug discovery. The advent of high-throughput automated imaging has enabled the capturing of a wide range of morphological features of cells or organisms in response to perturbations at the single-cell resolution. Concurrently, significant advances in machine learning and deep learning, especially in computer vision, have led to substantial improvements in analyzing large-scale high-content images at high throughput. These efforts have facilitated understanding of compound mechanism of action, drug repurposing, characterization of cell morphodynamics under perturbation, and ultimately contributing to the development of novel therapeutics. In this review, we provide a comprehensive overview of the recent advances in the field of morphological profiling. We summarize the image profiling analysis workflow, survey a broad spectrum of analysis strategies encompassing feature engineering- and deep learning-based approaches, and introduce publicly available benchmark datasets. We place a particular emphasis on the application of deep learning in this pipeline, covering cell segmentation, image representation learning, and multimodal learning. Additionally, we illuminate the application of morphological profiling in phenotypic drug discovery and highlight potential challenges and opportunities in this field.

Unraveling the Intertwining Factors Underlying the Assembly of High-Nuclearity Heterometallic Clusters.

Two closely related yet distinctly different cationic clusters, [Dy52Ni44(HEIDA)36(OH)138(OAc)24(H2O)30]10+ (1) and [Dy112Ni76(HEIDA)44(EIDA)24(IDA)4(OH)268(OAc)48(H2O)44]4+ (2) (HEIDA=N-(2-hydroxyethyl)iminodiacetate), each featuring a multi-shell core of Platonic and Archimedean polyhedra, were obtained. Depending on the specific conditions used for the co-hydrolysis of Dy3+ and Ni2+, the product can be crystallized out as one particular type of cluster or as a mixture of 1 and 2. How the reaction process was affected by the amount of hydrolysis-facilitating base and/or by the reaction temperature and duration was investigated. It has been found that a reaction at a high temperature and/or for an extended period favors the formation of the compact and thermodynamically more stable 1, while a brief reaction with a large amount of the base is good for the kinetic product 2. By tuning these intertwining conditions, the reaction can be regulated toward a particular product.

Engineered bacterial therapeutics for detecting and treating CRC.

Despite an overall decrease in occurrence, colorectal cancer (CRC) remains the third most common cause of cancer deaths in the USA. Detection of CRC is difficult in high-risk groups, including those with genetic predispositions, with disease traits, or from certain demographics. There is emerging interest in using engineered bacteria to identify early CRC development, monitor changes in the adenoma and CRC microenvironment, and prevent cancer progression. Novel genetic circuits for cancer therapeutics or functions to enhance existing treatment modalities have been tested and verified in vitro and in vivo. Inclusion of biocontainment measures would prepare strains to meet therapeutic standards. Thus, engineered bacteria present an opportunity for detection and treatment of CRC lesions in a highly sensitive and specific manner.

A Cubic Tinkertoy-like Heterometallic Cluster with a Record Magnetocaloric Effect.

Clusters showing a giant magnetocaloric effect (MCE) are of interest as molecular coolants for magnetic refrigeration. Herein, we report two heterometallic clusters, denoted as Gd152Ni14@Cl24 and Sm152Ni8, just to highlight their inorganic core motifs, obtained by ligand-controlled co-hydrolysis of Ni2+ and Ln3+ (Ln = Gd, Sm) in the presence of N-(2-hydroxyethyl)iminodiacetic acid (H2HEIDA). Both clusters display fascinating cubic Tinkertoy-like structures, with the core motifs being built of multiple metallic shells of Platonic and Archimedean polyhedra. The isothermal magnetic entropy change─a direct measurement of MCE─was determined to be 52.65 J·kg-1·K-1 at 2.5 K and 7.0 T for the Gd-containing cluster; this value is the highest known for any molecular clusters so far reported.

A tetravalent praseodymium complex with field-induced slow magnetic relaxation.

Herein the synthesis, structural characterization, and magnetic properties of a Pr(IV) complex [Pr(OSiPh3)4(L)] (1, L = 4,4'-dimethoxy-2,2'-bipyridine) are reported. The stability of the Pr(IV) complex significantly enhanced with the use of the bidentate ligand L. Slow magnetic relaxation was observed at low temperatures, indicating that the complex may be the first single-ion magnet with a tetravalent lanthanide ion being the magnetic center.

Biosynthesis of Dolastatin 10 in Marine Cyanobacteria, a Prototype for Multiple Approved Cancer Drugs.

Dolastatin 10, a potent tubulin-targeting marine anticancer natural product, provided the basis for the development of six FDA-approved antibody-drug conjugates. Through the screening of cyanobacterial Caldora penicillata environmental DNA libraries and metagenome sequencing, we identified its biosynthetic gene cluster. Functional prediction of 10 enzymes encoded in the 39 kb cluster supports the dolastatin 10 biosynthesis. The nonheme diiron monooxygenase DolJ was biochemically characterized to mediate the terminal thiazole formation in dolastatin 10.

Morphological Profiling for Drug Discovery in the Era of Deep Learning.

Morphological profiling is a valuable tool in phenotypic drug discovery. The advent of high-throughput automated imaging has enabled the capturing of a wide range of morphological features of cells or organisms in response to perturbations at the single-cell resolution. Concurrently, significant advances in machine learning and deep learning, especially in computer vision, have led to substantial improvements in analyzing large-scale high-content images at high-throughput. These efforts have facilitated understanding of compound mechanism-of-action (MOA), drug repurposing, characterization of cell morphodynamics under perturbation, and ultimately contributing to the development of novel therapeutics. In this review, we provide a comprehensive overview of the recent advances in the field of morphological profiling. We summarize the image profiling analysis workflow, survey a broad spectrum of analysis strategies encompassing feature engineering- and deep learning-based approaches, and introduce publicly available benchmark datasets. We place a particular emphasis on the application of deep learning in this pipeline, covering cell segmentation, image representation learning, and multimodal learning. Additionally, we illuminate the application of morphological profiling in phenotypic drug discovery and highlight potential challenges and opportunities in this field.

Substituent Positioning Effects on the Magnetic Properties of Sandwich-Type Erbium(III) Complexes with Bis(trimethylsilyl)-Substituted Cyclooctatetraenyl Ligands.

Lanthanide complexes with judiciously designed ligands have been extensively studied for their potential applications as single-molecule magnets. With the influence of ligands on their magnetic properties generally established, recent research has unearthed certain effects inherent to site differentiation due to the different types and varying numbers of substituents on the same ligand platform. Using two new sandwich-type Er(III) complexes with cyclooctatetraenyl (COT) ligands featuring two differently positioned trimethylsilyl (TMS) substituents, namely, [Li(DME)Er(COT1,5-TMS2)2]n (Er1) and [Na(DME)3][Er(COT1,3-TMS2)2] (Er2) [COT1,3-TMS2 and COT1,5-TMS2 donate 1,3- and 1,5-bis(trimethylsilyl)-substituted cyclooctatetraenyl ligands, respectively; DME = 1,2-dimethoxyethane], and with reference to previously reported [Li(DME)3][Er(COT1,4-TMS2)2] (A) and [K(DME)2][Er(COT1,4-TMS2)2] (B), any possible substituent position effects have been explored for the first time. The rearrangement of the TMS substituents from the starting COT1,4-TMS2 to COT1,3-TMS2 and COT1,5-TMS2, by way of formal migration of the TMS group, was thermally induced in the case of Er1, while for the formation of Er2, the use of Na+ in the placement of its Li+ and K+ congeners is essential. Both Er1 and Er2 display single-molecule magnetic behaviors with energy barriers of 170(3) and 172(6) K, respectively. Magnetic hysteresis loops, butterfly-shaped for Er1 and wide open for Er2, were observed up to 12 K for Er1 and 13 K for Er2. Studies of magnetic dynamics reveal the different pathways for relaxation of magnetization below 10 K, mainly by the Raman process for Er1 and by quantum tunneling of magnetization for Er2, leading to the order of magnitude difference in magnetic relaxation times and sharply different magnetic hysteresis loops.

Direct aromatic nitration by bacterial P450 enzymes.

Nitro aromatics have broad applications in industry, agriculture, and pharmaceutics. However, their industrial production is faced with many challenges including poor selectivity, heavy pollution and safety concerns. Nature provides multiple strategies for aromatic nitration, which opens the door for the development of green and efficient biocatalysts. Our group's efforts focused on a unique bacterial cytochrome P450 TxtE that originates from the biosynthetic pathway of phytotoxin thaxtomins, which can install a nitro group at C4 of l-Trp indole ring. TxtE is a Class I P450 and its reaction relies on a pair of redox partners ferredoxin and ferredoxin reductase for essential electron transfer. To develop TxtE as an efficient nitration biocatalyst, we created artificial self-sufficient P450 chimeras by fusing TxtE with the reductase domain of the bacterial P450BM3 (BM3R). We evaluated the catalytic performance of the chimeras with different lengths of the linker connecting TxtE and BM3R domains and identified one with a 14-amino-acid linker (TB14) to give the best activity. In addition, we demonstrated the broad substrate scope of the engineered biocatalyst by screening diverse l-Trp analogs. In this chapter, we provide a detailed procedure for the development of aromatic nitration biocatalysts, including the construction of P450 fusion chimeras, biochemical characterization, determination of catalytic parameters, and testing of enzyme-substrate scope. These protocols can be followed to engineer other P450 enzymes and illustrate the processes of biocatalytic development for the synthesis of nitro chemicals.

Recent progress in unraveling the biosynthesis of natural sunscreens mycosporine-like amino acids.

Exposure to ultraviolet (UV) rays is a known risk factor for skin cancer, which can be notably mitigated through the application of sun care products. However, escalating concerns regarding the adverse health and environmental impacts of synthetic anti-UV chemicals underscore a pressing need for the development of biodegradable and eco-friendly sunscreen ingredients. Mycosporine-like amino acids (MAAs) represent a family of water-soluble anti-UV natural products synthesized by various organisms. These compounds can provide a two-pronged strategy for sun protection as they not only exhibit a superior UV absorption profile but also possess the potential to alleviate UV-induced oxidative stresses. Nevertheless, the widespread incorporation of MAAs in sun protection products is hindered by supply constraints. Delving into the biosynthetic pathways of MAAs can offer innovative strategies to overcome this limitation. Here, we review recent progress in MAA biosynthesis, with an emphasis on key biosynthetic enzymes, including the dehydroquinate synthase homolog MysA, the adenosine triphosphate (ATP)-grasp ligases MysC and MysD, and the nonribosomal peptide synthetase (NRPS)-like enzyme MysE. Additionally, we discuss recently discovered MAA tailoring enzymes. The enhanced understanding of the MAA biosynthesis paves the way for not only facilitating the supply of MAA analogs but also for exploring the evolution of this unique family of natural sunscreens. ONE-SENTENCE SUMMARY: This review discusses the role of mycosporine-like amino acids (MAAs) as potent natural sunscreens and delves into recent progress in their biosynthesis.

Design, synthesis and evaluation of halogenated phenazine antibacterial prodrugs targeting nitroreductase enzymes for activation.

It is of great importance to develop new strategies to combat antibiotic resistance. Our lab has discovered halogenated phenazine (HP) analogues that are highly active against multidrug-resistant bacterial pathogens. Here, we report the design, synthesis, and study of a new series of nitroarene-based HP prodrugs that leverage intracellular nitroreductase (NTR) enzymes for activation and subsequent release of active HP agents. Our goals of developing HP prodrugs are to (1) mitigate off-target metal chelation (potential toxicity), (2) possess motifs to facilitate intracellular, bacterial-specific HP release, (3) improve water solubility, and (4) prevent undesirable metabolism (e.g., glucuronidation of HP's phenol). Following the synthesis of HP-nitroarene prodrugs bearing a sulfonate ester linker, NTR-promoted release experiments demonstrated prodrug HP-1-N released 70.1% of parent HP-1 after 16 hours (with only 6.8% HP-1 release without NTR). In analogous in vitro experiments, no HP release was observed for control sulfonate ester compounds lacking the critical nitro group. When compared to parent HP compounds, nitroarene prodrugs evaluated during these studies demonstrate similar antibacterial activities in MIC and zone of inhibition assays (against lab strains and clinical isolates). In conclusion, HP-nitroarene prodrugs could provide a future avenue to develop potent agents that target antibiotic resistant bacteria.

Electron Paramagnetic Resonance Spectra of Pentagonal Bipyramidal Gadolinium Complexes.

Gadolinium is a special case in spectroscopy because of the near isotropic nature of the 4f7 configuration of the +3 oxidation state. Gd3+ complexes have been studied in several symmetries to understand the underlying mechanisms of the ground state splitting. The abundance of information in Gd3+ spectra can be used as a probe for properties of the other rare earth ions in the same complexes. In this work, the zero-field splitting (ZFS) of a series of Gd3+ pentagonal bipyramidal complexes of the form [GdX1X2(Leq)5]n+ [n = 1, X = axial ligands: Cl-, -OtBu, -OArF5 or n = 3, X = tBuPO(NHiPr)2, Leq = equatorial ligand: Py, THF or H2O] with near fivefold symmetry axes along X1-Gd-X2 was investigated. The ZFS parameters were determined by fitting of room-temperature continuous wave electron paramagnetic resonance (EPR) spectra (at X-, K-, and Q-band) to a spin Hamiltonian incorporating extended Stevens operators compatible with C5 symmetry. Examination of the acquired parameters led to the conclusion that the ZFS is dominated by the B20 term and that the magnitude of B20 is almost entirely dependent on, and inversely proportional to, the donor strength of the axial ligands. Surveying the continuous shape measure and the X1-Gd-X2 angle of the complexes showed that there is some correlation between the proximity of each complex to D5h symmetry and the magnitude of the B65 parameter, but that the deformation of the X1-Gd-X2 angle is more significant than other distortions. Finally, the magnitude of B20 was found to be inversely proportional to the thermal barrier for the reversal of the magnetic moment (Ueff) of the corresponding isostructural Dy3+ complexes.

Chemical and genomic characterization of a potential probiotic treatment for stony coral tissue loss disease.

Considered one of the most devastating coral disease outbreaks in history, stony coral tissue loss disease (SCTLD) is currently spreading throughout Florida's coral reefs and the greater Caribbean. SCTLD affects at least two dozen different coral species and has been implicated in extensive losses of coral cover. Here we show Pseudoalteromonas sp. strain McH1-7 has broad-spectrum antibacterial activity against SCTLD-associated bacterial isolates. Chemical analyses indicated McH1-7 produces at least two potential antibacterials, korormicin and tetrabromopyrrole, while genomic analysis identified the genes potentially encoding an L-amino acid oxidase and multiple antibacterial metalloproteases (pseudoalterins). During laboratory trials, McH1-7 arrested or slowed disease progression on 68.2% of diseased Montastraea cavernosa fragments treated (n = 22), and it prevented disease transmission by 100% (n = 12). McH1-7 is the most chemically characterized coral probiotic that is an effective prophylactic and direct treatment for the destructive SCTLD as well as a potential alternative to antibiotic use.

Determinative Effect of Axial Linearity on Single-Molecule Magnet Performance in Dinuclear Dysprosium Complexes.

Two dichloride-bridged dinuclear dysprosium(III) complexes based on salen ligands, namely, [Dy(L1 )(µ-Cl)(thf)]2 (1; H2 L1 =N,N'-bis(3,5-di-tert-butylsalicylidene)phenylenediamine) and [Dy2 (L2 )2 (µ-Cl)2 (thf)2 ]2 (2; H2 L2 =N,N'-bis(3,5-di-tert-butylsalicylidene)ethylenediamine) are reported. These two complexes have two short Dy-O(PhO) bonds that exhibit angles of ∼90° for 1 and ∼143° for 2, leading to clear slow relaxation of the magnetization for 2 and not for 1. Compound 2 has a near-identical core to the recently reported compound [Dy2 (L3 )2 (µ-Cl)2 (thf)2 ] (3; H2 L3 =N-(2-pyridylmethyl)-N,N-bis(2'-hydroxy-3',5'-di-tert-butylbenzyl)amine). The only substantial difference is the relative angle of the two O(PhO) -Dy-O(PhO) vectors, which is collinear in 2 owing to inversion symmetry and ∼68° in 3 due to a molecular C2 axis. It is shown that this subtle structural difference leads to large differences in the dipolar ground states, giving rise to open magnetic hysteresis for 3 and not for 2.

Design, Synthesis, and Evaluation of Carbonate-Linked Halogenated Phenazine-Quinone Prodrugs with Improved Water-Solubility and Potent Antibacterial Profiles.

Pathogenic bacteria have devastating impacts on human health as a result of acquired antibiotic resistance and innate tolerance. Every class of our current antibiotic arsenal was initially discovered as growth-inhibiting agents that target actively replicating (individual, free-floating) planktonic bacteria. Bacteria are notorious for utilizing a diversity of resistance mechanisms to overcome the action of conventional antibiotic therapies and forming surface-attached biofilm communities enriched in (non-replicating) persister cells. To address problems associated with pathogenic bacteria, our group is developing halogenated phenazine (HP) molecules that demonstrate potent antibacterial and biofilm-eradicating activities through a unique iron starvation mode of action. In this study, we designed, synthesized, and investigated a focused collection of carbonate-linked HP prodrugs bearing a quinone trigger to target the reductive cytoplasm of bacteria for bioactivation and subsequent HP release. The quinone moiety also contains a polyethylene glycol group, which dramatically enhances the water-solubility properties of the HP-quinone prodrugs reported herein. We found carbonate-linked HP-quinone prodrugs 11, 21-23 to demonstrate good linker stability, rapid release of the active HP warhead following dithiothreitol (reductive) treatment, and potent antibacterial activities against methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus epidermidis, and Enterococcus faecalis. In addition, HP-quinone prodrug 21 induced rapid iron starvation in MRSA and S. epidermidis biofilms, illustrating prodrug action within these surface-attached communities. Overall, we are highly encouraged by these findings and believe that HP prodrugs have the potential to address antibiotic resistant and tolerant bacterial infections.

Heterologous Production of the C33-C45 Polyketide Fragment of Anticancer Apratoxins in a Cyanobacterial Host.

A polyketide synthase subcluster of cytotoxic apratoxin A was isolated from a Moorena bouillonii environmental DNA library and engineered with a thioesterase II domain for heterologous expression in the filamentous cyanobacterium Anabaena sp. PCC7120. Further engineering with a rhamnose-inducible promoter led to the production of (2R,3R,5R,7R)-3,7-dihydroxy-2,5,8,8-tetramethylnonanoic acid, a stereogenically rich chiral building block that is important to the efficient synthesis of apratoxin analogues, representing the first synthetic biology attempt for this type of polyketide fragment.

Cryptic Diversity of Black Band Disease Cyanobacteria in Siderastrea siderea Corals Revealed by Chemical Ecology and Comparative Genome-Resolved Metagenomics.

Black band disease is a globally distributed and easily recognizable coral disease. Despite years of study, the etiology of this coral disease, which impacts dozens of stony coral species, is not completely understood. Although black band disease mats are predominantly composed of the cyanobacterial species Roseofilum reptotaenium, other filamentous cyanobacterial strains and bacterial heterotrophs are readily detected. Through chemical ecology and metagenomic sequencing, we uncovered cryptic strains of Roseofilum species from Siderastrea siderea corals that differ from those on other corals in the Caribbean and Pacific. Isolation of metabolites from Siderastrea-derived Roseofilum revealed the prevalence of unique forms of looekeyolides, distinct from previously characterized Roseofilum reptotaenium strains. In addition, comparative genomics of Roseofilum strains showed that only Siderastrea-based Roseofilum strains have the genetic capacity to produce lasso peptides, a family of compounds with diverse biological activity. All nine Roseofilum strains examined here shared the genetic capacity to produce looekeyolides and malyngamides, suggesting these compounds support the ecology of this genus. Similar biosynthetic gene clusters are not found in other cyanobacterial genera associated with black band disease, which may suggest that looekeyolides and malyngamides contribute to disease etiology through yet unknown mechanisms.

Carbohydrate-Small Molecule Hybrids as Lead Compounds Targeting IL-6 Signaling.

In the past 25 years, a number of efforts have been made toward the development of small molecule interleukin-6 (IL-6) signaling inhibitors, but none have been approved to date. Monosaccharides are a diverse class of bioactive compounds, but thus far have been unexplored as a scaffold for small molecule IL-6-signaling inhibitor design. Therefore, in this present communication, we combined a structure-based drug design approach with carbohydrate building blocks to design and synthesize novel IL-6-signaling inhibitors targeting glycoprotein 130 (gp130). Of this series of compounds, LS-TG-2P and LS-TF-3P were the top lead compounds, displaying IC50 values of 6.9 and 16 µM against SUM159 cell lines, respectively, while still retaining preferential activity against the IL-6-signaling pathway. The carbohydrate moiety was found to improve activity, as N-unsubstituted triazole analogues of these compounds were found to be less active in vitro compared to the leads themselves. Thus, LS-TG-2P and LS-TF-3P are promising scaffolds for further development and study as IL-6-signaling inhibitors.

Biosynthesis of Lyngbyastatins 1 and 3, Cytotoxic Depsipeptides from an Okeania sp. Marine Cyanobacterium.

Lyngbyastatins (Lbns) 1 (1) and 3 (2) belong to a group of cyclic depsipeptides that inhibit cancer cell proliferation. These compounds have been isolated from different marine cyanobacterial collections, while further development of these compounds relies on their lengthy total synthesis. Biosynthetic studies of these compounds can provide viable strategies to access these compounds and develop new analogs. In this study, we report the identification and characterization of one Lbn biosynthetic gene cluster (BGC) from the marine cyanobacterium Okeania sp. VPG18-21. We initially identified 1 and 2 in the organic extract by mass spectrometry and performed the targeted isolation of these compounds, which feature a (2S,3R)-3-amino-2-methylpentanoic acid (MAP) and a (2S,3R)-3-amino-2-methylhexanoic acid (Amha) moiety, respectively. Parallel metagenomic sequencing of VPG18-21 led to the identification of a putative Lbn BGC that encodes six megaenzymes (LbnA-F), including one polyketide synthase (PKS, LbnE), four nonribosomal peptide synthetases (NRPSs, LbnB-D and -F), and one PKS-NRPS hybrid (LbnA). Bioinformatic analysis of these enzymes suggested that the BGC produces 1 and 2. Furthermore, our biochemical studies of three recombinant adenylation domains uncovered their substrate specificities, supporting the identity of the BGC. Finally, we identified near-complete Lbn-like BGCs in the genomes of two other marine cyanobacteria.

A roadmap for the functional annotation of protein families: a community perspective.

Over the last 25 years, biology has entered the genomic era and is becoming a science of 'big data'. Most interpretations of genomic analyses rely on accurate functional annotations of the proteins encoded by more than 500 000 genomes sequenced to date. By different estimates, only half the predicted sequenced proteins carry an accurate functional annotation, and this percentage varies drastically between different organismal lineages. Such a large gap in knowledge hampers all aspects of biological enterprise and, thereby, is standing in the way of genomic biology reaching its full potential. A brainstorming meeting to address this issue funded by the National Science Foundation was held during 3-4 February 2022. Bringing together data scientists, biocurators, computational biologists and experimentalists within the same venue allowed for a comprehensive assessment of the current state of functional annotations of protein families. Further, major issues that were obstructing the field were identified and discussed, which ultimately allowed for the proposal of solutions on how to move forward.