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The SARS-CoV-2 (COVID-19) pandemic was officially declared by the World Health Organization on 11 March, 2020. From the initial public reports from China in December 2019, the contagion blazed its way from ground zero in Wuhan, China into 165 countries. The health disaster caught the planet by surprise. At the time of this writing, 6.6 million COVID-19 cases were confirmed worldwide. Central and South America are the latest hotspots. Utilizing state-of-the-art science and technology, industry, government, and academic enterprises internationally are engaged in a monumental campaign to combat and eliminate the viral threat. Small-molecule therapeutics discovery largely by way of drug repurposing and accelerated vaccine development are at the forefront of this campaign. Within five months time, 500 unique therapeutic agents have advanced into development, >150 clinical trials initiated, and seven agents authorized for emergency use. This article presents a snapshot of these activities as of 29 May 2020.

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.

A broad variety of medicinal chemistry approaches can be used for the identification of hits, generation of leads, as well as to accelerate the development of high quality drug candidates. Structure-based drug design (SBDD) methods are becoming increasingly powerful, versatile and more widely used. This review summarizes current developments in structure-based virtual screening and receptor-based pharmacophores, highlighting achievements as well as challenges, along with the value of structure-based lead optimization, with emphasis on recent examples of successful applications for the identification of novel active compounds.

Hemoglobin was one of the first protein structures to be determined by X-ray crystallography and served as a basis for the two-state MWC model for the mechanism of allosteric proteins. Since then, there has been an ongoing debate about whether Hb allostery involves the unliganded tense T state and the liganded relaxed R state or whether it involves the T state and an ensemble of liganded relaxed states. In fact, the former model is inconsistent with many functional observations, as well as the recent discoveries of several relaxed-state Hb structures such as RR2, R3 and R2. One school of thought has suggested the R2 state to be the physiologically relevant relaxed end state, with the R state mediating the T-->R2 transition. X-ray studies have been performed on human carbonmonoxy Hb at a resolution of 2.8 A. The ensuing liganded quaternary structure is different from previously reported liganded Hb structures. The distal beta-heme pocket is the largest when compared with other liganded Hb structures, partly owing to rotation of betaHis63(E7) out of the distal pocket, creating a ligand channel to the solvent. The structure also shows unusually smaller alpha- and beta-clefts. Results from this study taken in conjunction with previous findings suggest that multiple liganded Hb states with different quaternary structures may be involved in ligand uptake, stabilization, transport and release.

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.

A series of tetrahydroisoquinoline-N-phenylamide derivatives were designed, synthesized, and tested for their relative binding affinity and antagonistic activity against androgen receptor (AR). Compound 1b (relative binding affinity, RBA = 6.4) and 1h (RBA = 12.6) showed higher binding affinity than flutamide (RBA = 1), a potent AR antagonist. These two compounds also exerted optimal antagonistic activity against AR in reporter assays. The derivatives were also tested for their activities against another nuclear receptor, farnesoid x receptor (FXR), with most compounds acting as weak antagonists, however, compound 1h behaved as a FXR agonist with activity slightly less than that of chenodeoxycholic acid (CDCA), a natural FXR agonist.

The energetics and hydrogen bonding pattern of water molecules bound to proteins were mapped by analyzing structural data (resolution better than 2.3A) for sets of uncomplexed and ligand-complexed proteins. Water-protein and water-ligand interactions were evaluated using hydropatic interactions (HINT), a non-Newtonian forcefield based on experimentally determined logP(octanol/water) values. Potential water hydrogen bonding ability was assessed by a new Rank algorithm. The HINT-derived binding energies and Ranks for second shell water molecules were -0.04 kcal mol(-1) and 0.0, respectively, for first shell water molecules -0.38 kcal mol(-1) and 1.6, for active site water molecules -0.45 kcal mol(-1) and 2.3, for cavity water molecules -0.55 kcal mol(-1) and 3.3, and for buried water molecules -0.56 kcal mol(-1) and 4.4. For the last four classes, similar energies indicate that internal and external water molecules interact with protein almost equally, despite different degrees of hydrogen bonding. The binding energies and Ranks for water molecules bridging ligand-protein were -1.13 kcal mol(-1) and 4.5, respectively. This energetic contribution is shared equally between protein and ligand, whereas Rank favors the protein. Lastly, by comparing the uncomplexed and complexed forms of proteins, guidelines were developed for prediction of the roles played by active site water molecules in ligand binding. A water molecule with high Rank and HINT score is unlikely to make further interactions with the ligand and is largely irrelevant to the binding process, while a water molecule with moderate Rank and high HINT score is available for ligand interaction. Water molecule displaced for steric reasons were characterized by lower Rank and HINT score. These guidelines, tested by calculating HINT score and Rank for 50 water molecules bound in the active site of four uncomplexed proteins (for which the structures of the liganded forms were also available), correctly predicted the ultimate roles (in the complex) for 76% of water molecules. Some failures were likely due to ambiguities in the structural data.

The liganded hemoglobin (Hb) high-salt crystallization condition described by Max Perutz has generated three different crystals of human adult carbonmonoxy hemoglobin (COHbA). The first crystal is isomorphous with the "classical" liganded or R Hb structure. The second crystal reveals a new liganded Hb quaternary structure, RR2, that assumes an intermediate conformation between the R form and another liganded Hb quaternary structure, R2, which was discovered more than a decade ago. Like the R2 structure, the diagnostic R state hydrogen bond between beta2His97 and alpha1Thr38 is missing in the RR2 structure. The third crystal adopts a novel liganded Hb conformation, which we have termed R3, and it shows substantial quaternary structural differences from the R, RR2, and R2 structures. The quaternary structure differences between T and R3 are as large as those between T and R2; however, the T --> R3 and T --> R2 transitions are in different directions as defined by rigid-body screw rotation. Moreover, R3 represents an end state. Compared to all known liganded Hb structures, R3 shows remarkably reduced strain at the alpha-heme, reduced steric contact between the beta-heme ligand and the distal residues, smaller alpha- and beta-clefts, and reduced alpha1-alpha2 and beta1-beta2 iron-iron distances. Together, these unique structural features in R3 should make it the most relaxed and/or greatly enhance its affinity for oxygen compared to the other liganded Hbs. The current Hb structure-function relationships that are now based on T --> R, T -->R --> R2, or T --> R2 --> R transitions may have to be reexamined to take into account the RR2 and R3 liganded structures.

The crystal structures of R- and T-state hemoglobin (Hb) Bassett have been determined to 2.15 and 1.80 A resolution, respectively. Physiologically, Hb Bassett (alphaAsp94--Ala) is characterized by a low affinity for oxygen, a reduced Bohr effect and low cooperativity, as well as being slightly unstable (compared with normal adult hemoglobin; HbA). Comparisons between the Hb Bassett structures and previously determined R- and T-state HbA structures revealed that this mutant shares similar tertiary and quaternary structures with other Hbs. However, this analysis did identify localized structural differences between R-state Hb Bassett and R-state HbA at the alpha1beta2 (alpha2beta1) dimer interface and at the beta-cleft. Specifically, the beta-FG corner has shifted closer to the alpha-C helix in the mutant R structure. In addition, four intersubunit hydrogen bonds found at the alpha1beta2 interfaces of native R-state Hb structures are abolished or weakened and subsequently replaced by two new intersubunit hydrogen bonds in R-state Hb Bassett. Remarkably, the newly formed hydrogen bonds in the R-state mutant structure are also observed in T-state Hb structures. At the beta-cleft, betaHis46, which is known to contribute to the Bohr effect in Hb, makes a unique hydrogen-bonding interaction with betaAsn139 in the R-state Hb Bassett. Unlike the R-state mutant, the T-state Hb Bassett structure does not display any significant structural changes at both the alpha1beta2 (alpha2beta1) dimer interface and the beta-cleft. Quite significantly, the mutation has led to removal of an interdimer repulsion involving alpha1Asp94 and beta2Asp99. The R- and T-state structures of Hb Bassett suggest a stereochemical basis for the observed functional properties of this mutant.

Naturally occurring five-membered heterocyclic aldehydes, including 5-hydroxymethyl-2-furfural, increase the oxygen affinity of hemoglobin (Hb) and strongly inhibit the sickling of homozygous sickle red blood (SS) cells. X-ray studies of Hb complexed with these compounds indicate that they form Schiff base adducts in a symmetrical fashion with the N-terminal alphaVal1 nitrogens of Hb. Interestingly, two cocrystal types were isolated during crystallization experiments with deoxygenated Hb (deoxyHb): one crystal type was composed of the low-affinity or tense (T) state Hb quaternary structure; the other crystal type was composed of high-affinity or relaxed state Hb (with a R2 quaternary structure). The R2 crystal appears to be formed as a result of the aldehydes binding to fully or partially ligated Hb in the deoxyHb solution. Repeated attempts to crystallize the compounds with liganded Hb failed, except on rare occasions when very few R state crystals were obtained. Oxygen equilibrium, high performance liquid chromatography (HPLC), antisickling, and X-ray studies suggest that the examined heterocyclic aldehydes may be acting to prevent polymerization of sickle hemoglobin (HbS) by binding to and stabilizing liganded Hb in the form of R2 and/or various relaxed state Hbs, as well as binding to and destabilizing unliganded T state Hb. The proposed mechanism may provide a general model for the antisickling effects of aldehyde containing small molecules that bind to N-terminal alphaVal1 nitrogens of Hb. The examined compounds also represent a new class of potentially therapeutic agents for treating sickle cell disease (SCD).

Structural water molecules within protein active sites are relevant for ligand-protein recognition because they modify the active site geometry and contribute to binding affinity. In this work an analysis of the interactions between 23 ligands and dimeric HIV-1 protease is reported. The X-ray structures of these complexes show the presence of four types of structural water molecules: water 301 (on the symmetry axis), water 313, water 313bis, and peripheral waters. Except for water 301, these are generally complemented with a symmetry-related set. The GRID program was used both for checking water locations and for placing water molecules that appear to be missing from the complexes due to crystallographic uncertainty. Hydropathic analysis of the energetic contributions using HINT indicates a significant improvement of the correlation between HINT scores and the experimentally determined binding constants when the appropriate bridging water molecules are taken into account. In the absence of water r2 = 0.30 with a standard error of +/- 1.30 kcal mol(-1) and when the energetic contributions of the constrained waters are included r2 = 0.61 with a standard error of +/- 0.98 kcal mol(-1). HINT was shown to be able to map quantitatively the contribution of individual structural waters to binding energy. The order of relevance for the various types of water is water 301 > water 313 > water 313bis > peripheral waters. Thus, to obtain the most reliable free energy predictions, the contributions of structural water molecules should be included. However, care must be taken to include the effects of water molecules that add information value and not just noise.

“Getting it right” refers to the careful modeling of all elements in the living system, i.e. biological macromolecules, ligands and water molecules. In addition, careful attention should be paid to the protonation state of ionizable functional groups on the ligands and residues at the active site. Computational technology based on the empirical HINT program is described to: (1) calculate free energy scores for ligand binding; (2) include the implicit and explicit effects of water in and around the ligand binding site; and (3) incorporate the effects of global and local pH in molecular models. This last point argues for the simultaneous consideration of a number of molecular models, each with different protonation profiles. Data from recent studies of protein–ligand systems (trypsin, thrombin, neuraminidase, HIV-1 protease and others) are used to illustrate the concepts in the paper. Also discussed are experimental factors related to accurate free energy predictions with this and other computational technologies.

One factor that can strongly influence predicted free energy of binding is the ionization state of functional groups on the ligands and at the binding site at which calculations are performed. This analysis is seldom performed except in very detailed computational simulations. In this work, we address the issues of (i) modeling the complexity resulting from the different ionization states of ligand and protein residues involved in binding, (ii) if, and how, computational methods can evaluate the pH dependence of ligand inhibition constants, and (iii) how to score the protonation-dependent models. We developed a new and fairly rapid protocol called “computational titration” that enables parallel modeling of multiple ionization ensembles for each distinct protonation level. Models for possible protonation combinations for site/ligand ionizable groups are built, and the free energy of interaction for each of them is quantified by the HINT (Hydropathic INTeractions) software. We applied this procedure to the evaluation of the binding affinity of nine inhibitors (six derived from 2,3-didehydro-2-deoxy-N-acetylneuraminic acid, DANA) of influenza virus neuraminidase (NA), a surface glycoprotein essential for virus replication and thus a pharmaceutically relevant target for the design of anti-influenza drugs. The three-dimensional structures of the NA enzyme-inhibitor complexes indicate considerable complexity as the ligand-protein recognition site contains several ionizable moieties. Each computational titration experiment reveals a peak HINT score as a function of added protons. This maximum HINT score indicates the optimum pH (or the optimum protonation state of each inhibitor-protein binding site) for binding. The pH at which inhibition is measured and/ or crystals were grown and analyzed can vary from this optimum. A protonation model is proposed for each ligand that reconciles the experimental complex structure with measured inhibition and the free energy of binding. Computational titration methods allow us to analyze the effect of pH in silico and may be helpful in improving ligand binding free energy prediction when protonation or deprotonation of the residues or ligand functional groups at the binding site might be significant.

This chapter reviews current understanding of the molecular and cellular basis of the pathophysiology of sickle-cell anemia, with emphasis on the biophysics of the intracellular polymerization of sickle hemogobin. Factors that modify the severity of the disease manifestations, primarily by altering polymerization tendency, are discussed. This framework is used to present an overview of the major rational approaches to the therapy of sickle-cell anemia based on inhibiting polymerization with agents that affect either the hemoglobin molecule or the sickle erythrocyte. Discussion of agents that may decrease vascular entrapment of sickle erythrocytes is also presented. Detailed review of the most important current approaches to therapy—pharmacologically elevating fetal hemoglobin—focuses primarily on hydroxyurea, the only drug currently FDA approved for treating sickle-cell anemia. A brief discussion of other approaches, including stem cell transplantation and gene therapy, concludes the chapter. Keywords: sickle-cell anemia; sickle hemoglobin (hemoglobin S); fetal hemoglobin (hemoglobin F); polymerization; potassium cyanate; vanillin; nitric oxide; hydroxyurea; arginine butyrate; 5-azacytidine; gene therapy; bone marrow transplantation; stem cell transplantation

Structure-based drug design is achievable by exploiting the three-dimensional information intrinsic in structural models of proteins that are involved in human disease. Many other tools (biochemistry, cellular and whole-animal biology, organic synthesis, pharmaceutics, and computational analysis) must by tightly integrated to fulfill the promise of the structure-based approach, and the use of this approach does not automatically guarantee success. However, the successes that have been achieved make it clear that structure-based methods provide an efficient, effective, and elegant approach to medicinal chemistry. Examples are described in this chapter of the use of structure-based methods in designing compounds that have entered human clinical trials and, in many cases, were launched as products. Some of the lessons that have been learned about best practices in the application of structure-based methods are summarized. Keywords: structure-based drug design; hemoglobin; antifolate targets; proteases; oxidoreductases; hydrolases; picornavirus uncoating; phosphoryl transferases

Deficiency of human erythrocyte isozyme (RPK) is, together with glucose-6-phosphate dehydrogenase deficiency, the most common cause of the nonspherocytic hemolytic anemia. To provide a molecular framework to the disease, we have solved the 2.7 A resolution crystal structure of human RPK in complex with fructose 1,6-bisphosphate, the allosteric activator, and phosphoglycolate, a substrate analogue, and we have functionally and structurally characterized eight mutants (G332S, G364D, T384M, D390N, R479H, R486W, R504L, and R532W) found in RPK-deficient patients. The mutations target distinct regions of RPK structure, including domain interfaces and catalytic and allosteric sites. The mutations affect to a different extent thermostability, catalytic efficiency, and regulatory properties. These studies are the first to correlate the clinical symptoms with the molecular properties of the mutant enzymes. Mutations greatly impairing thermostability and/or activity are associated with severe anemia. Some mutant proteins exhibit moderate changes in the kinetic parameters, which are sufficient to cause mild to severe anemia, underlining the crucial role of RPK for erythrocyte metabolism. Prediction of the effects of mutations is difficult because there is no relation between the nature and location of the replaced amino acid and the type of molecular perturbation. Characterization of mutant proteins may serve as a valuable tool to assist with diagnosis and genetic counseling.

The prediction of the binding affinity between a protein and ligands is one of the most challenging issues for computational biochemistry and drug discovery. While the enthalpic contribution to binding is routinely available with molecular mechanics methods, the entropic contribution is more difficult to estimate. We describe and apply a relatively simple and intuitive calculation procedure for estimating the free energy of binding for 53 protein-ligand complexes formed by 17 proteins of known three-dimensional structure and characterized by different active site polarity. HINT, a software model based on experimental LogP(o/w) values for small organic molecules, was used to evaluate and score all atom-atom hydropathic interactions between the protein and the ligands. These total scores (H(TOTAL)), which have been previously shown to correlate with DeltaG(interaction) for protein-protein interactions, correlate with DeltaG(binding) for protein-ligand complexes in the present study with a standard error of +/-2.6 kcal mol(-1) from the equation DeltaG(binding) = -0.001 95 H(TOTAL) - 5.543. A more sophisticated model, utilizing categorized (by interaction class) HINT scores, produces a superior standard error of +/-1.8 kcal mol(-1). It is shown that within families of ligands for the same protein binding site, better models can be obtained with standard errors approaching +/-1.0 kcal mol(-1). Standardized methods for preparing crystallographic models for hydropathic analysis are also described. Particular attention is paid to the relationship between the ionization state of the ligands and the pH conditions under which the binding measurements are made. Sources and potential remedies of experimental and modeling errors affecting prediction of DeltaG(binding) are discussed.

Human erythrocyte pyruvate kinase plays an important role in erythrocyte metabolism. Mutation on the gene results in pyruvate kinase deficiency and is an important cause of hereditary nonspherocytic hemolytic anemia. Because of difficulties in isolating the mutant enzymes from patients, these mutations have not been fully studied. In this study, a complementary DNA (cDNA) encoding the human erythrocyte pyruvate kinase was generated. The cDNA was cloned into several expression vectors, and the protein was expressed and purified. The tetrameric protein exhibited properties characteristic of authentic human erythrocyte pyruvate kinase, including response to substrate, phosphoenolpyruvate, activation by fructose 1,6-bisphosphate, and inhibition by adenosine triphosphate (ATP). The N-terminal segment of the protein was highly susceptible to proteolysis, but only 2 of the 4 subunits were cleaved and lacked 47 N-terminal amino acid residues. A mutant protein, R510Q, which is the most frequently occurring mutation among Northern European population, was also generated and purified. The mutant protein retained its binding capacity to and could be activated by fructose 1,6-bisphosphate and showed similar kinetics toward phosphoenolpyruvate and adenosine diphosphate as for the wild-type enzyme. Conversely, the mutant protein has a dramatically decreased stability toward heat and is more susceptible to ATP inhibition. The enzyme instability decreases the enzyme level in the cell, accounting for the clinically observed “pyruvate kinase deficiency” of patients who are homozygous for this mutation. This study provides the first detailed functional characterization of human erythrocyte pyruvate kinase. These findings will allow the establishment of a fine correlation between molecular abnormalities and the clinical expression of the disease.

Crystallographic studies of the intermediate states between unliganded and fully liganded hemoglobin (Hb) have revealed a large range of subtle but functionally important structural differences. Only one T state has been reported, whereas three other quaternary states (the R state, B state, and R2 or Y state) for liganded Hb have been characterized; other studies have defined liganded Hbs that are intermediate between the T and R states. The high-salt crystal structure of bovine carbonmonoxy (CO bovine) Hb has been determined at a resolution of 2.1 A and is described here. A detailed comparison with other crystallographically solved Hb forms (T, R, R2 or Y) shows that the quaternary structure of CO bovine Hb closely resembles R state Hb. However, our analysis of these structures has identified several important differences between CO bovine Hb and R state Hb. Compared with the R state structures, the beta-subunit N-terminal region has shifted closer to the central water cavity in CO bovine Hb. In addition, both the alpha- and beta-subunits in CO bovine Hb have more constrained heme environments that appear to be intermediate between the T and R states. Moreover, the distal pocket of the beta-subunit heme in CO bovine Hb shows significantly closer interaction between the bound CO ligand and the Hb distal residues Val 63(E11) and His 63(E7). The constrained heme groups and the increased steric contact involving the CO ligand and the distal heme residues relative to human Hb may explain in part the low intrinsic oxygen affinity of bovine Hb.

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.

Purpose: Preclinical studies indicate that RSR13 oxygenates and radiosensitizes hypoxic solid tumors by decreasing the oxygen (O 2 )-binding affinity of hemoglobin (Hb). A Phase I open-label, multicenter dose and frequency escalation study was conducted to assess the safety, tolerance, pharmacokinetics, and pharmacodynamic effect of daily RSR13 administration to cancer patients receiving concurrent palliative radiotherapy (RT). Methods and Materials: Eligibility criteria included the following: ECOG performance status ≤2; resting and exercise arterial oxygen saturation (SaO 2 ) ≥90%; an indication for palliative RT, 20–40 Gy in 10–15 fractions. RSR13 was administered i.v. via central vein over 60 min immediately before RT. Patients received supplemental O 2 via nasal cannula at 4 L/min during RSR13 infusion and RT. Plasma, red blood cell (RBC), and urine RSR13 concentrations were assayed. The pharmacodynamic effect of RSR13 on Hb-O 2 binding affinity was quantified by multipoint tonometry and expressed as an increase in p50, defined as the partial pressure of O 2 that results in 50% SaO 2 . The RSR13 dose in the first cohort was 75 mg/kg once a week for two doses; successive cohorts received higher, more frequent doses up to 100 mg/kg/day for 10 days during RT. Results: Twenty patients were enrolled in the study. Repeated daily doses of RSR13 were generally well tolerated. Two adverse events of note occurred: ( 1 ) A patient with pre-existing restrictive lung disease had transient persistent hypoxemia after the sixth RSR13 dose; ( 2 ) a patient with a recurrent glioma receiving high-dose corticosteroids had edema after the seventh RSR13 dose, likely due to the daily high-volume fluid infusions. Both patients recovered to baseline status with conservative management. Maximum pharmacodynamic effect occurred at the end of RSR13 infusion and was proportional to the RBC RSR13 concentration. After an RSR13 dose of 100 mg/kg, the peak increase in p50 averaged 8.1 mm Hg, consistent with the targeted physiologic effect, and then diminished with a half-life of approximately 5 h. Conclusions: RSR13 was well tolerated in daily doses up to 100 mg/kg administered for 10 days during RT. The combined administration of RSR13 with 4 L/min supplemental O 2 yielded pharmacodynamic conditions in which hypoxic tumor radiosensitization can occur. Ongoing Phase II and Phase III studies are evaluating the combination of RT and RSR13 for selected indications, including primary brain tumors, brain metastases, and non–small-cell lung cancer.

A practical computational method for the molecular modeling of free-energy changes associated with protein mutations is reported. The de novo generation, optimization, and thermodynamic analysis of a wide variety of deoxy and oxy hemoglobin mutants are described in detail. Hemoglobin is shown to be an ideal candidate protein for study because both the native deoxy and oxy states have been crystallographically determined, and a large and diverse population of its mutants has been thermodynamically characterized. Noncovalent interactions for all computationally generated hemoglobin mutants are quantitatively examined with the molecular modeling program HINT (Hydropathic INTeractions). HINT scores all biomolecular noncovalent interactions, including hydrogen bonding, acid-base, hydrophobic-hydrophobic, acid-acid, base-base, and hydrophobic-polar, to generate dimer-dimer interface "scores" that are translated into free-energy estimates. Analysis of 23 hemoglobin mutants, in both deoxy and oxy states, indicates that the effects of mutant residues on structurally bound waters (and visa versa) are important for generating accurate free-energy estimates. For several mutants, the addition/elimination of structural waters is key to understanding the thermodynamic consequences of residue mutation. Good agreement is found between calculated and experimental data for deoxy hemoglobin mutants (r = 0.79, slope = 0.78, standard error = 1.4 kcal mol(-1), n = 23). Less accurate estimates were initially obtained for oxy hemoglobin mutants (r = 0.48, slope = 0.47, standard error = 1.4 kcal mol(-1), n = 23). However, the elimination of three outliers from this data set results in a better correlation of r = 0.87 (slope = 0.72, standard error = 0.75, n = 20). These three mutations may significantly perturb the hemoglobin quaternary structure beyond the scope of our structural optimization procedure. The method described is also useful in the examination of residue ionization states in protein structures. Specifically, we find an acidic residue within the native deoxy hemoglobin dimer-dimer interface that may be protonated at physiological pH. The final analysis is a model design of novel hemoglobin mutants that modify cooperative free energy (deltaGc)--the energy barrier between the allosteric transition from deoxy to oxy hemoglobin.

To date, a number of p53-derived peptides have been evaluated in vitro for their ability to inhibit the carcinogenic p53–mdm2 interaction. Design of second-generation nonpeptidic compounds requires the reduction of large peptide structures down to small molecules maintaining the proper spatial arrangement of key functional groups. Molecular modeling software exists that can predict and rank intermolecular interactions from the p53–mdm2 complex crystal structure. Such analyses can yield a pharmacophore model suitable as a search query for a 3D chemical database to generate new lead compounds. As preliminary validation of this methodology, the Hydropathic INTeractions (HINT) program has been used to generate noncovalent interaction measurements between reported peptide inhibitors and mdm2. Quantitative structure–activity relationships were developed expressing peptide activity as a linear combination of hydropathic descriptors. In general, HINT measurements accurately modeled the effects of even single-atom alterations of the p53–peptide structure on activity, accounting for 70–90% of variation in experimental inhibition constants. These results surpassed those of a recently described molecular dynamics-based approach and required significantly less computation time. In conclusion, the HINT program can be integrated into the drug design cycle for next-generation p53–mdm2 complex inhibitors with confidence in its ability to simulate this noteworthy protein–protein interaction. Proteins 2001;45:169–175. © 2001 Wiley-Liss, Inc.

The structure of catalase from human erythrocytes (HEC) was determined in tetragonal crystals of space group I4(1) by molecular-replacement methods, using the orthorhombic crystal structure as a search model. It was then refined in a unit cell of dimensions a = b = 203.6 and c = 144.6 A, yielding R and R(free) of 0.196 and 0.244, respectively, for all data at 2.4 A resolution. A major difference of the HEC structure in the tetragonal crystal compared with the orthorhombic structure was the omission of a 20-residue N-terminal segment corresponding to the first exon of the human catalase gene. The overall structures were otherwise identical in both crystal forms. The NADPH-binding sites were empty in all four subunits and bound water molecules were observed at the active sites. The structure of the C-terminal segment, which corresponds to the last exon, remained undetermined. The tetragonal crystals showed a pseudo-4(1)22 symmetry in molecular packing. Two similar types of lattice contact interfaces between the HEC tetramers were observed; they were related by the pseudo-dyad axes.

A non-covalent interaction force field model derived from the partition coefficient of 1-octanol/water solubility is described. This model, HINT for Hydropathic INTeractions, is shown to include, in very empirical and approximate terms, all components of biomolecular associations, including hydrogen bonding, Coulombic interactions, hydrophobic interactions, entropy and solvation/desolvation. Particular emphasis is placed on: (1) demonstrating the relationship between the total empirical HINT score and free energy of association, deltaGinteraction; (2) showing that the HINT hydrophobic-polar interaction sub-score represents the energy cost of desolvation upon binding for interacting biomolecules; and (3) a new methodology for treating constrained water molecules as discrete independent small ligands. An example calculation is reported for dihydrofolate reductase (DHFR) bound with methotrexate (MTX). In that case the observed very tight binding, deltaGinteraction < or = -13.6 kcal mol(-1), is largely due to ten hydrogen bonds between the ligand and enzyme with estimated strength ranging between -0.4 and -2.3 kcal mol(-1). Four water molecules bridging between DHFR and MTX contribute an additional -1.7 kcal mol(-1) stability to the complex. The HINT estimate of the cost of desolvation is +13.9 kcal mol(-1).

Catalase (E.C. 1.11.1.6) was purified from human erythrocytes and crystallized in three different forms: orthorhombic, hexagonal and tetragonal. The structure of the orthorhombic crystal form of human erythrocyte catalase (HEC), with space group P2(1)2(1)2(1) and unit-cell parameters a = 84.9, b = 141.7, c = 232.5 A, was determined and refined with 2.75 A resolution data. Non-crystallographic symmetry restraints were employed and the resulting R value and R(free) were 0.206 and 0.272, respectively. The overall structure and arrangement of HEC molecules in the orthorhombic unit cell were very similar to those of bovine liver catalase (BLC). However, no NADPH was observed in the HEC crystal and a water was bound to the active-site residue His75. Conserved lattice interactions suggested a common growth mechanism for the orthorhombic crystals of HEC and BLC.

Pyridoxine 5′-phosphate oxidase (PNP Ox) catalyzes the terminal step in the biosynthesis of pyridoxal 5′-phosphate. The 53-kDa homodimeric enzyme contains a noncovalently bound flavin mononucleotide (FMN) on each monomer. Three crystal forms of Escherichia coli PNP Ox complexed with FMN have been obtained at room temperature. The first crystal form belongs to trigonal space group P3 1 21 or P3 2 21 with unit cell dimensions a = b = 64.67A, c = 125.64A, and has one molecule of the complex (PNP Ox–FMN) per asymmetric unit. These crystals grow very slowly to their maximum size in about 2 to 4 months and diffract to about 2.3 A. The second crystal form belongs to tetragonal space group P4 1 or P4 3 with unit cell dimensions a = b = 54.92A, c = 167.65A, and has two molecules of the complex per asymmetric unit. The crystals reach their maximum size in about 5 weeks and diffract to 2.8 A. A third crystal form with a rod-like morphology grows faster and slightly larger than the other two forms, but diffracts poorly and could not be characterized by X-ray analysis. The search for heavy-atom derivatives for the first two crystal forms to solve the structure is in progress.

Improving the delivery of oxygen to the tissues by decreasing the oxygen affinity of haemoglobin has been a major aim of several laboratories over recent years because this may reduce the consequences of anaemia and/or improve tissue oxygenation in cases of decreased blood perfusion. Within the same context, lowering the oxygen affinity may prove valuable in the application of native or recombinant haemoglobin solutions as a blood substitute. The shift of the oxygen equilibrium curve to the right is obtained by various modulators. Among them, the bezafibrate derivatives are considered as a most interesting group. These principles are of the utmost importance in thalassaemia and other haemoglobinopathies where the beneficial effects of the compensatory synthesis of fetal haemoglobin are diminished by the increased oxygen affinity of this pigment. In this paper we present the results of a study initiated to determine whether a potent oxygen affinity modifier, RSR-4, could satisfactorily decrease the oxygen affinity of fetal haemoglobin, thus improving tissue oxygenation. The experiments were carried out on whole blood and on purified haemoglobin solutions and showed that the effector markedly decreased the oxygen affinity of HbF (from 18.7 to 3.73 mmHg in whole blood). At the same time the cooperativity index (n50) and the oxygen saturation levels remained within normal limits under the conditions of the main experiment. These observations have important implications for the potential application of oxygen affinity modifiers in vivo.

The software program, HINT (Hydropathic INTeractions), which characterizes non-polar-non-polar, polar-polar, and non-polar-polar interactions, has been used to examine subunit interface associations involved in the hemoglobin allosteric transition at a residue and atomic level. HINT differs from many other computational programs in that it is based not on a statistical method or a force-field but employs parameters experimentally determined from solvent transfer experiments. The main focus of this study is to compare HINT scores that are based upon experimentally and thermodynamically derived measurements with experimentally determined thermodynamic results. The HINT analysis yields a good first-order approximation of experimentally measured energies for these interactions as determined by free energies of dimer-tetramer assembly for mutant hemoglobins. The results provide a framework for understanding subunit stabilities based upon individual atom interactions and repulsions. HINT, in agreement with previous analyses, indicates that: (1) the alpha1beta1 and alpha2beta2 subunit contacts are stabilized via several polar and many hydrophobic interactions with few repulsive contact areas in both the T (deoxyhemoglobin) and R (oxyhemoglobin) structures; (2) the alpha1alpha2 subunit contacts are primarily stabilized by polar salt bridge linkages in both T and R states; and (3) the alpha1beta2 and alpha2beta1 contacts have both strong positive and negative interactions in both T and R states with few hydrophobic interactions. The HINT scoring methodology provides a quantitative characterization of the major role of the alpha1beta2 and alpha2beta1 interfaces in the T-->R quaternary transition. HINT also confirms the stronger hydrogen bond formation in mutant Hb Rothschild (Trp 37beta-->Arg) with Asp94alpha1 that gives rise to a low-affinity (deoxy) hemoglobin. HINT shows that the stabilization of the alpha1beta2 interface with mutant Hb Ypsilanti (Asp99alpha-->Tyr) produces a high-affinity (oxy) hemoglobin by reducing hydrophobic-polar contacts in the R state. HINT interaction maps also identified specific sites for mutagenesis at the alpha1beta2 interface that can be explored to shift the allosteric equilibrium in either direction. In addition, the HINT program provides useful diagnostic data for checking the quality of refined crystallographic structures.

Four new series of monoaldehyde bisacids and bisaldehyde bisacids with varying chain lengths have been synthesized and evaluated as allosteric effectors of hemoglobin. Molecular modeling, oxygen equilibrium, and crystallographic studies were combined for structure/function studies. Crystallographic analyses of the bisaldehydes reveal that Schiff base interaction occurred exclusively between Val 1 alpha and Lys 99 alpha of the opposite alpha chain even though the two terminal Val 1 alpha nitrogens are ideally spaced to also form cross-links. The reason for the observed mode of binding appears to be the influence of chain direction set by key substitutions on the bisaldehyde molecule. Even longer chain derivatives that could overcome the direction set by the key functional groups bind in the same manner. These studies support the general conclusion that long flexible molecules prefer to bind along cavity walls, like double-sided molecular sticky tape, rather than span large open spaces with few chances for interaction. The cross-linked bisaldehydes bind at the same site when incubated under both allosteric states and exhibit reduced cooperativity with a significant decrease in oxygen affinity. The chain length acts as a molecular ratchet and dictates the degree of allosteric effect observed. The tighter the cross-link, the greater the constraint on the tense- (T-) state and the stronger the allosteric effect that is produced. The monoaldehyde bisacids bind in the same fashion with Schiff base formation at Val 1 alpha while the acid that replaces the second aldehyde moiety forms a salt bridge with Lys 99 alpha of the opposite subunit. This class of molecules has weaker allosteric effector activity as would be expected with replacement of one covalent bond by a salt bridge. The importance of Lys 99 alpha on the allosteric equilibrium is confirmed.

Monoaldehyde allosteric effectors of hemoglobin were designed, using molecular modeling software (GRID), to form a Schiff base adduct with the Val 1 alpha N-terminal nitrogens and interact via a salt bridge with Arg 141 alpha of the opposite subunit. The designed molecules were synthesized if not available. It was envisioned that the molecules, which are aldehyde acids, would produce a high-affinity hemoglobin with potential interest as antisickling agents similar to other aldehyde acids reported earlier. X-ray crystallographic analysis indicated that the aldehyde acids did bind as modeled de novo in symmetry-related pairs to the alpha subunit N-terminal nitrogens. However, oxygen equilibrium curves run on solutions obtained from T- (tense) state hemoglobin crystals of reacted effector molecules produced low-affinity hemoglobins. The shift in the allosteric equilibrium was opposite to that expected. We conclude that the observed shift in allosteric equilibrium was due to the acid group on the monoaldehyde aromatic ring that forms a salt bridge with the guanidinium ion of Arg 141 alpha on the opposite subunit. This added constraint to the T-state structure that ties two subunits across the molecular symmetry axis shifts the equilibrium further toward the T-state. We tested this idea by comparing aldehydes that form Schiff base interactions with the same Val 1 alpha residues but do not interact across the dimer subunit symmetry axis (a new one in this study with no acid group and others that have had determined crystal structures). The latter aldehydes shift the allosteric equilibrium toward the R-state. A hypothesis to predict the direction in shift of the allosteric equilibrium is made and indicates that it is not exclusively where the molecule binds but how it interacts with the protein to stabilize or destabilize the T- (tense) allosteric state.

Physical organic structural properties of small molecules and macromolecules such as bond count, branching and proximity between multiple polar fragments contribute significantly to measured hydrophobicity (log P). These structural properties are encoded in the Rekker and Leo methods of calculating log P as structural-dependent factors. Regardless of the size of the atom primitive set, methods predicting log P with only atom primitives can miss subtle structural detail within series of related compounds. The HINT (Hydropathic INTeractions) model for inter- and intramolecular noncovalent interactions calculates atom-based hydrophobic constants, but uses all Leo-type factors in the calculation rather than a large set of atom primitives. Two types of applications of HINT are discussed: evaluation of the binding of an inhibitor (A74704) to HIV-1 protease, where it is shown that modeling of the protonation state (i.e., Asp25, Asp125) in the protein can strongly influence perceived substrate binding; and the use of HINT to calculate a third (hydropathic) field for CoMFA can yield a statistically enhanced and predictive model for molecular design.

Three new routines (LOCK. KEY and LOCKSMITH) for the program HINT (Hydrophobic interactions) are described and demonstrated. The KEY routine uses receptor structure to model the hydropathic profile of the ideal substrate for the receptor. The LOCK routine uses substrate or drug structure to model the hydropathic character of the receptor. LOCKSMITH is an algorithm designed to highlight the significant hydropathic features from a collection of agents. Ten allosteric modifiers of hemoglobin that have been characterized biologically and with X-ray diffraction to determine their protein binding sites/conformations illustrate the KEY and LOCKSMITH routines: The LOCKSMITH composite map correctly identifies the structural features and conformation of the more active modifiers. In addition, many hydropathic features of the “ideal” drug predicted by the KEY map overlap with actual structural features of the most active hemoglobin allosteric modifiers.

The design, synthesis, and testing of several halomethyl analogues of choline and acetylcholine as potential cholinotoxins is described. The compounds were evaluated for their ability to inhibit high-affinity choline transport and their affinity toward postsynaptic muscarinic receptors. Among the analogues tested, bromomethyl and iodomethyl analogues of choline were found to be the most potent inhibitors of the high affinity choline transport system. Introduction of a beta-methyl group in the halomethyl analogues drastically reduced their potencies. The bromomethyl and iodomethyl analogues were further investigated for their effects on choline acetyltransferase activity, acetylcholinesterase activity and QNB binding. Neither compound possesses significant ability to alter any of the above cholinergic markers, except at very high concentrations. These results suggest that the bromomethyl and iodomethyl choline analogues may be used as specific inhibitors of the presynaptic high-affinity choline transport system.

Vanillin, a food additive, has been evaluated as a potential agent to treat sickle cell anemia. Earlier studies indicated that vanillin had moderate antisickling activity when compared with other aldehydes. We have determined by high performance liquid chromatography that vanillin reacts covalently with sickle hemoglobin (HbS) both in solution and in intact red blood cells. Hemoscan oxygen equilibrium curves show a dose- dependent left shift, particularly at low oxygen tensions. Rheologic evaluation (pO2 scan Ektacytometry) of vanillin-reacted HbS erythrocytes shows a dose-dependent inhibition of deoxygenation-induced cell sickling. Ektacytometry also suggests that vanillin may have a direct inhibitory effect on HbS polymer formation. Vanillin has no adverse effects on cell ion or water content. X-ray crystallographic studies with deoxyhemoglobin (HbA)-vanillin demonstrate that vanillin binds near His 103 alpha, Cys 104 alpha, and Gln 131 beta in the central water cavity. A secondary binding site is located between His 116 beta and His 117 beta. His 116 beta has been implicated as a polymer contact residue. Oxygen equilibrium, ektacytometry, and x-ray studies indicate that vanillin may be acting to decrease HbS polymerization by a dual mechanism of action; allosteric modulation to a high-affinity HbS molecule and by stereospecific inhibition of T state HbS polymerization. Because vanillin is a food additive on the GRAS (generally regarded as safe) list, and because it has little or no adverse effects at high dosages in animals, vanillin is a candidate for further evaluation as an agent for the treatment of sickle cell disease.

The protein-bound conformations of six new allosteric effectors similar to bezafibrate that markedly decrease the oxygen affinity of hemoglobin have been determined by X-ray crystallography. Comparisons are made with the bound conformations of three urea analogues reported by Lalezari, Perutz, and co-workers. All six new molecules bind at the same site previously observed for bezafibrate and exhibit a wide range of allosteric activity. Unlike the urea derivatives, which show two binding sites for the most potent derivatives, only one of the six new molecules (one with moderate allosteric activity) exhibits a second binding site. A new computer program, HINT (hydrophobic interactions), has been created and utilized to identify the major interactions between small molecules and the protein. The three strongest interactions identified by HINT involve Arg 141 alpha with the acid of the analogues, Lys 99 alpha with the bridging amide carbonyl, and the amide NH of the side chain of Asn 108 beta with the halogenated aromatic ring.

Three isomeric series of 2-(aryloxy)-2-methylpropionic acids were prepared and studied for their ability to decrease the oxygen affinity of human hemoglobin A. The isomeric aryloxy groups included 4-[[(aryloyl)amino]methyl]phenoxy, 4-(arylacetamido)phenoxy, and 4-[[(arylamino)carbonyl]methyl]phenoxy. A total of 20 compounds were synthesized and tested. Structure-activity relationships are presented. Several of the new compounds were found to be strong allosteric effectors of hemoglobin. The two most active compounds are 2-[4-[[(3,5-dichloroanilino)carbonyl]-methyl]phenoxy]- 2-methylpropionic acid and the corresponding 3,5-dimethyl derivative. The latter two compounds have been compared to other known potent allosteric effectors in the same assay and show greater activity. Both compounds also exhibit a right shift in the oxygen equilibrium curve when incubated with whole blood. The new compounds may be of interest in clinical or biological areas that require or would benefit from a reversal of depleted oxygen supply (i.e., ischemia, stroke, tumor radiotherapy, blood storage, blood substitutes, etc.). They are also structurally related to several marketed antilipidemic agents.

Department of General and Inorganic Chemistry, UniVersity of Parma, Via G.P. Usberti 17/A 43100, Parma, Italy, National Institute for Biosystems and Biostructures, Rome, Italy, Department of Medicinal Chemistry and Institute for Structural Biology & Drug DiscoVery, Virginia Commonwealth UniVersity, Richmond, Virginia 23298-0540, Department of Pharmaceutics, UniVersity of Parma, Via GP Usberti 27/A, 43100 Parma, Italy, High Performance Systems, CINECA Supercomputing Centre, Casalecchio di Reno, Bologna, Italy, Dulbecco Telethon Institute, Department of Chemistry, UniVersity of Modena and Reggio Emilia, Via Campi 183, 41100 Modena, Italy, Department of Mathematics and Natural Sciences, Pharmaceutical Institute, Christian-Albrechts-UniVersity, Gutenbergstrasse 76, 24118 Kiel, Germany, Departments of Biochemistry & Molecular Biology, Computer Science & Engineering, and Physics & Astronomy, Michigan State UniVersity, East Lansing, Michigan 48824-1319, Department of Molecular Biology, MB-5, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037-1000, Molecular Modeling and Bioinformatics Unit, Institute of Biomedical Research, Scientific Park of Barcelona, Department of Biochemistry and Molecular Biology, UniVersity of Barcelona, Josep Samitier 1-5, Barcelona 08028, Spain, Department of Experimental Medicine, UniVersity of Parma, Via Volturno, 39, 43100, Parma, Italy, Department of Chemical, Food, Pharmaceutical and Pharmacological Sciences, UniVersity of Piemonte Orientale “Amedeo AVogadro”, Via BoVio 6, 28100 NoVara, Italy, Institute of Pharmacy and Food Chemistry, UniVersity of Wurzburg, Am Hubland, D-97074 Wurzburg, Germany

This paper details comprehensive binding studies (solution and X-ray) of human hemoglobin A with a group of halogenated carboxylic acids that were investigated as potential antisickling agents. It is, to our knowledge, the first study to compare solution and crystal binding for a series of compounds under similar high-salt conditions used for cocrystallization. The compounds include [(3,4-dichlorobenzyl)oxy]acetic acid, [(p-bromobenzyl)oxy]acetic acid, clofibric acid, and bezafibrate. The location and stereochemistry of binding sites have been established by X-ray crystallography, while the number of binding sites and affinity constants were measured by using equilibrium dialysis. The solution binding studies were conducted with deoxygenated hemoglobin and carbonmonoxyhemoglobin with low (50 mM) and high (2 M) salt concentrations. It was concluded that the observed crystal structures are consistent with the binding observed in solution and that the number of binding sites is independent of salt concentration, while the binding constant increases with increasing salt concentration. The studies also reveal that relatively small changes in the chemical structure of a drug molecule can result in entirely different binding sites on the protein. Moreover, the X-ray studies provide a possible explanation for the multiplicity in function exhibited by these compounds as allosteric modulators and/or antisickling agents. Finally, the studies that these compounds bind differently to the R and T states of hemoglobin, an observation of special significance to the original design of these agents.

The stereochemistry associated with the amobarbital N-glucoside diastereomers (1a and 1b) that are excreted by humans in urine is unknown. Using X-ray crystallography, the absolute configuration of 1b was determined to be S (C-5 position of the barbiturate ring). Following oral administration of amobarbital to Caucasians and Orientals, from 5 to 25% of the dose of amobarbital was excreted in the urine as 1b. The other diastereomer, 1a, accounted for less than 0.1 to 0.2% of the dose in four individuals, with none detected in nine individuals. The rate constants, k f ,1b, determined from the urinary excretion of lb were lower than those previously reported for unresolved amobarbital N-glucosides. However, based on the urinary excretion of lb, the rate constants, K, for elimination of amobarbital in Caucasians and Orientals were similar to those previously determined from the serum levels of amobarbital and the urinary excretion of unresolved amobarbital N-glucosides. In previous studies of the N-glucosylation of amobarbital, it is likely that a single N-glucose diastereomer, lb, was being observed.

Erythrocyte pyruvate kinase (PK) is an important glycolytic enzyme, and manipulation of its regulatory behavior by allosteric modifiers is of interest for medicinal purposes. Human-erythrocyte PK was expressed in Rosetta cells and purified on an Ni-NTA column. A search of the small-molecules database of the National Cancer Institute (NCI), using the UNITY software, led to the identification of several compounds with similar pharmacophores as fructose-1,6-bisphosphate (FBP), the natural allosteric activator of the human kinases. The compounds were subsequently docked into the FBP binding site using the programs FlexX and GOLD, and their interactions with the protein were analyzed with the energy-scoring function of HINT. Seven promising candidates, compounds 1-7, were obtained from the NCI, and subjected to kinetics analysis, which revealed both activators and inhibitors of the R-isozyme of PK (R-PK). The allosteric effectors discovered in this study could prove to be lead compounds for developing medications for the treatment of hemolytic anemia, sickle-cell anemia, hypoxia-related diseases, and other disorders arising from erythrocyte PK malfunction.