Mechanistic insights into SARS-CoV-2 spike protein induction of the chemokine CXCL10.

During a SARS-CoV-2 infection, macrophages recognize viral components resulting in cytokine production. While this response fuels virus elimination, overexpression of cytokines can lead to severe COVID-19. Previous studies suggest that the spike protein (S) of SARS-CoV-2 can elicit cytokine production via the transcription factor NF-κB and the toll-like receptors (TLRs). In this study, we found that: (i) S and the S2 subunit induce CXCL10, a chemokine implicated in severe COVID-19, gene expression by human macrophage cells (THP-1); (ii) a glycogen synthase kinase-3 inhibitor attenuates this induction; (iii) S and S2 do not activate NF-κB but do activate the transcription factor IRF; (iv) S and S2 do not require TLR2 to elicit CXCL10 production or activate IRF; and (v) S and S2 elicit CXCL10 production by peripheral blood mononuclear cells (PBMCs). We also discovered that the cellular response, or lack thereof, to S and S2 is a function of the recombinant S and S2 used. While such a finding raises the possibility of confounding LPS contamination, we offer evidence that potential contaminating LPS does not underly induced increases in CXCL10. Combined, these results provide insights into the complex immune response to SARS-CoV-2 and suggest possible therapeutic targets for severe COVID-19.

Anti-Inflammatory and Therapeutic Effects of a Novel Small-Molecule Inhibitor of Inflammation in a Male C57BL/6J Mouse Model of Obesity-Induced NAFLD/MAFLD.

Purpose: Non-alcoholic fatty liver disease (NAFLD), recently renamed metabolic (dysfunction) associated fatty liver disease (MAFLD), is the most common chronic liver disease in the United States. Presently, there is an intense and ongoing effort to identify and develop novel therapeutics for this disease. In this study, we explored the anti-inflammatory activity of a new compound, termed IOI-214, and its therapeutic potential to ameliorate NAFLD/MAFLD in male C57BL/6J mice fed a high fat (HF) diet. Methods: Murine macrophages and hepatocytes in culture were treated with lipopolysaccharide (LPS) ± IOI-214 or DMSO (vehicle), and RT-qPCR analyses of inflammatory cytokine gene expression were used to assess IOI-214's anti-inflammatory properties in vitro. Male C57BL/6J mice were also placed on a HF diet and treated once daily with IOI-214 or DMSO for 16 weeks. Tissues were collected and analyzed to determine the effects of IOI-214 on HF diet-induced NAFL D/MAFLD. Measurements such as weight, blood glucose, serum cholesterol, liver/serum triglyceride, insulin, and glucose tolerance tests, ELISAs, metabolomics, Western blots, histology, gut microbiome, and serum LPS binding protein analyses were conducted. Results: IOI-214 inhibited LPS-induced inflammation in macrophages and hepatocytes in culture and abrogated HF diet-induced mesenteric fat accumulation, hepatic inflammation and steatosis/hepatocellular ballooning, as well as fasting hyperglycemia without affecting insulin resistance or fasting insulin, cholesterol or TG levels despite overall obesity in vivo in male C57BL/6J mice. IOI-214 also decreased systemic inflammation in vivo and improved gut microbiota dysbiosis and leaky gut. Conclusion: Combined, these data indicate that IOI-214 works at multiple levels in parallel to inhibit the inflammation that drives HF diet-induced NAFLD/MAFLD, suggesting that it may have therapeutic potential for NAFLD/MAFLD.

Evidence for investigating GSK-3 inhibitors as potential therapeutics for severe COVID-19.

A key component of severe COVID-19 is a "cytokine storm" i.e., the excessive expression of unneeded cytokines. Previous studies suggest that SARS-CoV-2 proteins can induce macrophages to secrete pro-inflammatory cytokines; a process that may involve Toll-like receptors (TLRs). Glycogen synthase kinase-3 (GSK-3) has been implicated in TLR signal transduction and a selective GSK-3 inhibitor, termed COB-187, dramatically attenuates cytokine expression induced by the TLR ligand lipopolysaccharide (LPS). In the present study, we provide evidence that the SARS-CoV-2 spike protein (S) and the S2 subunit (S2) induce production of CXCL10 (a chemokine elevated in severe COVID-19) by a human macrophage cell line. Further, we report that two clinically relevant GSK-3 inhibitors and COB-187 attenuate S and S2 protein-induced CXCL10 production. Combined, our observations provide impetus for investigating GSK-3 inhibitors as potential therapeutics for severe COVID-19.

A novel GSK-3 inhibitor binds to GSK-3ß via a reversible, time and Cys-199-dependent mechanism.

Glycogen synthase kinase-3 (GSK-3) has been implicated in numerous pathologies making GSK-3 an attractive therapeutic target. Our group has identified a compound termed COB-187 that is a potent and selective inhibitor of GSK-3. In this study, we probed the mechanism by which COB-187 inhibits GSK-3ß. Progress curves, generated via real-time monitoring of kinase activity, indicated that COB-187 inhibition of GSK-3ß is time-dependent and subsequent jump dilution assays revealed that COB-187 binding to GSK-3ß is reversible. Further, a plot of the kinetic constant (kobs) versus COB-187 concentration suggested that, within the range of concentrations studied, COB-187 binds to GSK-3ß via an induced-fit mechanism. There is a critical cysteine residue at the entry to the active site of GSK-3ß (Cys-199). We generated a mutant version of GSK-3ß wherein Cys-199 was substituted with an alanine. This mutation caused a dramatic decrease in the activity of COB-187; specifically, an IC50 in the nM range for wild type versus >100 µM for the mutant. A screen of COB-187 against 34 kinases that contain a conserved cysteine in their active site revealed that COB-187 is highly selective for GSK-3 indicating that COB-187's inhibition of GSK-3ß via Cys-199 is specific. Combined, these findings suggest that COB-187 inhibits GSK-3ß via a specific, reversible, time and Cys-199-dependent mechanism.

A small-molecule pan-class I glucose transporter inhibitor reduces cancer cell proliferation in vitro and tumor growth in vivo by targeting glucose-based metabolism.

BACKGROUND: Cancer cells drastically increase the uptake of glucose and glucose metabolism by overexpressing class I glucose transporters (GLUT1-4) to meet their energy and biomass synthesis needs and are very sensitive and vulnerable to glucose deprivation. Although targeting glucose uptake via GLUTs has been an attractive anticancer strategy, the relative anticancer efficacy of multi-GLUT targeting or single GLUT targeting is unclear. Here, we report DRB18, a synthetic small molecule, is a potent anticancer compound whose pan-class I GLUT inhibition is superior to single GLUT targeting. METHODS: Glucose uptake and MTT/resazurin assays were used to measure DRB18's inhibitory activities of glucose transport and cell viability/proliferation in human lung cancer and other cancer cell lines. Four HEK293 cell lines expressing GLUT1-4 individually were used to determine the IC50 values of DRB18's inhibitory activity of glucose transport. Docking studies were performed to investigate the potential direct interaction of DRB18 with GLUT1-4. Metabolomics analysis was performed to identify metabolite changes in A549 lung cancer cells treated with DRB18. DRB18 was used to treat A549 tumor-bearing nude mice. The GLUT1 gene was knocked out to determine how the KO of the gene affected tumor growth. RESULTS: DRB18 reduced glucose uptake mediated via each of GLUT1-4 with different IC50s, which match with the docking glidescores with a correlation coefficient of 0.858. Metabolomics analysis revealed that DRB18 altered energy-related metabolism in A549 cells by changing the abundance of metabolites in glucose-related pathways in vitro and in vivo. DRB18 eventually led to G1/S phase arrest and increased oxidative stress and necrotic cell death. IP injection of DRB18 in A549 tumor-bearing nude mice at 10 mg/kg body weight thrice a week led to a significant reduction in the tumor volume compared with mock-treated tumors. In contrast, the knockout of the GLUT1 gene did not reduce tumor volume. CONCLUSIONS: DRB18 is a potent pan-class I GLUT inhibitor in vitro and in vivo in cancer cells. Mechanistically, it is likely to bind the outward open conformation of GLUT1-4, reducing tumor growth through inhibiting GLUT1-4-mediated glucose transport and metabolisms. Pan-class I GLUT inhibition is a better strategy than single GLUT targeting for inhibiting tumor growth.

RNA drug discovery: Conformational restriction enhances specific modulation of the T-box riboswitch function.

Antibacterial drug resistance is a global health concern that requires multiple solution approaches including development of new antibacterial compounds acting at novel targets. Targeting regulatory RNA is an emerging area of drug discovery. The T-box riboswitch is a regulatory RNA mechanism that controls gene expression in Gram-positive bacteria and is an exceptional, novel target for antibacterial drug design. We report the design, synthesis and activity of a series of conformationally restricted oxazolidinone-triazole compounds targeting the highly conserved antiterminator RNA element of the T-box riboswitch. Computational binding energies correlated with experimentally-derived Kd values indicating the predictive capabilities for docking studies within this series of compounds. The conformationally restricted compounds specifically inhibited T-box riboswitch function and not overall transcription. Complex disruption, computational docking and RNA binding specificity data indicate that inhibition may result from ligand binding to an allosteric site. These results highlight the importance of both ligand affinity and RNA conformational outcome for targeted RNA drug design.

Isosteres of ester derived glucose uptake inhibitors.

Glucose transporters (GLUTs) facilitate glucose uptake and are overexpressed in most cancer cells. Inhibition of glucose transport has been shown to be an effective method to slow the growth of cancer cells both in vitro and in vivo. We have previously reported on the anticancer activity of an ester derived glucose uptake inhibitor. Due to the hydrolytic instability of the ester linkage we have prepared a series of isosteres of the ester moiety. Of all of the isosteres prepared, the amine linkage showed the most promise. Several additional analogues of the amine-linked compounds were also prepared to improve the overall activity.

Modulation of LPS-induced inflammatory cytokine production by a novel glycogen synthase kinase-3 inhibitor.

Sepsis is a serious condition that can lead to long-term organ damage and death. At the molecular level, the hallmark of sepsis is the elevated expression of a multitude of potent cytokines, i.e. a cytokine storm. For sepsis involving gram-negative bacteria, macrophages recognize lipopolysaccharide (LPS) shed from the bacteria, activating Toll-like-receptor 4 (TLR4), and triggering a cytokine storm. Glycogen synthase kinase-3 (GSK-3) is a highly active kinase that has been implicated in LPS-induced cytokine production. Thus, compounds that inhibit GSK-3 could be potential therapeutics for sepsis. Our group has recently described a novel and highly selective inhibitor of GSK-3 termed COB-187. In the present study, using THP-1 macrophages, we evaluated the ability of COB-187 to attenuate LPS-induced cytokine production. We found that COB-187 significantly reduced, at the protein and mRNA levels, cytokines induced by LPS (e.g. IL-6, TNF-α, IL-1ß, CXCL10, and IFN-ß). Further, the data suggest that the inhibition could be due, at least in part, to COB-187 reducing NF-κB (p65/p50) DNA binding activity as well as reducing IRF-3 phosphorylation at Serine 396. Thus, COB-187 appears to be a potent inhibitor of the cytokine storm induced by LPS.

Identification of a novel selective and potent inhibitor of glycogen synthase kinase-3.

Glycogen synthase kinase-3 (GSK-3) is a multitasking protein kinase that regulates numerous critical cellular functions. Not surprisingly, elevated GSK-3 activity has been implicated in a host of diseases including pathological inflammation, diabetes, cancer, arthritis, asthma, bipolar disorder, and Alzheimer's. Therefore, reagents that inhibit GSK-3 activity provide a means to investigate the role of GSK-3 in cellular physiology and pathophysiology and could become valuable therapeutics. Finding a potent inhibitor of GSK-3 that can selectively target this kinase, among over 500 protein kinases in the human genome, is a significant challenge. Thus there remains a critical need for the identification of selective inhibitors of GSK-3. In this work, we introduce a novel small organic compound, namely COB-187, which exhibits potent and highly selective inhibition of GSK-3. Specifically, this study 1) utilized a molecular screen of 414 kinase assays, representing 404 unique kinases, to reveal that COB-187 is a highly potent and selective inhibitor of GSK-3; 2) utilized a cellular assay to reveal that COB-187 decreases the phosphorylation of canonical GSK-3 substrates indicating that COB-187 inhibits cellular GSK-3 activity; and 3) reveals that a close isomer of COB-187 is also a selective and potent inhibitor of GSK-3. Taken together, these results demonstrate that we have discovered a region of chemical design space that contains novel GSK-3 inhibitors. These inhibitors will help to elucidate the intricate function of GSK-3 and can serve as a starting point for the development of potential therapeutics for diseases that involve aberrant GSK-3 activity.

Antitumor Activities of the Novel Isosteviol Derivative 10C Against Liver Cancer.

BACKGROUND/AIM: Hepatocellular carcinoma (HCC) is the most common type of liver cancer and the fifth most common primary malignancy with worldwide increasing incidence. The current study aimed to investigate the anticancer activities of novel isosteviol derivatives towards human HepG2 hepatocellular cancer cells and in an animal model of hepatocellular carcinoma. MATERIALS AND METHODS: Twelve isosteviol derivatives were screened for their anti-proliferative activities against HepG2 and IC50 was calculated for all designed derivatives. The impact of the potent isosteviol derivative 10C on HepG2 cells was further studied by MTT assay, Annexin V/PI staining, flow cytometry and western blotting. In vivo studies were performed to assess the anticancer effect of isosteviol derivative 10C on Diethyl Nitrosamine-induced liver cancer in female rats by evaluating the physiological processes. RESULTS: isosteviol derivative 10C induced growth inhibition with IC50 of 2 µM mainly through induction of apoptosis in HepG2 cells. Additionally, isosteviol derivative 10C induced G1 phase arrest, which was further confirmed by increased expression of cyclin dependent kinase inhibitor 1A (CDKN1A, p21). It also increased BAX, BID and PARP-1 and while it reduced pro-CASPASE-3 expression and phosphorylation levels of AKT in HepG2 cells. Furthermore, western blotting data showed that E-cadherin, ß-catenin, VEGF and COX-2 expressions were suppressed by isosteviol derivative 10C in HepG2 cells. The in vivo study demonstrated that dose-dependent treatment of isosteviol derivative 10C led to significant reduction in tumor size compared to the untreated group after the fourth injection with no significant effects on major physiological processes. CONCLUSION: Taken together, in vitro and in vivo studies revealed that isosteviol derivative 10C induced apoptosis in HepG2 cells, blocked angiogenic signaling and it did not induce any apparent toxicity towards the treated hosts which merits further investigation at clinical level.

Phenylmethimazole and a thiazole derivative of phenylmethimazole inhibit IL-6 expression by triple negative breast cancer cells.

Inhibition of interleukin-6 (IL-6) holds significant promise as a therapeutic approach for triple negative breast cancer (TNBC). We previously reported that phenylmethimazole (C10) reduces IL-6 expression in several cancer cell lines. We have identified a more potent derivative of C10 termed COB-141. In the present work, we tested the hypothesis that C10 and COB-141 inhibit TNBC cell expressed IL-6 and investigated the potential for classical IL-6 pathway induced signaling within TNBC cells. A panel of TNBC cell lines (MDA-MB-231, Hs578T, MDA-MB-468) was used. Enzyme linked immunosorbent assays (ELISA) revealed that C10 and COB-141 inhibit MDA-MB-231 cell IL-6 secretion, with COB-141 being ~6.5 times more potent than C10. Therefore, the remainder of the study focused on COB-141 which inhibited IL-6 secretion, and was found, via quantitative real time polymerase chain reaction (QRT-PCR), to inhibit IL-6 mRNA in the TNBC panel. COB-141 had little, if any, effect on metabolic activity indicating that the IL-6 inhibition is not via a toxic effect. Flow cytometric analysis and QRT-PCR revealed that the TNBC cell lines do not express the IL-6 receptor (IL-6Rα). Trans-AM assays suggested that COB-141 exerts its inhibitory effect, at least in part, by reducing NF-κB (p65/p50) DNA binding. In summary, COB-141 is a potent inhibitor of TNBC cell expressed IL-6 and the inhibition does not appear to be due to non-specific toxicity. The TNBC cell lines do not have an intact classical IL-6 signaling pathway. COB-141's inhibitory effect may be due, at least in part, to reducing NF-κB (p65/p50) DNA binding.

Simple modifications to methimazole that enhance its inhibitory effect on tumor necrosis factor-α-induced vascular cell adhesion molecule-1 expression by human endothelial cells.

The expression of vascular cell adhesion molecule-1 (VCAM-1) on the vascular endothelium can be increased by pro-inflammatory cytokines [e.g. tumor necrosis factor-α (TNF-α)]. VCAM-1 contributes to leukocyte adhesion to, and emigration from, the vasculature which is a key aspect of pathological inflammation. As such, a promising therapeutic approach for pathological inflammation is to inhibit the expression of VCAM-1. Methimazole [3-methyl-1, 3 imidazole-2 thione (MMI)] is routinely used for the treatment of Graves׳ disease and patients treated with MMI have decreased levels of circulating VCAM-1. In this study we used cultured human umbilical vein endothelial cells (HUVEC) to investigate the effect of MMI structural modifications on TNF-α induced VCAM-1 expression. We found that addition of a phenyl ring at the 4-nitrogen of MMI yields a compound that is significantly more potent than MMI at inhibiting 24h TNF-α-induced VCAM-1 protein expression. Addition of a para methoxy to the appended phenyl group increases the inhibition while substitution of a thiazole ring for an imidazole ring in the phenyl derivatives yields no clear difference in inhibition. Addition of the phenyl ring to MMI appears to increase toxicity as does substitution of a thiazole ring for an imidazole ring in the phenyl MMI derivatives. Each of the compounds reduced TNF-α-induced VCAM-1 mRNA expression and had a functional inhibitory effect, i.e. each inhibited monocytic cell adhesion to 24h TNF-α-activated HUVEC under fluid flow conditions. Combined, these studies provide important insights into the design of MMI-related anti-inflammatory compounds.

Synthesis and cytotoxic activity of MOM-ether analogs of isosteviol.

Lung cancer is one of the most common malignancies worldwide. In this Letter, novel MOM-ether analogs of isosteviol were designed and synthesized to be tested for anticancer activities against H1299 lung cancer cell lines. The effects of these derivatives were studied in H1299 human large cell lung carcinoma cells that are null for p53 and compared to normal counterparts NL-20 normal lung epithelial cells. The initial screening of twelve MOM-ether analogs of isosteviol derivatives on H1299 lung cancer cells by MTT assay revealed that two derivatives (an ester and a carbamate) were the most potent in reducing cell viability. The IC50 values for these derivatives were determined to be 14 and 21 µM respectively. We compared the cytotoxicity of the best derivatives in H1299 lung cancer cells and NL-20 normal lung epithelial cells. Both derivatives showed lower cytotoxic effects on NL-20 normal lung epithelial cells. Moreover, both derivatives induced apoptosis in H1299 lung cancer cells more than NL-20 normal lung epithelial cells.

Defining the putative inhibitory site for a selective negative allosteric modulator of human α4ß2 neuronal nicotinic receptors.

Neuronal nicotinic receptors (nAChRs) have been implicated in several diseases and disorders such as autism spectrum disorders, Alzheimer's disease, Parkinson's disease, epilepsy, and nicotine addiction. To understand the role of nAChRs in these conditions, it would be beneficial to have selective molecules that target specific nAChRs in vitro and in vivo. Our laboratory has previously identified a novel allosteric site on human α4ß2 nAChRs using a series of computational and in vitro approaches. At this site, we have identified negative allosteric modulators that selectively inhibit human α4ß2 nAChRs, a subtype implicated in nicotine addiction. This study characterizes the allosteric site via site-directed mutagenesis. Three amino acids (Phe118, Glu60, and Thr58) on the ß2 subunit were shown to participate in the inhibitory properties of the selective antagonist KAB-18 and provided insights into its antagonism of human α4ß2 nAChRs. SAR studies with KAB-18 analogues and various mutant α4ß2 nAChRs also provided information concerning how different physiochemical features influence the inhibition of nAChRs through this allosteric site. Together, these studies identify the amino acids that contribute to the selective antagonism of human α4ß2 nAChRs at this allosteric site. Finally, these studies define the physiochemical features of ligands that influence interaction with specific amino acids in this allosteric site.

A small-molecule inhibitor of glucose transporter 1 downregulates glycolysis, induces cell-cycle arrest, and inhibits cancer cell growth in vitro and in vivo.

The functional and therapeutic importance of the Warburg effect is increasingly recognized, and glycolysis has become a target of anticancer strategies. We recently reported the identification of a group of novel small compounds that inhibit basal glucose transport and reduce cancer cell growth by a glucose deprivation-like mechanism. We hypothesized that the compounds target Glut1 and are efficacious in vivo as anticancer agents. Here, we report that a novel representative compound WZB117 not only inhibited cell growth in cancer cell lines but also inhibited cancer growth in a nude mouse model. Daily intraperitoneal injection of WZB117 at 10 mg/kg resulted in a more than 70% reduction in the size of human lung cancer of A549 cell origin. Mechanism studies showed that WZB117 inhibited glucose transport in human red blood cells (RBC), which express Glut1 as their sole glucose transporter. Cancer cell treatment with WZB117 led to decreases in levels of Glut1 protein, intracellular ATP, and glycolytic enzymes. All these changes were followed by increase in ATP-sensing enzyme AMP-activated protein kinase (AMPK) and declines in cyclin E2 as well as phosphorylated retinoblastoma, resulting in cell-cycle arrest, senescence, and necrosis. Addition of extracellular ATP rescued compound-treated cancer cells, suggesting that the reduction of intracellular ATP plays an important role in the anticancer mechanism of the molecule. Senescence induction and the essential role of ATP were reported for the first time in Glut1 inhibitor-treated cancer cells. Thus, WZB117 is a prototype for further development of anticancer therapeutics targeting Glut1-mediated glucose transport and glucose metabolism.

Fused ring aziridines as a facile entry into triazole fused tricyclic and bicyclic heterocycles.

The intramolecular dipolar cycloaddition of an azide with an alkyne has provided a useful entry into triazole fused tricyclic heterocycles containing both the triazole ring and the oxazolidin-2-one ring system. The requisite azido-alkynes have been prepared via a two-step sequence from fused ring aziridines. A series of 6-12 membered rings containing both the oxazolidinone and triazole rings have been prepared. These ring systems have been designed as conformationally restrained analogs of RNA-binding oxazolidinones.

3D-QSAR and 3D-QSSR models of negative allosteric modulators facilitate the design of a novel selective antagonist of human α4ß2 neuronal nicotinic acetylcholine receptors.

Subtype selective molecules for α4ß2 neuronal nicotinic acetylcholine receptors (nAChRs) have been sought as novel therapeutics for nicotine cessation. α4ß2 nAChRs have been shown to be involved in mediating the addictive properties of nicotine while other subtypes (i.e., α3ß4 and α7) are believed to mediate the undesired effects of potential CNS drugs. To obtain selective molecules, it is important to understand the physiochemical features of ligands that affect selectivity and potency on nAChR subtypes. Here we present novel QSAR/QSSR models for negative allosteric modulators of human α4ß2 nAChRs and human α3ß4 nAChRs. These models support previous homology model and site-directed mutagenesis studies that suggest a novel mechanism of antagonism. Additionally, information from the models presented in this work was used to synthesize novel molecules; which subsequently led to the discovery of a new selective antagonist of human α4ß2 nAChRs.

Ligand-induced changes in T box antiterminator RNA stability.

The T box antiterminator RNA element is an important component of the T box riboswitch that controls the transcription of vital genes in many Gram-positive bacteria. A series of 1,4-disubstituted 1,2,3-triazoles was screened in a fluorescence-monitored thermal denaturation assay to identify ligands that altered the stability of antiterminator model RNA. Several ligands were identified that significantly increased or decreased the melting temperature (T(m) ) of the RNA. The results indicate that this series of triazole ligands can alter the stability of antiterminator model RNA in a structure-dependent manner.

Synthesis of a functionalized oxabicyclo[2.2.1]-heptene-based chemical library.

The 7-oxabicyclo[2.2.1]heptene ring system is a common structural motif in many pharmacologically interesting molecules. We recognized the potential to employ this highly oxygenated and conformationally-restricted scaffold in diversity-oriented synthesis to generate a library of non-chiral but topologically complex compounds. Herein, we report the synthesis and biological evaluation of two 96-member tricyclic libraries containing the oxabicyclo[2.2.1]heptene framework using acetal formation as the key step.

Impact of fermentation, drying, roasting and Dutch processing on flavan-3-ol stereochemistry in cacao beans and cocoa ingredients.

This paper reports a systematic study of the level of flavan-3-ol monomers during typical processing steps as cacao beans are dried, fermented and roasted and the results of Dutch-processing. Methods have been used that resolve the stereoisomers of epicatechin and catechin. In beans harvested from unripe and ripe cacao pods, we find only (-)-epicatechin and (+)-catechin with (-)-epicatechin being by far the predominant isomer. When beans are fermented there is a large loss of both (-)-epicatechin and (+)-catechin, but also the formation of (-)-catechin. We hypothesize that the heat of fermentation may, in part, be responsible for the formation of this enantiomer. When beans are progressively roasted at conditions described as low, medium and high roast conditions, there is a progressive loss of (-)-epicatechin and (+)-catechin and an increase in (-)-catechin with the higher roast levels. When natural and Dutch-processed cacao powders are analyzed, there is progressive loss of both (-)-epicatechin and (+)-catechin with lesser losses of (-)-catechin. We thus observe that in even lightly Dutch-processed powder, the level of (-)-catechin exceeds the level of (-)-epicatechin. The results indicate that much of the increase in the level of (-)-catechin observed during various processing steps may be the result of heat-related epimerization from (-)-epicatechin. These results are discussed with reference to the reported preferred order of absorption of (-)-epicatechin > (+)-catechin > (-)-catechin. These results are also discussed with respect to the balance that must be struck between the beneficial impact of fermentation and roasting on chocolate flavor and the healthful benefits of chocolate and cocoa powder that result in part from the flavan-3-ol monomers.