Permeation Of Small Molecules Research Articles

DNA-Origami-Armored DNA Condensates.

DNA condensates, formed by liquid-liquid phase separation (LLPS), emerge as promising soft matter assemblies for creating artificial cells. The advantages of DNA condensates are their molecular permeability through the surface due to their membrane-less structure and their fluidic property. However, they face challenges in the design of their surface, e. g., unintended fusion and less regulation of permeable molecules. Addressing them, we report surface modification of DNA condensates with DNA origami nanoparticles, employing a Pickering-emulsion strategy. We successfully constructed core-shell structures with DNA origami coatings on DNA condensates and further enhanced the condensate stability toward fusion via connecting DNA origamis by responding to DNA input strands. The 'armoring' prevented the fusion of DNA condensates, enabling the formation of multicellular-like structures of DNA condensates. Moreover, the permeability was altered through the state change from coating to armoring the DNA condensates. The armored DNA condensates have significant potential for constructing artificial cells, offering increased surface stability and selective permeability for small molecules while maintaining compartmentalized space and multicellular organization.

Comparative analysis of classic network vs. nanogel junction network in konjac glucomannan/kappa carrageenan hybrid hydrogels

The three-dimensional network architecture of hydrogels significantly influences their mechanical and physical properties; therefore, understanding them is essential for designing optimized hydrogel-based biomaterials. This study presents a comparative analysis of two hybrid hydrogels composed of konjac glucomannan (KGM) and kappa carrageenan (KCAR) with the same stiffness (5.2–5.7 kPa and 1.6–1.7 kPa) thus similar cross-linking density but different network architectures: a classic network formed by extended polysaccharide interactions and a nanogel junction network where nanoscale cross-linked KCAR (KCAR-NGs) links KGM chains. The mechanical behavior, dissolution, and diffusion characteristics were examined, revealing that the classic network demonstrates superior tensile resistance, elongation, and solvent-induced swelling resistance, leading to slower dissolution rates and higher viscosity. Conversely, the nanogel junction network offers higher permeability for small molecules and faster dissolution, suggesting a more open network structure. These findings highlight the nanogel-based hydrogels' advantages for biomedical applications requiring stability, permeability, and rapid dissolution without high temperatures or chelating agents. This study underscores the potential of nanogel junction networks to balance hydrogel stiffness and permeability, advancing the design of hydrogel-based biomaterials.

Prediction of Mycobacterium tuberculosis cell wall permeability using machine learning methods.

Tuberculosis (TB) caused by the bacteria Mycobacterium tuberculosis (M. tb), continues to pose a significant worldwide health threat. The advent of drug-resistant strains of the disease highlights the critical need for novel treatments. The unique cell wall of M. tb provides an extra layer of protection for the bacteria and hence only compounds that can penetrate this barrier can reach their targets within the bacterial cell wall. The creation of a reliable machine learning (ML) model to predict the mycobacterial cell wall permeability of small molecules is presented in this work and four ML algorithms, including Random Forest, Support Vector Machines (SVM), k-nearest Neighbour (k-NN) and Logistic Regression were trained on a dataset of 5368 compounds. RDKit and Mordred toolkits were used to calculate features. To determine the most effective model, various performance metrics were used such as accuracy, precision, recall, F1 score, and area under the receiver operating characteristic curve. The best-performing model was further refined with hyperparameter tuning and tenfold cross-validation. The SVM model with filtering outperformed the other machine learning models and demonstrated 80.26% and 81.13% accuracy on the test and validation datasets, respectively. The study also provided insights into the molecular descriptors that play the most important role in predicting the ability of a molecule to pass the M. tb cell wall, which could guide future compound design. The model is available at https://github.com/PGlab-NIPER/MTB_Permeability .

Stability of Immobilized Chemosensor‐Filled Vesicles on Anti‐Fouling Polymer Brush Surfaces

AbstractThe characterization of phospholipid membrane permeability for small molecules is crucial for many applications in drug discovery and biomedical research in general. Here, chemosensor‐laden vesicles offer an attractive platform for permeability assays. In this work, the stability of immobilized chemosensor‐filled vesicles is explored on anti‐fouling polymer brush surfaces for potential use in monitoring small molecule membrane permeability. The study focuses on the development of a method for immobilizing sensor‐loaded vesicles into arbitrary patterns on surfaces and characterizing their stability under changing temperatures and compositions. As substrate to enable intact, long‐term stable vesicle immobilization reactive polymer brushes with anti‐fouling properties are used. Utilizing microchannel cantilever spotting, biotin moieties are introduced on the polymer brush surface, enabling stable tethering of vesicles through streptavidin‐biotin interactions. The immobilized vesicles are monitored through fluorescence microscopy for their response to analytes under changing environmental parameters and vesicle composition. A higher stability of immobilized vesicles compared to free‐floating ones is observed, with permeability only at elevated temperatures. By tuning vesicle compositions, permeability at lower temperatures can be raised again. Overall, the study provides insights into a novel approach for vesicle immobilization, showcasing the potential of surface‐bound vesicles for applications in microfluidic systems and as biosensors in various assays.

Liposomal Copermeation Assay Reveals Unexpected Membrane Interactions of Commonly Prescribed Drugs.

The permeation of small molecules across biological membranes is a crucial process that lies at the heart of life. Permeation is involved not only in the maintenance of homeostasis at the cell level but also in the absorption and biodistribution of pharmacologically active substances throughout the human body. Membranes are formed by phospholipid bilayers that represent an energy barrier for permeating molecules. Crossing this energy barrier is assumed to be a singular event, and permeation has traditionally been described as a first-order kinetic process, proportional only to the concentration gradient of the permeating substance. For a given membrane composition, permeability was believed to be a unique property dependent only on the permeating molecule itself. We provide experimental evidence that this long-held view might not be entirely correct. Liposomes were used in copermeation experiments with a fluorescent probe, where simultaneous permeation of two substances occurred over a single phospholipid bilayer. Using an assay of six commonly prescribed drugs, we have found that the presence of a copermeant can either enhance or suppress the permeation rate of the probe molecule, often more than 2-fold in each direction. This can have significant consequences for the pharmacokinetics and bioavailability of commonly prescribed drugs when used in combination and provide new insight into so-far unexplained drug-drug interactions as well as changing the perspective on how new drug candidates are evaluated and tested.

Two Toxoplasma gondii putative pore-forming proteins, GRA47 and GRA72, influence small molecule permeability of the parasitophorous vacuole.

Toxoplasma gondii is a parasite that poses significant health risks to those with impaired immunity. It replicates inside host cells shielded by the PVM, which controls nutrient and waste exchange with the host. GRA72, previously identified as essential in the absence of the GRA17 nutrient channel, is implicated in forming an alternative nutrient channel. Here we found that GRA47 associates with GRA72 and is also important for the PVM's permeability to small molecules. Removal of GRA47 leads to distorted vacuoles and impairs small molecule transport across the PVM, resembling the effects of GRA17 and GRA72 deletions. Structural models suggest GRA47 and GRA72 form distinct pore structures, with a pore-lining histidine critical to their function. Toxoplasma strains lacking GRA47 or those with a histidine mutation have impaired growth and reduced virulence in mice, highlighting these proteins as potential targets for new treatments against toxoplasmosis.

Microbeam Radiation Therapy Opens a Several Days’ Vessel Permeability Window for Small Molecules in Brain Tumor Vessels

PurposeSynchrotron microbeam radiation therapy (MRT), based on an inhomogeneous geometric and microscopic irradiation pattern of the tissues with high dose and high dose rate X-rays, enhances the permeability of brain tumor vessels. This study attempts to determine time and size range of the permeability window induced by MRT in the blood brain (tumor) barrier. Methods and MaterialsRats-bearing 9L gliomas were exposed to MRT, either unidirectional (tumor dose 406 Gy), or bidirectional (crossfired) (2 × 203 Gy). We measured vessel permeability to molecules of 3 sizes (Gd-DOTA, Dotarem®, 0.56 kDa; gadolinium-labeled albumin, ∼74 kDa; gadolinium-labeled IgG, 160 kDa) by daily in vivo magnetic resonance imaging (MRI), from 1 day before to 10 days post-irradiation. ResultsAn equivalent tumor dose of bidirectional MRT delivered from two orthogonal directions increased tumor vessel permeability for the smallest molecule tested more effectively than unidirectional MRT. Bidirectional MRT also affected the permeability of normal contralateral vessels to a different extent than unidirectional MRT. Conversely, bidirectional MRT did not modify the permeability of normal or tumor vessels for both larger molecules (74 and 160 kDa). ConclusionsHigh-dose bidirectional (cross-fired) MRT induced a significant increase in tumor vessel permeability for small molecules between the first and the seventh day after irradiation, while permeability of vessels in normal brain tissue remained stable. Such a permeability window could facilitate an efficient and safe delivery of intravenous small molecules (≤0.56 kDa) to tumoral tissues. A permeability window was not achieved by molecules larger than gado-grafted albumin (74 kDa). Vascular permeability for molecules between these two sizes has not been determined.

In Silico Study on the Contribution of the Follicular Route to Dermal Permeability of Small Molecules

PurposeThis study investigates in silico the contribution of the hair follicle to the overall dermal permeability of small molecules, as published experimental work provides inconclusive information on whether the follicular route favours the permeation of hydrophobic or hydrophilic permeants.MethodA study is conducted varying physico-chemical parameters of permeants such as lipophilicity, molecular weight and protein binding. The simulated data is compared to published experimental data to discuss how those properties can modulate the contribution of the hair follicle to the overall dermal permeation.ResultsThe results indicate that the contribution of the follicular route to dermal permeation can range from negligible to notable depending on the combination of lipophilic/hydrophilic properties of the substance filling the follicular route and the permeant.ConclusionCharacterisation of the substance filling the follicular route is required for analysing the experimental data of dermal permeation of small molecules, as changes between in vivo and in vitro due to handling of samples and cessation of vital functions can modify the contribution of the follicular route to overall dermal permeation, hence hindering data interpretation.

Pericytes Enrich the Basement Membrane and Reduce Neutrophil Transmigration in an InVitro Model of Peripheral Inflammation at the Blood-Brain Barrier.

Sepsis is the most lethal and expensive condition treated in intensive care units. Sepsis survivors frequently suffer long-term cognitive impairment, which has been linked to the breakdown of the blood-brain barrier (BBB) during a sepsis-associated "cytokine storm". Because animal models poorly recapitulate sepsis pathophysiology, human models are needed to understand sepsis-associated brain injury and to develop novel therapeutic strategies. With the concurrent emergence of tissue chip technologies and the maturation of protocols for human induced pluripotent stem cell (hiPSC), we can now develop advanced invitro models of the human BBB and immune system to understand the relationship between systemic inflammation and brain injury. Here, we present a BBB model of the primary barrier developed on the μSiM (microphysiological system enabled by an ultrathin silicon nanomembrane) tissue chip platform. The model features isogenically matched hiPSC-derived extended endothelial culture method brain microvascular endothelial cell-like cells (EECM-BMEC-like cells) and brain pericyte-like cells (BPLCs) in a back-to-back coculture separated by the ultrathin (100 nm) membrane. Both endothelial monocultures and cocultures with pericytes responded to sepsis-like stimuli, with increased small-molecule permeability, although no differences were detected between culture conditions. Conversely, BPLC coculture reduced the number of neutrophils that crossed the EECM-BMEC-like cell monolayer under sepsis-like stimulation. Interestingly, this barrier protection was not seen when the stimulus originated from the tissue side. Our studies are consistent with the reported role for pericytes in regulating leukocyte trafficking during sepsis but indicate that EECM-BMEC-like cells alone are sufficient to maintain the restrictive small-molecule permeability of the BBB.

Development and validation of PAMPA-BBB QSAR model to predict brain penetration potential of novel drug candidates.

Efficiently circumventing the blood-brain barrier (BBB) poses a major hurdle in the development of drugs that target the central nervous system. Although there are several methods to determine BBB permeability of small molecules, the Parallel Artificial Membrane Permeability Assay (PAMPA) is one of the most common assays in drug discovery due to its robust and high-throughput nature. Drug discovery is a long and costly venture, thus, any advances to streamline this process are beneficial. In this study, ∼2,000 compounds from over 60 NCATS projects were screened in the PAMPA-BBB assay to develop a quantitative structure-activity relationship model to predict BBB permeability of small molecules. After analyzing both state-of-the-art and latest machine learning methods, we found that random forest based on RDKit descriptors as additional features provided the best training balanced accuracy (0.70 ± 0.015) and a message-passing variant of graph convolutional neural network that uses RDKit descriptors provided the highest balanced accuracy (0.72) on a prospective validation set. Finally, we correlated in vitro PAMPA-BBB data with in vivo brain permeation data in rodents to observe a categorical correlation of 77%, suggesting that models developed using data from PAMPA-BBB can forecast in vivo brain permeability. Given that majority of prior research has relied on in vitro or in vivo data for assessing BBB permeability, our model, developed using the largest PAMPA-BBB dataset to date, offers an orthogonal means to estimate BBB permeability of small molecules. We deposited a subset of our data into PubChem bioassay database (AID: 1845228) and deployed the best performing model on the NCATS Open Data ADME portal (https://opendata.ncats.nih.gov/adme/). These initiatives were undertaken with the aim of providing valuable resources for the drug discovery community.

Dissecting the Permeability of the Escherichia coli Cell Envelope to a Small Molecule Using Tailored Intensiometric Fluorescent Protein Sensors.

Membranes provide a highly selective barrier that defines the boundaries of any cell while providing an interface for communication and nutrient uptake. However, despite their central physiological role, our capacity to study or even engineer the permeation of distinct solutes across biological membranes remains rudimentary. This especially applies to Gram-negative bacteria, where the outer and inner membrane impose two permeation barriers. Addressing this analytical challenge, we exemplify how the permeability of the Escherichia coli cell envelope can be dissected using a small-molecule-responsive fluorescent protein sensor. The approach is exemplified for the biotechnologically relevant macrolide rapamycin, for which we first construct an intensiometric rapamycin detector (iRapTor) while comprehensively probing key design principles in the iRapTor scaffold. Specifically, this includes the scope of minimal copolymeric linkers as a function of topology and the concomitant need for gate post residues. In a subsequent step, we apply iRapTors to assess the permeability of the E. coli cell envelope to rapamycin. Despite its lipophilic character, rapamycin does not readily diffuse across the E. coli envelope but can be enhanced by recombinantly expressing a nanopore in the outer membrane. Our study thus provides a blueprint for studying and actuating the permeation of small molecules across the prokaryotic cell envelope.

Analysis of reproducibility and robustness of a renal proximal tubule microphysiological system OrganoPlate 3-lane 40 for in vitro studies of drug transport and toxicity.

Microphysiological systems are an emerging area of in vitro drug development, and their independent evaluation is important for wide adoption and use. The primary goal of this study was to test reproducibility and robustness of a renal proximal tubule microphysiological system, OrganoPlate 3-lane 40, as an in vitro model for drug transport and toxicity studies. This microfluidic model was compared with static multiwell cultures and tested using several human renal proximal tubule epithelial cell (RPTEC) types. The model was characterized in terms of the functional transport for various tubule-specific proteins, epithelial permeability of small molecules (cisplatin, tenofovir, and perfluorooctanoic acid) versus large molecules (fluorescent dextrans, 60-150 kDa), and gene expression response to a nephrotoxic xenobiotic. The advantages offered by OrganoPlate 3-lane 40 as compared with multiwell cultures are the presence of media flow, albeit intermittent, and increased throughput compared with other microfluidic models. However, OrganoPlate 3-lane 40 model appeared to offer only limited (eg, MRP-mediated transport) advantages in terms of either gene expression or functional transport when compared with the multiwell plate culture conditions. Although OrganoPlate 3-lane 40 can be used to study cellular uptake and direct toxic effects of small molecules, it may have limited utility for drug transport studies. Overall, this study offers refined experimental protocols and comprehensive comparative data on the function of RPETCs in traditional multiwell culture and microfluidic OrganoPlate 3-lane 40, information that will be invaluable for the prospective end-users of in vitro models of the human proximal tubule.

Genome-wide CRISPR screen identifies genes synthetically lethal with GRA17, a nutrient channel encoding gene in Toxoplasma.

Toxoplasma gondii is a parasite that replicates within a specialized compartment called the parasitophorous vacuole (PV), which is surrounded by the PV membrane (PVM). To obtain essential nutrients, Toxoplasma must transport molecules across the PVM, a process mediated by the secreted parasite proteins GRA17 and GRA23. These proteins form pores in the PVM through which small molecules can diffuse in and out of the PV. GRA17 and GRA23 are synthetically lethal, suggesting that at least one pore type is essential for parasite survival. In the 'nutrient sensitized' Δgra17 strain it is likely that other Toxoplasma genes become essential, because they mediate nutrient acquisition from the host or are involved in the trafficking of GRA23 to the PVM. To identify these genes, a genome-wide loss-of-function screen was performed in wild-type and Δgra17 parasites, which identified multiple genes that were synthetically sick/lethal with GRA17. Several of these genes were involved in the correct localization of GRAs, including GRA17/GRA23, to the PVM. One of the top hits, GRA72, was predicted to form a pore on the PVM, and its deletion led to the formation of enlarged "bubble vacuoles" with reduced PVM small molecule permeability, similar to what was previously observed for Δgra17 parasites. Furthermore, Δgra72 parasites had reduced in vitro growth and virulence in mice. These findings suggest that in the absence of GRA17, other genes become essential, likely because they play a role in the proper localization of GRA23 (and other GRAs) or because they determine host-derived nutrient acquisition at the PVM.

Predictions from First-Principles of Membrane Permeability to Small Molecules: How Useful Are They in Practice?

Predicting from first-principles the rate of passive permeation of small molecules across the biological membrane represents a promising strategy for screening lead compounds upstream in the drug-discovery and development pipeline. One popular avenue for the estimation of permeation rates rests on computer simulations in conjunction with the inhomogeneous solubility-diffusion model, which requires the determination of the free-energy change and position-dependent diffusivity of the substrate along the translocation pathway through the lipid bilayer. In this Perspective, we will clarify the physical meaning of the membrane permeability inferred from such computer simulations, and how theoretical predictions actually relate to what is commonly measured experimentally. We will also examine why these calculations remain both technically challenging and overly computationally expensive, which has hitherto precluded their routine use in nonacademic settings. We finally synopsize possible research directions to meet these challenges, increase the predictive power of physics-based rates of passive permeation, and, by ricochet, improve their practical usefulness.

Visualization of Molecular Permeation into a Multi-compartment Phospholipid Vesicle.

Passive permeation of small molecules into vesicles with multiple compartments is a critical event in many chemical and biological processes. We consider the translocation of the peptide NAF-144-67 labeled with a fluorescent fluorescein dye across membranes of rhodamine-labeled 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) into liposomes with internal vesicles. Time-resolved microscopy revealed a sequential absorbance of the peptide in both the outer and inner micrometer vesicles that developed over a time period of minutes to hours, illustrating the spatial and temporal progress of the permeation. There is minimal perturbation of the membrane structure and no evidence for pore formation. On the basis of molecular dynamics simulations of NAF-144-67, we extended a local defect model to migration processes that include multiple compartments. The model captures the long residence time of the peptide within the membrane and the rate of permeation through the liposome and its internal compartments. Imaging experiments confirm the semi-quantitative description of the permeation of the model by activated diffusion and open the way for studies of more complex systems.

Lipid packing in biological membranes governs protein localization and membrane permeability

Plasma membrane (PM) heterogeneity has long been implicated in various cellular functions. However, mechanistic principles governing functional regulations of lipid environment are not well understood due to the inherent complexities associated with the relevant length and timescales that limit both direct experimental measurements and their interpretation. In this context, computer simulations hold immense potential to investigate molecular-level interactions and mechanisms that lead to PM heterogeneity and its functions. Herein, we investigate spatial and dynamic heterogeneity in model membranes with coexisting liquid ordered and liquid disordered phases and characterize the membrane order in terms of the local topological changes in lipid environment using the nonaffine deformation framework. Furthermore, we probe the packing defects in these membranes, which can be considered as the conjugate of membrane order assessed in terms of the nonaffine parameter. In doing so, we formalize the connection between membrane packing and local membrane order and use that to explore the mechanistic principles behind their functions. Our observations suggest that heterogeneity in mixed phase membranes is a consequence of local lipid topology and its temporal evolution, which give rise to disparate lipid packing in ordered and disordered domains. This in turn governs the distinct nature of packing defects in these domains that can play a crucial role in preferential localization of proteins in mixed phase membranes. Furthermore, we observe that lipid packing also leads to contrasting distribution of free volume in the membrane core region in ordered and disordered membranes, which can lead to distinctive membrane permeability of small molecules. Our results, thus, indicate that heterogeneity in mixed phase membranes closely governs the membrane functions that may emerge from packing-related basic design principles.

Explaining Blood-Brain Barrier Permeability of Small Molecules by Integrated Analysis of Different Transport Mechanisms.

The blood-brain barrier (BBB) represents a major obstacle to delivering drugs to the central nervous system (CNS), resulting in the lack of effective treatment for many CNS diseases including brain cancer. To accelerate CNS drug development, computational prediction models could save the time and effort needed for experimental evaluation. Here, we studied BBB permeability focusing on active transport (influx and efflux) as well as passive diffusion using previously published and self-curated data sets. We created prediction models based on physicochemical properties, molecular substructures, or their combination to understand which mechanisms contribute to BBB permeability. Our results show that features that predicted passive diffusion over membranes overlap with features that explain endothelial permeation of approved CNS-active drugs. We also identified physical properties and molecular substructures that positively or negatively predicted BBB transport. These findings provide guidance toward identifying BBB-permeable compounds by optimally matching physicochemical and molecular properties to BBB transport mechanisms.

Transdermal drug delivery in horses: An in vitro comparison of skin structure and permeation of two model drugs at various anatomical sites.

Oral and parenteral drug delivery in horses can be difficult. Equine-specific transdermal drug formulations offer improved ease of treatment; development of such formulations requires a deeper understanding of the structural and chemical tissue barrier of horse skin. To compare the structural composition and barrier properties of equine skin. Six warmblood horses (two males, four females) with no skin diseases. Routine histological and microscopic analyses were carried out with image analysis for skin from six different anatomical locations. In vitro drug permeation was analysed using a standard Franz diffusion cell protocol coupled with reversed phase-high-performance liquid chromatography detailing flux, lag times and tissue partitioning ratios of two model drug compounds. Epidermal and dermal thicknesses varied between sites. The dermal and epidermal thicknesses of the croup were 1764 ± 115 μm and 36 ± 3.6 μm, respectively, and were significantly different (p < 0.05) from the inner thigh thicknesses which were 824 ± 35 μm and 49 ± 3.6 μm. Follicular density and size also varied. The highest flux for the model hydrophilic molecule (caffeine) was for the flank (3.22 ± 0.36 μg/cm2 /h), while that for the lipophilic molecule (ibuprofen) was for the inner thigh (0.12 ± 0.02 μg/cm2 /h). Anatomical location differences in equine skin structure and small molecule permeability were demonstrated. These results can aid in the development of transdermal therapies for horses.

Size and Charge Effects on Crossover of Flow Battery Reactants Evaluated by Quinone Permeabilities Through Nafion

Organic reactants are promising candidates for long-lifetime redox flow batteries, and synthetic chemistry unlocks a wide design space for new molecules. Minimizing crossover of these molecules through ion exchange membranes is one important design consideration, but the ways in which the crossover rate depends on the structure of the crossing species remain unclear. Here, we contribute a systematic evaluation of size- and charge-based effects on dilute-solution small molecule permeability through the Nafion NR212 cation exchange membrane. We found that increasing the magnitude of charge number z with the same sign as membrane fixed charges, achieved here by successive sulfonation of quinone redox cores, results in more than an order of magnitude permeability reduction per sulfonate. Size-based effects, understood by comparing the Stokes radii of the quinones studied, also reduces permeability with increasing effective molecule size, but doubling the effective size of the redox reactants resulted in a permeability decrease of less than a factor of three.

A Metabolic‐Tag‐Based Method for Assessing the Permeation of Small Molecules Across the Mycomembrane in Live Mycobacteria**

AbstractThe general lack of permeability of small molecules observed for Mycobacterium tuberculosis (Mtb) is most ascribed to its unique cell envelope. More specifically, the outer mycomembrane is hypothesized to be the principal determinant for access of antibiotics to their molecular targets. We describe a novel assay that combines metabolic tagging of the peptidoglycan, which sits directly beneath the mycomembrane, click chemistry of test molecules, and a fluorescent labeling chase step, to measure the permeation of small molecules. We showed that the assay workflow was robust and compatible with high‐throughput analysis in mycobacteria by testing a small panel of azide‐tagged molecules. The general trend is similar across the two types of mycobacteria with some notable exceptions. We anticipate that this assay platform will lay the foundation for medicinal chemistry efforts to understand and improve uptake of both existing drugs and newly‐discovered compounds into mycobacteria.