Small Molecules For Treatment Research Articles

P.42Discovery of novel small molecule treatment options for FSHD

There is wide support for the pathogenesis model in which gain-of-function of the DUX4 gene in skeletal muscle cells underlies FSHD progression. DUX4 is a transcription factor whose expression is normally restricted to early embryonic development. Expression of DUX4 in muscle tissue of FSHD patients initiates a transcription cascade ultimately resulting in overt pathology. Drug discovery efforts in FSHD have been hampered by the very low and stochastic expression of DUX4 in a small percentage of myonuclei. Because the regulatory pathways involved in the activation of DUX4 are largely unknown, we implemented a phenotypic approach to identify novel small molecules and drug targets to repress DUX4. In order to build a predictive and translatable assay, we used primary patient-derived myocytes to develop and validate a high-content screening assay allowing automated sensitive detection and quantification of endogenous, non-stimulated DUX4 protein and myotube fusion efficiency. Our assay confirmed that the expression of DUX4 is dynamically regulated during myogenic differentiation and showed that effects on fusion are associated with potentially false-positive signals on the DUX4 readout. The excellent assay performance enabled high-throughput screening, resulting in the discovery and optimization of multiple hit series with confirmed activity in secondary assays. RNA-seq studies with selected compounds revealed normalization of the FSHD transcription signature. We applied a number of chemoproteomic approaches to deconvolute the mode of action of the phenotypic hit series, resulting in the discovery of novel targets to repress DUX4. In order to support the pharmacokinetic/pharmacodynamic strategy, we developed a xenograft animal model in which we could demonstrate in vivo repression of DUX4 after oral dosing of a selected lead compound from one of the series. Together, these results form the basis for building a diverse FSHD-focused product pipeline.

Devil in the detail of newborn screening for cystic fibrosis

The recognition that the sweat chloride concentration was elevated in people with cystic fibrosis (CF) was fundamental to its diagnosis and treatment.1 A sweat chloride >60 mmol/L was diagnostic of...

In vivo identification of small molecules mediating Gpr126/Adgrg6 signaling during Schwann cell development.

Gpr126/Adgrg6, an adhesion family G protein-coupled receptor (aGPCR), is required for the development of myelinating Schwann cells in the peripheral nervous system. Myelin supports and insulates vertebrate axons to permit rapid signal propagation throughout the nervous system. In mammals and zebrafish, mutations in Gpr126 arrest Schwann cells at early developmental stages. We exploited the optical and pharmacological tractability of larval zebrafish to uncover drugs that mediate myelination by activating Gpr126 or functioning in parallel. Using a fluorescent marker of mature myelinating glia (Tg[mbp:EGFP-CAAX]), we screened hypomorphic gpr126 mutant larvae for restoration of myelin basic protein (mbp) expression along peripheral nerves following small molecule treatment. Our screens identified five compounds sufficient to promote mbp expression in gpr126 hypomorphs. Using an allelic series of gpr126 mutants, we parsed the ability of small molecules to restore mbp, suggesting differences in drug efficacy dependent on Schwann cell developmental state. Finally, we identify apomorphine hydrochloride as a direct small molecule activator of Gpr126 using combined in vivo/in vitro assays and show that aporphine class compounds promote Schwann cell development in vivo. Our results demonstrate the utility of in vivo screening for aGPCR modulators and identify small molecules that interact with the gpr126-mediated myelination program.

Small Molecule Modulation of the Integrated Stress Response Governs the Keratoconic Phenotype In Vitro.

PurposeThe degenerative corneal disease keratoconus is a leading indicator for corneal transplant with an unknown etiology. We recently identified the activation of the integrated stress response (ISR) in ex vivo human corneas and in vitro cell culture. Utilizing small molecules to modulate the ISR we sought to investigate the effects of stimulating the ISR in healthy cells to recapitulate aspects of the in vitro keratoconic phenotype and whether relieving the ISR signaling would recover the disease phenotype.MethodsCorneal fibroblasts were extracted from patients undergoing corneal transplant or unaffected cadaverous donor limbal rings. Cells were exposed to the DNA damage-inducible protein (GADD34) inhibitor SAL003 to stimulate the ISR, or Trans-ISRIB to relieve ISR signaling pathway. Collagen production was assessed through hydroxyproline production, Sirius Red incorporation, or quantitative (q)PCR. Western blotting, hydroxyproline, and qPCR were used to assess components of the ISR pathway and collagen production.ResultsISR stimulation through SAL003 resulted in significant decrease of hydroxyproline and COL1A1 transcription and eventual apoptosis in normal fibroblasts. Patient (KC) fibroblast production of hydroxyproline was increased in response to ISRIB, while matrix metalloproteinase (MMP)9 production was lowered. The prospective biomarker of keratoconus prolactin-inducible factor was also upregulated in KC fibroblast cultures in response to ISRIB. Inflammatory markers TNFα and IL-1β were unaffected.ConclusionsActivation of the ISR is sufficient to recapitulate many key aspects of the KC phenotype in unaffected cells in vitro. Inhibition of the ISR also relieves many of the hallmarks of KC in affected cells. Therefore, targeting of the ISR through small molecules is a potential therapeutic path for small molecule treatment of keratoconus.

Fine-Tuning the Treatment of Psoriatic Arthritis: Focus on the IL-23 Pathway

The symposium ‘Fine-tuning the treatment of PsA: Focus on the IL-23 pathway’ took place during the 2019 European League Against Rheumatism (EULAR) Annual Congress in Madrid, Spain. The presentations covered the rationale for targeting IL-23 in psoriatic arthritis (PsA), details of the IL-23 pathway relevant to psoriatic disease, practical implications and consequences of targeting IL-23, and experiences of targeting IL-23 in psoriasis from the dermatologists’ perspective. Dr Stefan Siebert set the scene by outlining the pathophysiology of psoriatic diseases, particularly PsA, describing disease heterogeneity, explaining the role of inflammation, and highlighting the rationale for targeting the IL-12/23 pathway. He summarised key findings on the IL-12/23 inhibitor ustekinumab in PsA from clinical trials and real-world data available to date. Delving deeper into the IL-23 pathway, Prof Georg Schett explained the function of IL-23 and its role in inflammatory disease and autoimmunity. After briefly describing the history of the relatively recent discovery of this cytokine, Prof Schett discussed preclinical and clinical studies underlying today’s understanding of IL-23 and why it is an appropriate target in PsA. Multiple biologic or small-molecule treatments for PsA have been investigated in clinical trials. Prof Peter Taylor discussed the practical implications of targeting IL-23 and provided more details about the specific effects of targeting not only IL-23 (with risankizumab, tildrakizumab, or guselkumab) but also IL-12/23 (with ustekinumab) and IL-17 (with ixekizumab, secukinumab, or brodalumab). In the final presentation, Prof Lluís Puig described clinical experience of targeting IL-23 in psoriasis and provided an overview of findings from several clinical trials, including: VOYAGE 1 and 2 (guselkumab versus the TNF inhibitor [TNFi] adalimumab); NAVIGATE (guselkumab versus ustekinumab); and the head-to-head ECLIPSE study (guselkumab versus secukinumab). The symposium concluded with a lively panel discussion in which the speakers addressed a variety of questions and comments from the audience.

Beyond cystic fibrosis transmembrane conductance regulator therapy: a perspective on gene therapy and small molecule treatment for cystic fibrosis.

Cystic fibrosis (CF) is a life-limiting disease caused by defective or deficient cystic fibrosis transmembrane conductance regulator (CFTR) activity. The recent advent of the FDA-approved CFTR modulator drug ivacaftor, alone or in combination with lumacaftor or tezacaftor, has enabled treatment of the majority of patients suffering from CF. Even before the identification of the CFTR gene, gene therapy was put forward as a viable treatment option for this genetic condition. However, initial enthusiasm has been hampered as CFTR gene delivery to the lungs has proven to be more challenging than expected. This review covers the contemporary clinical and scientific knowledge base for small molecule CFTR modulator drug therapy, gene delivery vectors and CRISPR/Cas9 gene editing and highlights the prospect of these technologies for future treatment options.

Exploration of Target Synergy in Cancer Treatment by Cell-Based Screening Assay and Network Propagation Analysis.

Computational approaches have previously been introduced to predict compounds with activity against multiple targets or compound combinations with synergistic functional effects. By contrast, there are no computational studies available that explore combinations of targets that might act synergistically upon small molecule treatment. Herein, we introduce an approach designed to identify synergistic target pairs on the basis of cell-based screening data and compounds with known target annotations. The targets involved in forming synergistic pairs were analyzed through a novel network propagation algorithm for rationalizing possible common synergy mechanisms. This algorithm enabled further analysis of each synergistic target pair and the identification of "interactors", i.e., proteins with higher propagation scores than would be expected by adding the individual contributions of each target in the synergistic pair. We detected 137 synergistic target pairs including 51 unique targets. A global network analysis of these 51 targets made it possible to derive a subnetwork of proteins with significant synergy. Furthermore, interactors were identified for 87 synergistic target pairs upon individual analysis of the network propagation of each pair. These interactors were associated with pathways related to cancer and apoptosis, membrane transport, and steroid metabolism and provided possible explanations of synergistic effects.

Emerging small-molecule treatments for multiple sclerosis: focus on B cells.

Multiple sclerosis (MS) is a major cause of disability in young adults. Following an unknown trigger (or triggers), the immune system attacks the myelin sheath surrounding axons, leading to progressive nerve cell death. Antibodies and small-molecule drugs directed against B cells have demonstrated good efficacy in slowing progression of the disease. This review focusses on small-molecule drugs that can affect B-cell biology and may have utility in disease management. The risk genes for MS are examined from the drug target perspective. Existing small-molecule therapies for MS with B-cell actions together with new drugs in development are described. The potential for experimental molecules with B-cell effects is also considered. Small molecules can have diverse actions on B cells and be cytotoxic, anti-inflammatory and anti-viral. The current B cell–directed therapies often kill B-cell subsets, which can be effective but lead to side effects and toxicity. A deeper understanding of B-cell biology and the effect on MS disease should lead to new drugs with better selectivity, efficacy, and an improved safety profile. Small-molecule drugs, once the patent term has expired, provide a uniquely sustainable form of healthcare.

Transcriptome Profiling and Molecular Therapeutic Advances in Cystic Fibrosis: Recent Insights.

In cystic fibrosis (CF), mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene disrupt the capacity of the encoded protein to function as a channel to transport chloride ions and water across cell membranes. The consequences are deleterious, system-wide, and immensely variable, even among patients with the same CFTR genotype. This underscores the need to characterize the mechanisms contributing to CF pathophysiology. Gene replacement and gene editing therapies have been pursued intensively and are expected to provide a one-time treatment for CF. However, gene replacement therapy is limited by the lack of efficient vectors to deliver functional copies of CFTR to cells without immunological complications, while gene editing technologies such as CRISPR/Cas9 are still in their infancy, mainly useful in somatic cells and limited by off-target insertions. Small molecule treatments targeted at potentiating or correcting CFTR have shown clinical benefits, but they are limited to a few CFTR mutations and insufficient to overcome challenges related to clinical heterogeneity. Transcriptome profiling approaches have emerged as robust tools capable of characterizing phenotypic variability and revealing novel molecular targets with therapeutic potential for CF. We summarize current insights gained through transcriptome profiling approaches in CF studies and recent advances in molecular therapeutics.

Length doesn't matter-telomere damage triggers cellular senescence in the ageing heart.

Telomere shortening induces cellular senescence in proliferative cells. Yet, it is presently unclear how it is triggered in post‐mitotic cells such as cardiac myocytes. A new study by Anderson et al (2019) reports that during ageing of the heart, cellular senescence develops independently of telomere length, but is evoked by DNA damage, which preferentially accumulates at the telomere. Removal of senescent cells using senolytic drugs ameliorated cardiac hypertrophy and fibrosis and may inform novel approaches to improve the conditions for the ageing heart.

Small-Molecules Selectively Modulate Iron-Deficiency Signaling Networks in Arabidopsis

Plant growth requires optimal levels of iron (Fe). Fe is used for energy production, numerous enzymatic processes, and is indispensable for cellular metabolism. Recent studies have established the mechanism involved in Fe uptake and transport. However, our knowledge of Fe sensing and signaling is limited. Dissecting Fe signaling may be useful for crop improvement by Fe fortification. Here, we report two small-molecules, R3 and R6 [where R denotes repressor of IRON-REGULATED TRANSPORTER 1 (IRT1)], identified through a chemical screening, whose use blocked activation of the Fe-deficiency response in Arabidopsis thaliana. Physiological analysis of plants treated with R3 and R6 showed that these small molecules drastically attenuated the plant response to Fe starvation. Small-molecule treatment caused severe chlorosis and strongly reduced chlorophyll levels in plants. Fe content in shoots was decreased considerably by small-molecule treatments especially in Fe deficiency. Small-molecule treatments attenuated the Fe-deficiency-induced expression of the Fe uptake gene IRT1. Analysis of FER-LIKE IRON-DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT) and subgroup Ib basic helix-loop-helix (bHLH) gene (bHLH38/39/100/101) expression showed that R3 affects the FIT-network, whereas R6 affects both the FIT and Ib bHLH networks. An assessment of the effects of the structural analogs of R3 and R6 on the induction of Fe-dependent chlorosis revealed the functional motif of the investigated chemicals. Our findings suggest that small-molecules selectively modulate the distinct signaling routes that operate in response to Fe-deficiency. R3 and R6 likely interrupt the activity of key upstream signaling regulators whose activities are required for the activation of the Fe-starvation transcriptional cascade in Arabidopsis roots.

Activation of TAp73 and inhibition of TrxR by Verteporfin for improved cancer therapy in TP53 mutant pancreatic tumors.

Aim:TAp73 is a tumor suppressor, which compensates for p53 loss and induces apoptosis in tumors in response to genotoxic stress or small-molecule treatments. Pancreatic ductal adenocarcinoma has a late onset of the disease, responds poorly to the existing therapies and has a very low survival rates.Result:Here, using drug-repurposing approach, we found that protoporphyrin IX (PpIX) and benzoporphyrin derivative (BPD) monoacid ring A activate TAp73 and induce apoptosis in pancreatic cancer cells. PpIX and BPD induce reactive oxygen species and inhibit thioredoxin reductase 1.Conclusion:Thus, PpIX and BPD target cancer cells’ vulnerabilities namely activate TAp73 tumor suppressor and inhibit oncogenic Trx1. Our findings may contribute to faster repurposing of PpIX and BPD to treat pancreatic tumors.

Identification and Characterization of Menadione and Benzethonium Chloride as Potential Treatments of Pierce's Disease of Grapevines.

Xylella fastidiosa infects a wide range of plant hosts and causes Pierce's disease (PD) of grapevines. The type 1 multidrug resistance (MDR) efflux system is essential for pathogenicity and survival of bacterial pathogens in planta. X. fastidiosa, with a single MDR system, is significantly more vulnerable to inhibition by small-molecule treatments than most bacterial pathogens that typically carry redundant MDR systems. A high-throughput cell viability assay using a green fluorescent protein-marked strain of X. fastidiosa Temecula 1 was developed to screen two Prestwick combinatorial small-molecule libraries of drugs and phytochemicals (1,600 chemicals in total) approved by the Food and Drug Administration and European Medicines Agency for cell growth inhibition. The screens revealed 215 chemicals that inhibited bacterial growth by >50% at 50 µM concentrations. Seven chemicals proved to lyse X. fastidiosa cells at 25 µM, including four phytochemicals. Menadione (2-methyl-1,4-naphthoquinone, vitamin K) from the phytochemical library and benzethonium chloride (a topical disinfectant) from the chemical library both showed significant bactericidal activity against X. fastidiosa. Both menadione and benzethonium chloride foliar spray (15 and 5 mM, respectively) and soil drench (5 and 25 mM, respectively) treatments were equally effective in reducing PD symptoms by 54 to 59% and revealed that the effects of both chemical treatments became systemic. However, menadione was phytotoxic when applied as a foliar spray at effective concentrations, causing significant loss of photosynthetic capacity.

New Generation of Selective Androgen Receptor Degraders: Our Initial Design, Synthesis, and Biological Evaluation of New Compounds with Enzalutamide-Resistant Prostate Cancer Activity.

In our effort to find small-molecule treatments of advanced prostate cancers (PCs), a novel series of indolyl and indolinyl propanamides (series II and III) were discovered as selective androgen receptor degraders (SARDs). Initial studies of androgen receptor (AR) antagonist (1) and agonist (2) propanamides yielded a tertiary aniline (3) with novel SARD activity but poor metabolic stability. Cyclization to II and III produced submicromolar AR antagonism and protein degradation selective to AR and AR splice variant (AR SV). II and III maintained potency against enzalutamide-resistant (Enz-R) mutant ARs and PC cells and were efficacious in Enz-R xenografts, suggesting their potential to treat advanced PCs. Design, synthesis, and biological activity of novel SARDs that could potentially be used for the treatment of a wide spectrum of PCs including castration-resistant, Enz-R, and/or AR SV-dependent advanced PCs that are often untreatable with known hormone therapies are discussed.

Revealing Nanoscale Morphology of the Primary Cilium Using Super-Resolution Fluorescence Microscopy

Super-resolution (SR) microscopy has been used to observe structural details beyond the diffraction limit of ∼250 nm in a variety of biological and materials systems. By combining this imaging technique with both computer-vision algorithms and topological methods, we reveal and quantify the nanoscale morphology of the primary cilium, a tiny tubular cellular structure (∼2–6 μm long and 200–300 nm in diameter). The cilium in mammalian cells protrudes out of the plasma membrane and is important in many signaling processes related to cellular differentiation and disease. After tagging individual ciliary transmembrane proteins, specifically Smoothened, with single fluorescent labels in fixed cells, we use three-dimensional (3D) single-molecule SR microscopy to determine their positions with a precision of 10–25 nm. We gain a dense, pointillistic reconstruction of the surfaces of many cilia, revealing large heterogeneity in membrane shape. A Poisson surface reconstruction algorithm generates a fine surface mesh, allowing us to characterize the presence of deformations by quantifying the surface curvature. Upon impairment of intracellular cargo transport machinery by genetic knockout or small-molecule treatment of cells, our quantitative curvature analysis shows significant morphological differences not visible by conventional fluorescence microscopy techniques. Furthermore, using a complementary SR technique, two-color, two-dimensional stimulated emission depletion microscopy, we find that the cytoskeleton in the cilium, the axoneme, also exhibits abnormal morphology in the mutant cells, similar to our 3D results on the Smoothened-measured ciliary surface. Our work combines 3D SR microscopy and computational tools to quantitatively characterize morphological changes of the primary cilium under different treatments and uses stimulated emission depletion to discover correlated changes in the underlying structure. This approach can be useful for studying other biological or nanoscale structures of interest.

Erin Reineke.

Erin Reineke.

Proteomic Profiling of Enteroid Cultures Skewed toward Development of Specific Epithelial Lineages.

Recently, 3D small intestinal organoids (enteroids) have been developed from cultures of intestinal stem cells which differentiate in vitro to generate all the differentiated epithelial cell types associated with the intestine and mimic the structural properties of the intestine observed in vivo. Small-molecule drug treatment can skew organoid epithelial cell differentiation toward particular lineages, and these skewed enteroids may provide useful tools to study specific epithelial cell populations, such as goblet and Paneth cells. However, the extent to which differentiated epithelial cell populations in these skewed enteroids represent their in vivo counterparts is not fully understood. This study utilises label-free quantitative proteomics to determine whether skewing murine enteroid cultures toward the goblet or Paneth cell lineages results in changes in abundance of proteins associated with these cell lineages in vivo. Here, proteomics data confirms that skewed enteroids recapitulate important features of the in vivo gut environment, demonstrating that they can serve as useful models for the investigation of normal and disease processes in the intestine. Furthermore, comparison of mass spectrometry data with histology data contained within the Human Protein Atlas identifies putative novel markers for goblet and Paneth cells.

Small Molecule Inhibition of MicroRNA miR-21 Rescues Chemosensitivity of Renal-Cell Carcinoma to Topotecan.

Chemical probes of microRNA (miRNA) function are potential tools for understanding miRNA biology that also provide new approaches for discovering therapeutics for miRNA-associated diseases. MicroRNA-21 (miR-21) is an oncogenic miRNA that is overexpressed in most cancers and has been strongly associated with driving chemoresistance in cancers such as renal cell carcinoma (RCC). Using a cell-based luciferase reporter assay to screen small molecules, we identified a novel inhibitor of miR-21 function. Following structure-activity relationship studies, an optimized lead compound demonstrated cytotoxicity in several cancer cell lines. In a chemoresistant-RCC cell line, inhibition of miR-21 via small molecule treatment rescued the expression of tumor-suppressor proteins and sensitized cells to topotecan-induced apoptosis. This resulted in a >10-fold improvement in topotecan activity in cell viability and clonogenic assays. Overall, this work reports a novel small molecule inhibitor for perturbing miR-21 function and demonstrates an approach to enhancing the potency of chemotherapeutics specifically for cancers derived from oncomir addiction.

Along the Central Dogma-Controlling Gene Expression with Small Molecules.

The central dogma of molecular biology, that DNA is transcribed into RNA and RNA translated into protein, was coined in the early days of modern biology. Back in the 1950s and 1960s, bacterial genetics first opened the way toward understanding life as the genetically encoded interaction of macromolecules. As molecular biology progressed and our knowledge of gene control deepened, it became increasingly clear that expression relied on many more levels of regulation. In the process of dissecting mechanisms of gene expression, specific small-molecule inhibitors played an important role and became valuable tools of investigation. Small molecules offer significant advantages over genetic tools, as they allow inhibiting a process at any desired time point, whereas mutating or altering the gene of an important regulator would likely result in a dead organism. With the advent of modern sequencing technology, it has become possible to monitor global cellular effects of small-molecule treatment and thereby overcome the limitations of classical biochemistry, which usually looks at a biological system in isolation. This review focuses on several molecules, especially natural products, that have played an important role in dissecting gene expression and have opened up new fields of investigation as well as clinical venues for disease treatment.

Molecular Defects in Cardiac Myofilament Ca2+-Regulation Due to Cardiomyopathy-Linked Mutations Can Be Reversed by Small Molecules Binding to Troponin.

The inherited cardiomyopathies, hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) are relatively common, potentially life-threatening and currently untreatable. Mutations are often in the contractile proteins of cardiac muscle and cause abnormal Ca2+ regulation via troponin. HCM is usually linked to higher myofilament Ca2+-sensitivity whilst in both HCM and DCM mutant tissue there is often an uncoupling of the relationship between troponin I (TnI) phosphorylation by PKA and modulation of myofilament Ca2+-sensitivity, essential for normal responses to adrenaline. The adrenergic response is blunted, and this may predispose the heart to failure under stress. At present there are no compounds or interventions that can prevent or treat sarcomere cardiomyopathies. There is a need for novel therapies that act at a more fundamental level to affect the disease process. We demonstrated that epigallocatechin-3 gallate (EGCG) was found to be capable of restoring the coupled relationship between Ca2+-sensitivity and TnI phosphorylation in mutant thin filaments to normal in vitro, independent of the mutation (15 mutations tested). We have labeled this property “re-coupling.” The action of EGCG in vitro to reverse the abnormality caused by myopathic mutations would appear to be an ideal pharmaceutical profile for treatment of inherited HCM and DCM but EGCG is known to be promiscuous in vivo and is thus unsuitable as a therapeutic drug. We therefore investigated whether other structurally related compounds can re-couple myofilaments without these off-target effects. We used the quantitative in vitro motility assay to screen 40 compounds, related to C-terminal Hsp90 inhibitors, and found 23 that can re-couple mutant myofilaments. There is no correlation between re-couplers and Hsp90 inhibitors. The Ca2+-sensitivity shift due to TnI phosphorylation was restored to 2.2 ± 0.01-fold (n = 19) compared to 2.0 ± 0.24-fold (n = 7) in wild-type thin filaments. Many of these compounds were either pure re-couplers or pure desensitizers, indicating these properties are independent; moreover, re-coupling ability could be lost with small changes of compound structure, indicating the possibility of specificity. Small molecules that can re-couple may have therapeutic potential.HIGHLIGHTS - Inherited cardiomyopathies are common diseases that are currently untreatable at a fundamental level and therefore finding a small molecule treatment is highly desirable.- We have identified a molecular level dysfunction common to nearly all mutations: uncoupling of the relationship between troponin I phosphorylation and modulation of myofilament Ca2+-sensitivity, essential for normal responses to adrenaline.- We have identified a new class of drugs that are capable of both reducing Ca2+-sensitivity and/or recouping the relationship between troponin I phosphorylation and Ca2+-sensitivity.- The re-coupling phenomenon can be explained on the basis of a single mechanism that is testable.- Measurements with a wide range of small molecules of varying structures can indicate the critical molecular features required for recoupling and allows the prediction of other potential re-couplers.