Targeting Kca3.1 Channels in Cancer.

The Kca3.1 channels, previously designated as IK1 or SK4 channels and encoded by the KCNN4 gene, are activated by a rise of the intracellular Ca2+ concentration. These K+ channels are widely expressed in many organs and involved in many pathologies. In particular, Kca3.1 channels have been studied intensively in the context of cancer. They are not only a marker and a valid prognostic tool for cancer patients, but have an important share in driving cancer progression. Their function is required for many characteristic features of the aggressive cancer cell behavior such as migration, invasion and metastasis as well as proliferation and therapy resistance. In the context of cancer, another property of Kca3.1 is now emerging. These channels can be a target for novel small molecule-based imaging probes, as it has been validated in case of fluorescently labeled senicapoc-derivatives. The aim of this review is (i) to give an overview on the role of Kca3.1 channels in cancer progression and in shaping the cancer microenvironment, (ii) discuss the potential of using Kca3.1 targeting drugs for cancer imaging, (iii) and highlight the possibility of combining molecular dynamics simulations to image inhibitor binding to Kca3.1 channels in order to provide a deeper understanding of Kca3.1 channel pharmacology. Alltogether, Kca3.1 is an attractive therapeutic target so that senicapoc, originally developed for the treatment of sickle cell anemia, should be repurposed for the treatment of cancer patients.

Rational Drug Design of Peptide-Based Therapies for Sickle Cell Disease.

Sickle cell disease (SCD) is a group of inherited disorders affecting red blood cells, which is caused by a single mutation that results in substitution of the amino acid valine for glutamic acid in the sixth position of the ß-globin chain of hemoglobin. These mutant hemoglobin molecules, called hemoglobin S, can polymerize upon deoxygenation, causing erythrocytes to adopt a sickled form and to suffer hemolysis and vaso-occlusion. Until recently, only two drug therapies for SCD, which do not even fully address the manifestations of SCD, were approved by the United States (US) Food and Drug Administration. A third treatment was newly approved, while a monoclonal antibody preventing vaso-occlusive crises is also now available. The complex nature of SCD manifestations provides multiple critical points where drug discovery efforts can be and have been directed. These notwithstanding, the need for new therapeutic approaches remains high and one of the recent efforts includes developments aimed at inhibiting the polymerization of hemoglobin S. This review focuses on anti-sickling approaches using peptide-based inhibitors, ranging from individual amino acid dipeptides investigated 30-40 years ago up to more promising 12- and 15-mers under consideration in recent years.

Rational design of pyridyl derivatives of vanillin for the treatment of sickle cell disease.

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.

Potential of Three Ethnomedicinal Plants as Antisickling Agents.

Sickle cell disease (SCD) is a genetic blood disorder that affects the shape and transportation of red blood cells (RBCs) in blood vessels, leading to various clinical complications. Many drugs that are available for treating the disease are insufficiently effective, toxic, or too expensive. Therefore, there is a pressing need for safe, effective, and inexpensive therapeutic agents from indigenous plants used in ethnomedicines. The potential of aqueous extracts of Cajanus cajan leaf and seed, Zanthoxylum zanthoxyloides leaf, and Carica papaya leaf in sickle cell disease management was investigated in vitro using freshly prepared 2% sodium metabisulfite for sickling induction. The results indicated that the percentage of sickled cells, which was initially 91.6% in the control, was reduced to 29.3%, 41.7%, 32.8%, 38.2%, 47.6%, in the presence of hydroxyurea, C. cajan seed, C. cajan leaf, Z. zanthoxyloides leaf, and C. papaya leaf extracts, respectively, where the rate of polymerization inhibition was 6.5, 5.9, 8.0, 6.6, and 6.0 (×10-2) accordingly. It was also found that the RBC resistance to hemolysis was increased in the presence of the tested agents as indicated by the reduction of the percentage of hemolyzed cells from 100% to 0%. The phytochemical screening results indicated the presence of important phytochemicals including tannins, saponins, alkaloids, flavonoids, and glycosides in all the plant extracts. Finally, gas chromatography-mass spectrometry analysis showed the presence of important secondary metabolites in the plants. These results suggest that the plant extracts have some potential to be used as alternative antisickling therapy to hydroxyurea in SCD management.

GBT440 increases haemoglobin oxygen affinity, reduces sickling and prolongs RBC half-life in a murine model of sickle cell disease.

A major driver of the pathophysiology of sickle cell disease (SCD) is polymerization of deoxygenated haemoglobin S (HbS), which leads to sickling and destruction of red blood cells (RBCs) and end-organ damage. Pharmacologically increasing the proportion of oxygenated HbS in RBCs may inhibit polymerization, prevent sickling and provide long term disease modification. We report that GBT440, a small molecule which binds to the N-terminal α chain of Hb, increases HbS affinity for oxygen, delays in vitro HbS polymerization and prevents sickling of RBCs. Moreover, in a murine model of SCD, GBT440 extends the half-life of RBCs, reduces reticulocyte counts and prevents ex vivo RBC sickling. Importantly, oral dosing of GBT440 in animals demonstrates suitability for once daily dosing in humans and a highly selective partitioning into RBCs, which is a key therapeutic safety attribute. Thus, GBT440 has the potential for clinical use as a disease-modifying agent in sickle cell patients.

Aryloxyalkanoic Acids as Non-Covalent Modifiers of the Allosteric Properties of Hemoglobin.

Hemoglobin (Hb) modifiers that stereospecifically inhibit sickle hemoglobin polymer formation and/or allosterically increase Hb affinity for oxygen have been shown to prevent the primary pathophysiology of sickle cell disease (SCD), specifically, Hb polymerization and red blood cell sickling. Several such compounds are currently being clinically studied for the treatment of SCD. Based on the previously reported non-covalent Hb binding characteristics of substituted aryloxyalkanoic acids that exhibited antisickling properties, we designed, synthesized and evaluated 18 new compounds (KAUS II series) for enhanced antisickling activities. Surprisingly, select test compounds showed no antisickling effects or promoted erythrocyte sickling. Additionally, the compounds showed no significant effect on Hb oxygen affinity (or in some cases, even decreased the affinity for oxygen). The X-ray structure of deoxygenated Hb in complex with a prototype compound, KAUS-23, revealed that the effector bound in the central water cavity of the protein, providing atomic level explanations for the observed functional and biological activities. Although the structural modification did not lead to the anticipated biological effects, the findings provide important direction for designing candidate antisickling agents, as well as a framework for novel Hb allosteric effectors that conversely, decrease the protein affinity for oxygen for potential therapeutic use for hypoxic- and/or ischemic-related diseases.

Identification of a novel class of covalent modifiers of hemoglobin as potential antisickling agents.

Aromatic aldehydes and ethacrynic acid (ECA) exhibit antipolymerization properties that are beneficial for sickle cell disease therapy. Based on the ECA pharmacophore and its atomic interaction with hemoglobin, we designed and synthesized several compounds - designated as KAUS (imidazolylacryloyl derivatives) - that we hypothesized would bind covalently to ßCys93 of hemoglobin and inhibit sickling. The compounds surprisingly showed weak allosteric and antisickling properties. X-ray studies of hemoglobin in complex with representative KAUS compounds revealed an unanticipated mode of Michael addition between the ß-unsaturated carbon and the N-terminal αVal1 nitrogen at the α-cleft of hemoglobin, with no observable interaction with ßCys93. Interestingly, the compounds exhibited almost no reactivity with the free amino acids, L-Val, L-His and L-Lys, but showed some reactivity with both glutathione and L-Cys. Our findings provide a molecular level explanation for the compounds biological activities and an important framework for targeted modifications that would yield novel potent antisickling agents.

Antisickling and toxicological evaluation of the leaves of Scoparia dulcis Linn (Scrophulariaceae).

BACKGROUND: Scoparia dulcis Linn (Scrophulariaceae) together with other medicinal plants serve as antisickling remedies in Africa. This study was aimed at investigating the antisickling activity of the leaves of the plant as well as establishing the toxicological profile. METHOD: Chemical tests were employed in phytochemical investigations. Evaluation of the antisickling activity involved the inhibition of sodium metabisulphite-induced sickling of the HbSS red blood cells obtained from confirmed sickle cell patients who were not in crises. Concentrations of the crude extract and its fractions were tested with normal saline and p-hydroxybenzoic acid serving as controls. Acute toxicological evaluation was carried out in mice while 30-day assessment was done in rats. RESULTS: Phytochemical screening revealed the presence of alkaloids, tannins, flavonoids and saponins. Percentage sickling inhibitions of the aqueous methanol extracts of S. dulcis were significant all through the period of assay p < 0. 05 compared to normal saline, but not significant with PHBA. The fractions had less activity compared to the crude extracts. The LD 50 of the extract in mice was above 8000 mg/kg body weight when administered orally. Toxicological evaluations at 250 and 500 mg/kg showed mild congestion in virtually all the target organs. CONCLUSION: The antisickling results confirmed traditional usage of Scoparia dulcis in the management of Sickle cell disorders and a candidate for further investigations.

Developing a pill to treat sickle cell disease.

A newly identified epigenetic modifier increases fetal hemoglobin in preclinical studies.

A molecular glue degrader of the WIZ transcription factor for fetal hemoglobin induction.

Sickle cell disease (SCD) is a prevalent, life-threatening condition attributable to a heritable mutation in ß-hemoglobin. Therapeutic induction of fetal hemoglobin (HbF) can ameliorate disease complications and has been intently pursued. However, safe and effective small-molecule inducers of HbF remain elusive. We report the discovery of dWIZ-1 and dWIZ-2, molecular glue degraders of the WIZ transcription factor that robustly induce HbF in erythroblasts. Phenotypic screening of a cereblon (CRBN)-biased chemical library revealed WIZ as a previously unknown repressor of HbF. WIZ degradation is mediated by recruitment of WIZ(ZF7) to CRBN by dWIZ-1, as resolved by crystallography of the ternary complex. Pharmacological degradation of WIZ was well tolerated and induced HbF in humanized mice and cynomolgus monkeys. These findings establish WIZ degradation as a globally accessible therapeutic strategy for SCD.

Crystallographic analysis of human hemoglobin elucidates the structural basis of the potent and dual antisickling activity of pyridyl derivatives of vanillin.

Vanillin has previously been studied clinically as an antisickling agent to treat sickle-cell disease. In vitro investigations with pyridyl derivatives of vanillin, including INN-312 and INN-298, showed as much as a 90-fold increase in antisickling activity compared with vanillin. The compounds preferentially bind to and modify sickle hemoglobin (Hb S) to increase the affinity of Hb for oxygen. INN-312 also led to a considerable increase in the solubility of deoxygenated Hb S under completely deoxygenated conditions. Crystallographic studies of normal human Hb with INN-312 and INN-298 showed that the compounds form Schiff-base adducts with the N-terminus of the α-subunits to constrain the liganded (or relaxed-state) Hb conformation relative to the unliganded (or tense-state) Hb conformation. Interestingly, while INN-298 binds and directs its meta-positioned pyridine-methoxy moiety (relative to the aldehyde moiety) further down the central water cavity of the protein, that of INN-312, which is ortho to the aldehyde, extends towards the surface of the protein. These studies suggest that these compounds may act to prevent sickling of SS cells by increasing the fraction of the soluble high-affinity Hb S and/or by stereospecific inhibition of deoxygenated Hb S polymerization.

Pyridyl derivatives of benzaldehyde as potential antisickling agents.

Compounds that bind to sickle hemoglobin (Hb S) producing an allosteric shift to the high-affinity Hb S that does not polymerize are being developed to treat sickle cell anemia (SCA). In this study, three series of pyridyl derivatives of substituted benzaldehydes (Classes I-III) that combine structural features of two previously determined potent antisickling agents, vanillin and pyridoxal, were synthesized. When analyzed with normal human whole blood, the compounds form Schiff-base adducts with Hb and left shift the oxygen equilibrium curve (OEC) to the more soluble high-affinity Hb, more than vanillin or pyridoxal. Generally, Class-I compounds with an aromatic aldehyde located ortho to the pyridyl substituent are the most potent, followed by the Class-II compounds with the aldehyde at the meta-position. Class-III compounds with the aldehyde at the para position show the weakest activity. The structure-activity studies of these pyridyl derivatives of substituted benzaldehydes demonstrate significant allosteric potency that may be useful for treating SCA.

Structural Basis of Antisickling Effects of Selected FDA Approved Drugs: A Drug Repurposing Study.

INTRODUCTION: Sickle cell disease is characterized by a point mutation involving substitution of glutamic acid at position 6 to valine. Encoded in this hydrophobic mutation is both an intrinsic capacity for the beta globin molecules to assemble into thermodynamically favoured polymeric states as well as a rational way of interrupting the aggregation. METHODS: In this work, starting with a theoretical model that employs occlusive binding onto the beta globin aggregation surface and using a range of computational methods and an effective energy for screening, a number of FDA approved drugs with computed aggregation inhibitory activities were identified. RESULTS AND CONCLUSION: The validity of the model was confirmed using sickling tests, after which pharmacophore models as well the structural basis for the observed antisickling effects were identified.

Rational modification of vanillin derivatives to stereospecifically destabilize sickle hemoglobin polymer formation.

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.

Tunable delocalization of unpaired electrons of nitroxide radicals for sickle-cell disease drug improvements.

Hydroxyurea is a drug recently approved to treat sickle cell diseases. Hydroxyurea benefits the patients by increasing the level of fetal hemoglobin via a nitroxide radical pathway. Here, we report an unpaired-electron-delocalization approach to tune the stability of nitroxide radicals. In this approach, the substitution by an unsaturated alkyl group containing conjugated C=C double bonds for the hydrogen on the nitrogen atom attached to the hydroxyl of hydroxyurea can significantly increase its ability to generate nitroxide radical. Furthermore, the increase can be remarkably enhanced by increasing the number of conjugated C=C double bonds. For a hydroxyurea derivative that contains two conjugated C=C double bonds, the reaction rate to generate its radical is 118 times faster than that of hydroxyurea, and for a hydroxyurea derivative containing 20 conjugated C=C double bonds, the reaction rate to form its radical is 238 times faster than that of hydroxyurea. For this reason, hydroxyurea derivatives with conjugated C=C double bonds may constitute new potential drugs for the treatment of sickle-cell diseases.

Identification of Aptamers That Bind to Sickle Hemoglobin and Inhibit Its Polymerization.

The pathophysiology of sickle cell disease (SCD) is dependent on the polymerization of deoxygenated sickle hemoglobin (HbS), leading to erythrocyte deformation (sickling) and vaso-occlusion within the microvasculature. Following deoxygenation, there is a delay time before polymerization is initiated, during which nucleation of HbS monomers occurs. An agent with the ability to extend this delay time or slow polymerization would therefore hold a therapeutic, possibly curative, potential. We used the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method to screen for HbS-binding RNA aptamers modified with nuclease-resistant 2'-fluoropyrimidines. Polymerization assays were employed to identify aptamers with polymerization-inhibitory properties. Two noncompeting aptamers, DE3A and OX3B, were found to bind hemoglobin, significantly increase the delay time, and reduce the rate of polymerization of HbS. These modifiable, nuclease-resistant aptamers are potential new therapeutic agents for SCD.

Mass spectrometric characterization of efaproxiral (RSR13) and its implementation into doping controls using liquid chromatography-atmospheric pressure ionization-tandem mass spectrometry.

Efaproxiral (2-[4-[[(3,5-dimethylanilino)carbonyl]methyl]phenoxyl]-2-methylpropionic acid, formerly referred to as RSR13) is prohibited in sports according to the World Anti-Doping Agency (WADA). The drug as well as structurally related compounds and a stable isotope-labeled derivative have been synthesized to elucidate the fragmentation pathway of efaproxiral, using electrospray ionization (ESI) and tandem mass spectrometry by employing a novel linear ion trap--orbitrap hybrid mass spectrometer--in positive and negative ionization modes. The elimination of 2-methyl acrylic acid (-86 u) has been identified as a major fragmentation process in both charge states. Negative ionization and collision-induced dissociation (CID) caused an additional release of carbon dioxide (-44 u), and positive ionization the loss of formic acid (-46 u). Efaproxiral was incorporated into an existing screening procedure for doping controls using solid-phase extraction (SPE) followed by liquid chromatography-tandem mass spectrometry, enabling a limit of detection of 2.5 ng/ml and interday precisions ranging from 7.9 to 13.0%.

The reaction of deoxy-sickle cell hemoglobin with hydroxyurea.

In addition to its capacity to increase fetal hemoglobin levels, other mechanisms are implicated in hydroxyurea's ability to provide beneficial effects to patients with sickle cell disease. We hypothesize that the reaction of hemoglobin with hydroxyurea may play a role. It is shown that hydroxyurea reacts with deoxy-sickle cell hemoglobin (Hb) to form methemoglobin (metHb) and nitrosyl hemoglobin (HbNO). The products of the reaction as well as the kinetics are followed by absorption spectroscopy and electron paramagnetic resonance (EPR) spectroscopy. Analysis of the kinetics shows that the reaction can be approximated by a pseudo-first order rate constant of 3.7x10(-4) (1/(s.M)) for the disappearance of deoxy-sickle cell hemoglobin. Further analysis shows that HbNO is formed at an observed average rate of 5.25x10(-5) (1/s), three to four times slower than the rate of formation of metHb. EPR spectroscopy is used to show that the formation of HbNO involves the specific transfer of NO from the NHOH group of hydroxyurea. The potential importance of this reaction is discussed in the context of metHb and HbNO being able to increase the delay time for sickle cell hemoglobin polymerization and HbNO's vasodilating capabilities through conversion to S-nitrosohemoglobin.

Quantum chemical design of hydroxyurea derivatives for the treatment of sickle-cell anemia.

Treatment of sickle-cell anemia by hydroxyurea has been shown to decrease patient mortality by 40%. In a rate-limiting step, hydroxyurea reacts with hemoglobin to form the nitroxide radical, which then decomposes to yield nitric oxide (NO). In this paper, we examine derivatives of hydroxyurea and their radicals by quantum chemical methods to identify derivatives that generate NO-producing radicals at a faster rate than hydroxyurea. The molecules are treated with Hartree-Fock theory, correlated wave function methods such as perturbation theory and coupled-cluster methods, and density functional theory. We observe that the inclusion of the correlation energy is important for an accurate comparison of the energy changes associated with modifications of the hydroxyurea molecule and its radical. The computational results are compared with available experimental data. All 19 derivatives of hydroxyurea, including a new medication for asthma Zileuton, manifest changes in their electronic energies that mark them as candidates for a faster formation of NO-producing radicals.

High-resolution crystal structure of deoxy hemoglobin complexed with a potent allosteric effector.

The crystal structure of human deoxy hemoglobin (Hb) complexed with a potent allosteric effector (2-[4-[[(3,5-dimethylanilino)carbonyl]methyl]phenoxy]-2-methylpropionic acid) = RSR-13) is reported at 1.85 A resolution. Analysis of the hemoglobin:effector complex indicates that two of these molecules bind to the central water cavity of deoxy Hb in a symmetrical fashion, and that each constrains the protein by engaging in hydrogen bonding and hydrophobic interactions with three of its four subunits. Interestingly, we also find that water-mediated interactions between the bound effectors and the protein make significant contributions to the overall binding. Physiologically, the interaction of RSR-13 with Hb results in increased oxygen delivery to peripheral tissues. Thus, this compound has potential therapeutic application in the treatment of hypoxia, ischemia, and trauma-related blood loss. Currently, RSR-13 is in phase III clinical trials as a radiosensitizing agent in the treatment of brain tumors. A detailed structural analysis of this compound complexed with deoxy Hb has important implications for the rational design of future analogs.