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Previously, we identified that AP-1 transcription factor FOSL1 is required to maintain cancer stem cells (CSCs) in HNSCC, and an AP-1 inhibitor, T-5224, can eliminate HNSCC CSCs. However, its potency is relatively low, and furthermore, whether T-5224 eradicates CSCs through targeting FOSL1 and whether FOSL1 serves as an effective target for eliminating CSCs in HNSCC, require further validation. We first found that T-5224 can bind to FOSL1 directly. As a proof-of-principle, several cereblon (CRBN)-recruiting PROTACs were designed and synthesized using T-5224 as a warhead for more effective of targeting FOSL1. The top compound can potently degrade FOSL1 in HNSCC, thereby effectively eliminating CSCs to suppress HNSCC tumorigenesis, with around 30 to 100-fold improved potency over T-5224. In summary, our study further validates FOSL1 as an effective target for eliminating CSCs in HNSCC and suggests that PROTACs may provide a unique molecular tool for the development of novel molecules for targeting FOSL1., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

It has been demonstrated that the KDM3 family of histone demethylases (KDM3A and KDM3B) epigenetically control the functional properties of colorectal cancer stem cells (CSCs) through Wnt/β-catenin signaling. Meanwhile, a broad-spectrum histone demethylase inhibitor, IOX1, suppresses Wnt-induced colorectal tumorigenesis predominantly through inhibiting the enzymatic activity of KDM3. In this work, several cereblon (CRBN)-recruiting PROTACs with various linker lengths were designed and synthesized using IOX1 as a warhead to target KDM3 proteins for degradation. Two of the synthesized PROTACs demonstrated favorable degradation profile and selectivity towards KDM3A and KDM3B. Compound 4 demonstrated favorable in vitro metabolic profile in liver enzymes as well as no hERG-associated cardiotoxicity. Compound 4 also showed dramatic ability in suppressing oncogenic Wnt signaling to eliminate colorectal CSCs and inhibit tumor growth, with around 10- to 35-fold increased potency over IOX1. In summary, this study suggests that PROTACs provide a unique molecular tool for the development of novel small molecules from the IOX1 skeleton for selective degradation of KDM3 to eliminate colorectal CSCs via suppressing oncogenic Wnt signaling., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

For the last two decades, the aromatic aldehyde 5-hydroxymethyl-furfural (5-HMF) has been the subject of several investigations for its pharmacologic potential. In 2004, the Safo group reported that 5-HMF has potent antisickling activity by targeting and ameliorating the primary pathophysiology of hypoxia-induced sickling of erythrocytes (red blood cells [RBC]). Following the encouraging outcome of the preclinical and phase I/II clinical studies of 5-HMF for the treatment of sickle cell disease (SCD), there have been multiple studies suggesting 5-HMF has several other biological or pharmacologic activities, including anti-allergic, antioxidant, anti-hypoxic, anti-ischemic, cognitive improvement, anti-tyrosinase, anti-proliferation, cytoprotective, and anti-inflammatory activities. The wide range of its effects makes 5-HMF a potential candidate for treating a variety of diseases including cognitive disorders, gout, allergic disorders, anemia, hypoxia, cancers, ischemia, hemorrhagic shock, liver fibrosis, and oxidative injury. Several of these therapeutic claims are currently under investigation and, while promising, vary in terms of the strength of their evidence. This review presents the research regarding the therapeutic potential of 5-HMF in addition to its sources, physicochemical properties, safety, absorption, distribution, metabolism, and excretion (ADME) profiles., (© 2024 The Author(s). Medicinal Research Reviews published by Wiley Periodicals LLC.)

The number of opioid-related overdose deaths and individuals that have suffered from opioid use disorders have significantly increased over the last 30 years. FDA approved maintenance therapies to treat opioid use disorder may successfully curb drug craving and prevent relapse but harbor adverse effects that reduce patient compliance. This has created a need for new chemical entities with improved patient experience. Previously our group reported a novel lead compound, NAT, a mu-opioid receptor antagonist that potently antagonized the antinociception of morphine and showed significant blood-brain barrier permeability. However, NAT belongs to thiophene containing compounds which are known structural alerts for potential oxidative metabolism. To overcome this, 15 NAT derivatives with various substituents at the 5'-position of the thiophene ring were designed and their structure-activity relationships were studied. These derivatives were characterized for their binding affinity, selectivity, and functional activity at the mu opioid receptor and assessed for their ability to antagonize the antinociceptive effects of morphine in vivo. Compound 12 showed retention of the basic pharmacological attributes of NAT while improving the withdrawal effects that were experienced in opioid-dependent mice. Further studies will be conducted to fully characterize compound 12 to examine whether it would serve as a new lead for opioid use disorder treatment and management., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Despite the availability of numerous pain medications, the current array of Food and Drug Administration-approved options falls short in adequately addressing pain states for numerous patients and consequently worsens the opioid crisis. Thus, it is imperative for basic research to develop novel and nonaddictive pain medications. Toward addressing this clinical goal, nalfurafine (NLF) was chosen as a lead and its structure-activity relationship (SAR) systematically studied through design, syntheses, and in vivo characterization of 24 analogues. Two analogues, 21 and 23 , showed longer durations of action than NLF in a warm-water tail immersion assay, produced in vivo effects primarily mediated by KOR and DOR, penetrated the blood-brain barrier, and did not function as reinforcers. Additionally, 21 produced fewer sedative effects than NLF. Taken together, these results aid the understanding of NLF SAR and provide insights for future endeavors in developing novel nonaddictive therapeutics to treat pain.

While agonists of mu (MOR) and kappa (KOR) opioid receptors have analgesic effects, they produce euphoria and dysphoria, respectively. Other side effects include respiratory depression and addiction for MOR agonists and sedation for KOR agonists. We reported that 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-{[4'-(2'-cyanopyridyl)]carboxamido}cmorphinan (NCP) displayed potent KOR full agonist and MOR partial agonist activities (58%) with 6.5x KOR-over-MOR selectivity in vitro Herein, we characterized pharmacological effects of NCP in rodents. In mice, NCP exerted analgesic effects against inflammatory pain in both the formalin test and the acetic acid writhing test, with A 50 values of 47.6 and 14.4 microg/kg (s.c.), respectively. The analgesic effects in the acetic acid writhing test were mediated by the KOR. NCP at doses much higher than those effective in reducing inflammatory pain did not produce antinociception in the hot plate and tail flick tests, inhibit compound 48/80-induced scratching, cause conditioned place aversion (CPA) or preference, impair rotarod performance, inhibit locomotor activity, cause respiratory depression, or precipitate morphine withdrawal. However, NCP (10~100 microg/kg) inhibited gastrointestinal transit with a maximum of ~40% inhibition. In MOR knockout mice, NCP caused CPA, demonstrating that its lack of CPA is due to combined actions on the MOR and KOR. Following s.c. injection, NCP penetrated into the mouse brain. In rats trained to self-administer heroin, NCP (1~320 microg/kg/infusion) did not function as a reinforcer. Thus, NCP produces potent analgesic effects via KOR without side effects except constipation. Therefore, dual full KOR/partial MOR agonists with moderate KOR-over-MOR selectivity may be promising as non-addictive analgesics for inflammatory pain. Significance Statement Developing non-addictive analgesics is crucial for reducing opioid overdose deaths, minimizing drug misuse, and promoting safer pain management practices. Herein, pharmacology of a potential non-addictive analgesic, NCP, is reported. NCP has full KOR agonist / partial MOR agonist activities with a 6.5 x selectivity for KOR over MOR. Unlike MOR agonists, analgesic doses of NCP do not lead to self-administration or respiratory depression. Furthermore, NCP does not produce aversion, hypolocomotion, or motor incoordination, side effects typically associated with KOR activation., (Copyright © 2024 American Society for Pharmacology and Experimental Therapeutics.)

While there are approved therapeutics to treat opioid overdoses, the need for treatments to reverse overdoses due to ultrapotent fentanyls remains unmet. This may be due in part to an adrenergic mechanism of fentanyls in addition to their stereotypical mu-opioid receptor (MOR) effects. Herein, we report our efforts to further understanding of the functions these distinct mechanisms impart. Employing the known MOR neutral antagonist phenylfentanil as a lead, 17 analogues were designed based on the concept of isosteric replacement. To probe mechanisms of action, these analogues were pharmacologically evaluated in vitro and in vivo, while in silico modeling studies were also conducted on phenylfentanil. While it did not indicate MOR involvement in vivo, phenylfentanil yielded respiratory minute volumes similar to those caused by fentanyl. Taken together with molecular modeling studies, these results indicated that respiratory effects of fentanyls may also correlate to inhibition of both α1A- and α1B-adrenergic receptors.

Opioid Use Disorder (OUD) can be described as intense preoccupation with using or obtaining opioids despite the negative consequences associated with their use. As the number of OUD cases in the U.S. increase, so do the number of opioid-related overdose deaths. In 2022, opioid-related overdose became the No. 1 cause of death for individuals in the U.S. between the ages of 25 and 64 years of age. Because of the introduction of highly potent synthetic opioids (e.g. fentanyl) to the illicit drug market, there is an urgent need for therapeutics that successfully reduce the number of overdoses and can help OUD patients maintain sobriety. Most abused opioids stimulate the mu-opioid receptor (MOR) and activation of this receptor can lead to positive (e.g., euphoria) consequences. However, the negative side effects of MOR stimulation can be fatal (e.g., sedation, respiratory depression). Therefore, the MOR is an attractive target for developing medications to treat OUD. Current FDA drugs include MOR agonists that aid in detoxification and relapse prevention, and MOR antagonists that also serve as maintenance therapies or reverse overdose. These medications are limited by their abuse potential, adverse effects, or pharmacological profiles which leaves ample room for research into designing new chemical entities with optimal physiological effects. These includes, orthosteric ligands that target the primary binding site of the MOR, allosteric ligands that positively, negatively, or "silently" modulate receptor function, and lastly, bitopic ligands target both the orthosteric and allosteric sites simultaneously., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The synthetic allosteric effector of hemoglobin, TD-7 has been investigated as a potential therapeutic agent for the treatment of sickle cell disease. The pharmacologic activity of TD-7 is due to formation of a Schiff-base interaction between its aldehyde group and the two N-terminal αVal1 amines of hemoglobin, effectively inhibiting sickling of red blood cells. However, TD-7 faces a challenge in terms of poor oral bioavailability due to rapid in-vivo oxidative metabolism of its aldehyde functional group. To address this shortcoming, researches have explored the use of a L-cysteine ethyl ester group to cap the aldehyde group to form a thiazolidine aromatic aldehyde prodrug complex, resulting in the improvement of the metabolic stability of this class of compounds. This report details the synthesis of a thiazolidine prodrug of TD-7, referred to as Pro-7, along with a comprehensive investigation of Pro-7 functional and biological properties. In an in-vitro Hb modification and Hb oxygen affinity studies using normal whole blood, as well as erythrocyte sickling inhibition using sickle whole blood, Pro-7 exhibited a gradual onset but progressive increase in all activities. Additionally, in-vivo pharmacokinetic studies conducted with Sprague Dawley rats demonstrated that Pro-7 can undergo hydrolysis to release TD-7. However, the blood concentration of TD-7 did not reach the desired therapeutic level. These findings suggest that the incorporation of the L-cysteine ethyl ester group to TD-7 represents a promising strategy to enhance the metabolic stability of aromatic aldehydes that could lead to the development of a more effective drug for the treatment of sickle cell disease.

X-ray crystallography and structure-based drug discovery have played a major role in the discovery of antisickling agents that target hemoglobin (Hb) for the treatment of sickle cell disease (SCD). Sickle cell disease, the most common inherited hematologic disorder, occurs as a result of a single point mutation of βGlu6 in normal human adult hemoglobin (HbA) to βVal6 in sickle hemoglobin (HbS). The disease is characterized by polymerization of HbS and sickling of red blood cells (RBCs), leading to several secondary pathophysiologies, including but not limited to vaso-occlusion, hemolytic anemia, oxidative stress, inflammation, stroke, pain crisis, and organ damage. Despite the fact that SCD was the first disease to have its molecular basis established, the development of therapies was for a very long time a challenge and took several decades to find therapeutic agents. The determination of the crystal structure of Hb by Max Perutz in the early 60s, and the pioneering X-ray crystallography research by Donald J. Abraham in the early 80s, which resulted in the first structures of Hb in complex with small molecule allosteric effectors of Hb, gave much hope that structure-based drug discovery (SBDD) could be used to accelerate development of antisickling drugs that target the primary pathophysiology of hypoxia-induced HbS polymerization to treat SCD. This article, which is dedicated to Donald J. Abraham, briefly reviews structural biology, X-ray crystallography and structure-based drug discovery from the perspective of Hb. The review also presents the impact of X-ray crystallography in SCD drug development using Hb as a target, emphasizing the major and important contributions by Don Abraham in this field., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Donkor, Pagare, Mughram and Safo.)

The search for selective opioid ligands with desired pharmacological potency and improved safety profile has always been an area of interest. Our previous effort yielded a potent opioid modulator, NAN, a 6α- N -7'-indolyl-substituted naltrexamine derivative, which exhibited promising pharmacological activities both in vitro and in vivo. However, significant human ether-a-go-go-related gene (hERG) liability limited its further development. Therefore, a systematic structural modification on NAN was conducted in order to alleviate hERG toxicity while preserving pharmacological properties, which led to the discovery of 2'-methylindolyl derivative compound 21 . Compared to NAN, compound 21 manifested overall improved pharmacological profiles. Follow-up hERG channel inhibition evaluation revealed a seven-fold decreased potency of compound 21 compared to NAN. Furthermore, several fundamental drug-like property evaluations suggested a reasonable ADME profile of 21 . Collectively, compound 21 appeared to be a promising opioid modulator for further development as a novel therapeutic agent toward opioid use disorder treatments.

Mu opioid receptor (MOR) selective antagonists and partial agonists have clinical utility for the treatment of opioid use disorders (OUDs). However, the development of many has suffered due to their poor pharmacokinetic properties and/or rapid metabolism. Our recent efforts to identify MOR modulators have provided 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(isoquinoline-3-carboxamido)morphinan (NAQ), a low-efficacy partial agonist, that showed sub-nanomolar binding affinity to the MOR ( K i 0.6 nM) with selectivity over the delta opioid receptor (δ/μ 241) and the kappa opioid receptor (κ/μ 48). Its potent inhibition of the analgesic effect of morphine (AD 50 0.46 mg/kg) and precipitation of significantly less withdrawal symptoms even at 100-fold greater dose than naloxone represents a promising molecule for further development as a novel OUD therapeutic agent. Therefore, further in vitro and in vivo characterization of its pharmacokinetics and pharmacodynamics properties was conducted to fully understand its pharmaceutical profile. NAQ showed favorable in vitro ADMET properties and no off-target binding to several classes of GPCRs, enzymes, and ion channels. Following intravenous administration, 1 mg/kg dose of NAQ showed a similar in vivo pharmacokinetic profile to naloxone; however, orally administered 10 mg/kg NAQ demonstrated significantly improved oral bioavailability over both naloxone and naltrexone. Abuse liability assessment of NAQ in rats demonstrated that NAQ functioned as a less potent reinforcer than heroin. Chronic 5 day NAQ pretreatment decreased heroin self-administration in a heroin-vs-food choice procedure similar to the clinically used MOR partial agonist buprenorphine. Taken together, these studies provide evidence supporting NAQ as a promising lead to develop novel OUD therapeutics., Competing Interests: The authors declare no competing financial interest., (© 2022 American Chemical Society.)

The Wnt/β-catenin signaling pathway plays extensive roles in cancer initiation, proliferation, and development, and has been implicated in the regulation of stem cells in the intestinal crypt, widely accepted as responsible for colorectal cancer (CRC) origination. This pathway has been a target of interest for many years for chemotherapeutic development of CRC due to its implication in most cases. Previously, a series of naphthoquinone analogs have been identified to inhibit the Wnt/β-catenin. It was postulated that these compounds exhibit their inhibitory activity via binding to β-catenin at the β-catenin/TCF4 interaction interface. In this study, we aimed to further define the critical pharmacophore for these compounds and verify their mechanisms of action for their abilities to inhibit the Wnt/β-catenin signaling pathway. Interestingly, our data suggested two of the compounds, compounds 3 and 6, may potently inhibit the Wnt/β-catenin signaling pathway via inhibition of the TCF4/DNA interaction, a novel finding compared to previous studies on these compounds. Our computational studies suggested that the compounds bound within the DNA binding HMG-box domain of TCF4 to elicit their inhibitory action. These compounds inhibited Wnt signaling in a dose dependent manner, suppressed Wnt direct target genes and demonstrated unforeseen degradation of the TCF4 protein. Thus, this study revealed a potentially novel mechanism of action of the chloro-naphthoquinone as possibly a multi-targeting scaffold, which warrants further investigation in future drug discovery on the 'undruggable" TCF proteins and an aberrantly activated Wnt/β-catenin signaling pathway., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The Wnt/β-catenin pathway has a crucial role in the proliferation and differentiation of normal cells as well as the self-renewal and pluripotency of stem cells, including cancer stem cells (CSCs). Targeting this pathway with small-molecule chemotherapeutics, discovered via conventional efforts, has proved difficult. Recently, computer-aided drug discovery efforts have produced promising chemotherapeutics. A concerted effort to develop inhibitors of this pathway through more efficient and cost-effective drug discovery methods could lead to a significant increase in clinically relevant therapeutics. Herein, patents from 2010 to 2020 are reviewed to identify those that have disclosed composition of matter for small-molecule inhibitors of the Wnt/ β-catenin pathway for cancer. We believe that such efforts will provide insights for future therapeutic candidate discovery and development in this field., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The μ opioid receptor (MOR) has been an intrinsic target to develop treatment of opioid use disorders (OUD). Herein, we report our efforts on developing centrally acting MOR antagonists by structural modifications of 17-cyclopropylmethyl-3,14-dihydroxy-4,5α-epoxy-6β-[(4'-pyridyl) carboxamido] morphinan (NAP), a peripherally acting MOR-selective antagonist. An isosteric replacement concept was applied and incorporated with physiochemical property predictions in the molecular design. Three analogs, namely, 25 , 26 , and 31 , were identified as potent MOR antagonists in vivo with significantly fewer withdrawal symptoms than naloxone observed at similar doses. Furthermore, brain and plasma drug distribution studies supported the outcomes of our design strategy on these compounds. Taken together, our isosteric replacement of pyridine with pyrrole, furan, and thiophene provided insights into the structure-activity relationships of NAP and aided the understanding of physicochemical requirements of potential CNS acting opioids. These efforts resulted in potent, centrally efficacious MOR antagonists that may be pursued as leads to treat OUD.

The functional interactions between opioid and chemokine receptors have been implicated in the pathological process of chronic pain. Mounting studies have indicated the possibility that a MOR-CXCR4 heterodimer may be involved in nociception and related pharmacologic effects. Herein we have synthesized a series of bivalent ligands containing both MOR agonist and CXCR4 antagonist pharmacophores with an aim to investigate the functional interactions between these two receptors. In vitro studies demonstrated reasonable recognition of designed ligands at both respective receptors. Further antinociceptive testing in mice revealed compound 1a to be the most promising member of this series. Additional molecular modeling studies corroborated the findings observed. Taken together, we identified the first bivalent ligand 1a showing promising antinociceptive effect by targeting putative MOR-CXCR4 heterodimers, which may serve as a novel chemical probe to further develop more potent bivalent ligands with potential application in analgesic therapies for chronic pain management., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Introduction: Sickle cell disease (SCD) is a debilitating inherited disorder that affects millions worldwide. Four novel SCD therapeutics have been approved, including the hemoglobin (Hb) modulator Voxelotor., Areas Covered: This review provides an overview of discovery efforts toward modulating Hb allosteric behavior as a treatment for SCD, with a focus on aromatic aldehydes that increase Hb oxygen affinity to prevent the primary pathophysiology of hypoxia-induce erythrocyte sickling., Expert Opinion: The quest to develop small molecules, especially aromatic aldehydes, to modulate Hb allosteric properties for SCD began in the 1970s; however, early promise was dogged by concerns that stalled support for research efforts. Persistent efforts eventually culminated in the discovery of the anti-sickling agent 5-HMF in the 2000s, and reinvigorated interest that led to the discovery of vanillin analogs, including Voxelotor, the first FDA approved Hb modulator for the treatment of SCD. With burgeoning interest in the field of Hb modulation, there is a growing landscape of intellectual property, including drug candidates at various stages of preclinical and clinical investigations. Hb modulators could provide not only the best chance for a highly effective oral therapy for SCD, especially in the under-developed world, but also a way to treat a variety of other human conditions.

Background: Aromatic aldehydes, with their ability to increase the oxygen affinity of sickle hemoglobin, have become important therapeutic agents for sickle cell disease (SCD). One such compound, voxelotor, was recently approved for SCD treatment. Methyl 6-((2-formyl-3-hydroxyphenoxy)methyl) picolinate (PP10) is another promising aromatic aldehyde, recently reported by our group. Like voxelotor, PP10 exhibits O 2 -dependent antisickling activity, but, unlike voxelotor, PP10 shows unique O 2 -independent antisickling effect. PP10, however, has limited solubility. This study therefore aimed to develop oral and parenteral formulations to improve PP10 solubility and bioavailability., Methods: Oral drug tablets with 2-hydroxypropyl beta cyclodextrin (HP-β-CD), polyvinylpyrrolidone, or Eudragit L100-55 PP10-binary system, and an intravenous (IV) formulation with d-α-tocopherol polyethylene glycol 1000 succinate (TPGS) or HP-β-CD, were developed. The pharmacokinetic behavior of the formulations was studied in Sprague-Dawley rats. PP10, a methylester, and its acid metabolite were also studied in vitro with sickle whole blood to determine their effect on Hb modification, Hb oxygen affinity, and sickle red blood cell inhibition., Results: Aqueous solubility of PP10 was enhanced ~5 times with the HP-β-CD binary system, while the TPGS aqueous micelle formulation was superior, with a drug concentration of 0.502 ± 0.01 mg/mL and a particle size of 26 ± 3 nm. The oral tablets showed relative and absolute bioavailabilities of 173.4% and 106.34%, respectively. The acid form of PP10 appeared to dominate in vivo, although both PP10 forms demonstrated pharmacologic effect., Conclusion: Oral and IV formulations of PP10 were successfully developed using HP-β-CD binary system and TPGS aqueous micelles, respectively, resulting in significantly improved solubility and bioavailability.

GABA (γ-amino butyric acid) is an important inhibitory neurotransmitter in the central nervous system. Attenuation of GABAergic neurotransmission plays an important role in the etiology of several neurological disorders including epilepsy, Alzheimer's disease, Huntington's chorea, migraine, Parkinson's disease, neuropathic pain, and depression. Increase in the GABAergic activity may be achieved through direct agonism at the GABA A receptors, inhibition of enzymatic breakdown of GABA, or by inhibition of the GABA transport proteins (GATs). These functionalities make GABA receptor modulators and GATs attractive drug targets in brain disorders associated with decreased GABA activity. There have been several reports of development of GABA modulators (GABA receptors, GABA transporters, and GABAergic enzyme inhibitors) in the past decade. Therefore, the focus of the present review is to provide an overview on various design strategies and synthetic approaches toward developing GABA modulators. Furthermore, mechanistic insights, structure-activity relationships, and molecular modeling inputs for the biologically active derivatives have also been discussed. Summary of the advances made over the past few years in the clinical translation and development of GABA receptor modulators is also provided. This compilation will be of great interest to the researchers working in the field of neuroscience. From the light of detailed literature, it can be concluded that numerous molecules have displayed significant results and their promising potential, clearly placing them ahead as potential future drug candidates.

Aromatic aldehydes elicit their antisickling effects primarily by increasing the affinity of hemoglobin (Hb) for oxygen (O 2 ). However, challenges related to weak potency and poor pharmacokinetic properties have hampered their development to treat sickle cell disease (SCD). Herein, we report our efforts to enhance the pharmacological profile of our previously reported compounds. These compounds showed enhanced effects on Hb modification, Hb-O 2 affinity, and sickling inhibition, with sustained pharmacological effects in vitro . Importantly, some compounds exhibited unusually high antisickling activity despite moderate effects on the Hb-O 2 affinity, which we attribute to an O 2 -independent antisickling activity, in addition to the O 2 -dependent activity. Structural studies are consistent with our hypothesis, which revealed the compounds interacting strongly with the polymer-stabilizing αF-helix could potentially weaken the polymer. In vivo studies with wild-type mice demonstrated significant pharmacologic effects. Our structure-based efforts have identified promising leads to be developed as novel therapeutic agents for SCD.

Sickle cell disease (SCD) results from a hemoglobin (Hb) mutation βGlu6 → βVal6 that changes normal Hb (HbA) into sickle Hb (HbS). Under hypoxia, HbS polymerizes into rigid fibers, causing red blood cells (RBCs) to sickle; leading to numerous adverse pathological effects. The RBC sickling is made worse by the low oxygen (O 2 ) affinity of HbS, due to elevated intra-RBC concentrations of the natural Hb effector, 2,3-diphosphoglycerate. This has prompted the development of Hb modifiers, such as aromatic aldehydes, with the intent of increasing Hb affinity for O 2 with subsequent prevention of RBC sickling. One such molecule, Voxelotor was recently approved by U.S. FDA to treat SCD. Here we report results of a novel aromatic aldehyde, VZHE-039, that mimics both the O 2 -dependent and O 2 -independent antisickling properties of fetal hemoglobin. The latter mechanism of action-as elucidated through crystallographic and biological studies-is likely due to disruption of key intermolecular contacts necessary for stable HbS polymer formation. This dual antisickling mechanism, in addition to VZHE-039 metabolic stability, has translated into significantly enhanced and sustained pharmacologic activities. Finally, VZHE-039 showed no significant inhibition of several CYPs, demonstrated efficient RBC partitioning and high membrane permeability, and is not an efflux transporter (P-gp) substrate.

Antagonists for the serotonin receptor 2B (5-HT 2B ) have clinical applications towards migraine, anxiety, irritable bowl syndrome, and MDMA abuse; however, few selective 5-HT 2B antagonists have been identified. Previous studies from these labs identified a natural product, 5-hydroxy-2-(2-phenylethyl)chromone (5-HPEC, 2) as the first non-nitrogenous ligand for the 5-HT 2B receptor. Studies on 5-HPEC optimization led to the identification of 5-hydroxy-2-(3-phenylpropyl)chromone (5-HPPC, 3), which showed a tenfold improvement in binding affinity over 2 at 5-HT 2B . This study aimed to further improve receptor pharmacology of this unique scaffold. Guided by molecular modeling studies modifications at the C-3' and C-4' positions of 3 were made to probe their effects on ligand binding affinity and efficacy. Among the derivatives synthesized 5-hydroxy-2-(3-(3-cyanophenyl)propyl)chromone (5-HCPC, 3d) showed the most promise with a multifold improvement in binding affinity (pK i  = 7.1 ± 0.07) over 3 with retained antagonism., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Cancer is often associated with alterations in the chaperome, a collection of chaperones, cochaperones, and other cofactors. Changes in the expression levels of components of the chaperome, in the interaction strength among chaperome components, alterations in chaperome constituency, and in the cellular location of chaperome members, are all hallmarks of cancer. Here we aim to provide an overview on how chemical biology has played a role in deciphering such complexity in the biology of the chaperome in cancer and in other diseases. The focus here is narrow and on pathologic changes in the chaperome executed by enhancing the interaction strength between components of distinct chaperome pathways, specifically between those of HSP90 and HSP70 pathways. We will review chemical tools and chemical probe-based assays, with a focus on HSP90. We will discuss how kinetic binding, not classical equilibrium binding, is most appropriate in the development of drugs and probes for the chaperome in disease. We will then present our view on how chaperome inhibitors may become potential drugs and diagnostics in cancer., (Copyright © 2020 Cold Spring Harbor Laboratory Press; all rights reserved.)

The intracellular polymerization and the concomitant sickling processes, central to the pathology of sickle cell disease, can be mitigated by increasing the oxygen affinity of sickle hemoglobin (HbS). Attempts to develop azolylacryloyl derivatives to covalently interact with βCys93 and destabilize the low-O 2 -affinity T-state (deoxygenated) HbS to the polymer resistant high-O 2 -affinity R-state (liganded) HbS were only partially successful. This was likely due to the azolylacryloyls carboxylate moiety directing the compounds to also bind in the central water cavity of deoxygenated Hb and stabilizing the T-state. We now report a second generation of KAUS compounds (KAUS-28, KAUS-33, KAUS-38, and KAUS-39) without the carboxylate moiety designed to bind exclusively to βCys93. As expected, the compounds showed reactivity with both free amino acid l-Cys and the Hb βCys93. At 2 mM concentrations, the compounds demonstrated increased Hb affinity for oxygen (6% to 15%) in vitro , while the previously reported imidazolylacryloyl carboxylate derivative, KAUS-15 only showed 4.5% increase. The increased O 2 affinity effects were sustained through the experimental period of 12 h for KAUS-28, KAUS-33, and KAUS-38, suggesting conserved pharmacokinetic profiles. When incubated at 2 mM with red blood cells from patients with homozygous SS, the compounds inhibited erythrocyte sickling by 5% to 9%, respectively in correlation with the increase Hb-O 2 affinity. These values compare to 2% for KAUS-15. When tested with healthy mice, KAUS-38 showed very low toxicity., (This journal is © The Royal Society of Chemistry 2019.)

Increasing the affinity of hemoglobin for oxygen represents a feasible and promising therapeutic approach for sickle cell disease by mitigating the primary pathophysiological event, i.e. the hypoxia-induced polymerization of sickle hemoglobin (Hb S) and the concomitant erythrocyte sickling. Investigations on a novel synthetic antisickling agent, SAJ-310, with improved and sustained antisickling activity have previously been reported. To further enhance the biological effects of SAJ-310, a structure-based approach was employed to modify this compound to specifically inhibit Hb S polymer formation through interactions which perturb the Hb S polymer-stabilizing αF-helix, in addition to primarily increasing the oxygen affinity of hemoglobin. Three compounds, TD-7, TD-8 and TD-9, were synthesized and studied for their interactions with hemoglobin at the atomic level, as well as their functional and antisickling activities in vitro. X-ray crystallographic studies with liganded hemoglobin in complex with TD-7 showed the predicted mode of binding, although the interaction with the αF-helix was not as strong as expected. These findings provide important insights and guidance towards the development of molecules that would be expected to bind and make stronger interactions with the αF-helix, resulting in more efficacious novel therapeutics for sickle cell disease.

Glucose Regulated Protein 170 (GRP170), also called Oxygen Regulated Protein 150 (ORP150), is a major molecular chaperone resident in the endoplasmic reticulum (ER). It belongs to the heat shock protein (HSP70) super family and can be induced by conditions such as hypoxia, ischemia and interferences in calcium homeostasis. It was recently reported that GRP170 may act as a nucleotide exchange factor (NEF) for GRP78 or binding immunoglobulin protein (BiP), and the ER canonical HSP70. However, little is known about the mechanism underlying its NEF activity. In this study, two homology models of GRP170 were constructed based on the X-ray crystal structures of ADP and ATP bound HSP110, a cytosolic homolog of GRP170, in order to characterize the differences in the binding modes of both ligands. It was observed that the differences in the binding modes of ADP and ATP led to a conformation change in the substrate binding domain which could potentially influence the binding of its substrates such as BiP. Our findings help understand the effect of nucleotide binding on the function of this chaperone protein as a NEF as well as the structural differences between GRP170 and its family members., (Published by Elsevier Inc.)

Hypoxia-induced polymerization of sickle hemoglobin (Hb S) is the principal phenomenon that underlays the pathophysiology and morbidity associated with sickle cell disease (SCD). Opportunely, as an allosteric protein, hemoglobin (Hb) serves as a convenient and potentially critical druggable target. Consequently, molecules that prevent Hb S polymerization (Hb modifiers), and the associated erythrocyte sickling have been investigated-and retain significant interest-as a viable therapeutic strategy for SCD. This group of molecules, including aromatic aldehydes, form high oxygen affinity Schiff-base adducts with Hb S, which are resistant to polymerization. Here, we report the design and synthesis of novel potent antisickling agents (SAJ-009, SAJ-310 and SAJ-270) based on the pharmacophore of vanillin and INN-312, a previously reported pyridyl derivative of vanillin. These novel derivatives exhibited superior in vitro binding and pharmacokinetic properties compared to vanillin, which translated into significantly enhanced allosteric and antisickling properties. Crystal structure studies of liganded Hb in the R2 quaternary state in complex with SAJ-310 provided important insights into the allosteric and antisickling properties of this group of compounds. While these derivatives generally show similar in vitro biological potency, significant structure-dependent differences in their biochemical profiles would help predict the most promising candidates for successful in vivo pre-clinical translational studies and inform further structural modifications to improve on their pharmacologic properties., (Published by Elsevier Ltd.)

Candidate drugs to counter intracellular polymerization of deoxygenated sickle hemoglobin (Hb S) continue to represent a promising approach to mitigating the primary cause of the pathophysiology associated with sickle cell disease (SCD). One such compound is the naturally occurring antisickling agent, 5-hydroxymethyl-2-furfural (5-HMF), which has been studied in the clinic for the treatment of SCD. As part of our efforts to develop novel efficacious drugs with improved pharmacologic properties, we structurally modified 5-HMF into 12 ether and ester derivatives. The choice of 5-HMF as a pharmacophore was influenced by a combination of its demonstrated attractive hemoglobin modifying and antisickling properties, well-known safety profiles, and its reported nontoxic major metabolites. The derivatives were investigated for their time- and/or dose-dependent effects on important antisickling parameters, such as modification of hemoglobin, corresponding changes in oxygen affinity, and inhibition of red blood cell sickling. The novel test compounds bound and modified Hb and concomitantly increased the protein affinity for oxygen. Five of the derivatives exhibited 1.5- to 4.0-fold higher antisickling effects than 5-HMF. The binding mode of the compounds with Hb was confirmed by X-ray crystallography and, in part, helps explain their observed biochemical properties. Our findings, in addition to the potential therapeutic application, provide valuable insights and potential guidance for further modifications of these (and similar) compounds to enhance their pharmacologic properties.

Scavenger receptor A (SRA), as an immune regulator, has been shown to play important roles in lipid metabolism, cardiovascular diseases, and pathogen recognition. Several natural product inhibitors of SRA have been studied for their potential application in modulating SRA functions. To understand the binding mode of these inhibitors on SRA, we conducted systematic molecular modeling studies in order to identify putative binding domain(s) that may be responsible for their recognition to the receptor as well as their inhibitory activity. Treatment of SRA with one of the natural product inhibitors, rhein, led to significant dissociation of SRA oligomers to its trimer and dimer forms, which further supported our hypothesis on their putative mechanism of action. Such information is believed to shed light on design of more potent inhibitors for the receptor in order to develop potential therapeutics through immune system modulation., (Published by Elsevier Inc.)

Scavenger receptor A (SRA) has been known as an immunosuppressive factor and therefore therapeutic inhibition of SRA may be potentially exploited for cancer immunotherapy. Our previously work suggested that rhein may act as an inhibitor of SRA in reversing immunosuppression of SRA during T cells activation. Herein, three deconstruction analogs of rhein, compound 1, 2, and 3, were further studied as inhibitors of SRA. These three compounds, particularly compound 1, also known as a natural product danthron, enhanced T cells activation, indicated by increased transcriptional activation of interleukin 2 (Il2) gene, production of IL-2 protein, and proliferation of T cells. Additionally, the interaction between these compounds and SRA was studied by molecular modeling. Compound 1 showed a favorable binding mode with the cysteine rich domain of SRA protein compared to compound 2 and 3. Collectively, those results would provide insight for future design and development of next generation rhein derivatives as SRA inhibitors., (Published by Elsevier Ltd.)

The fundamental pathophysiology of sickle cell disease is predicated by the polymerization of deoxygenated (T-state) sickle hemoglobin (Hb S) into fibers that distort red blood cells into the characteristic sickle shape. The crystal structure of deoxygenated Hb S (DeoxyHb S) and other studies suggest that the polymer is initiated by a primary interaction between the mutation βVal6 from one Hb S molecule, and a hydrophobic acceptor pocket formed by the residues βAla70, βPhe85 and βLeu88 of an adjacent located Hb S molecule. On the contrary, oxygenated or liganded Hb S does not polymerize or incorporate in the polymer. In this paper we present the crystal structure of carbonmonoxy-ligated sickle Hb (COHb S) in the quaternary classical R-state at 1.76Å. The overall structure and the pathological donor and acceptor environments of COHb S are similar to those of the isomorphous CO-ligated R-state normal Hb (COHb A), but differ significantly from DeoxyHb S as expected. More importantly, the packing of COHb S molecules does not show the typical pathological interaction between βVal6 and the βAla70, βPhe85 and βLeu88 hydrophobic acceptor pocket observed in DeoxyHb S crystal. The structural analysis of COHb S, COHb A and DeoxyHb S provides atomic level insight into why liganded hemoglobin does not form a polymer., (Copyright © 2016 Elsevier Inc. All rights reserved.)