Evaluation of the Binding Affinity of Nitrosylcobalamin to Intrinsic Factor as a Predictive Model for Cobalamin Binding Protein Interactions: A Comparative Study with Hydroxocobalamin.

Regiospecific replacement of a single phosphate (PO2) by a dithiophosphate (PS2) group in an RNA can dramatically increase its binding affinity for a target protein. Thus, complexes between antithrombin and anti-VEGF RNA aptamers with single dithiophosphate moieties and thrombin and VEGF, respectively, display equilibrium dissociation constants KD of ca. 1 pM, 1000-fold tighter than the native RNA complexes (ca. 1 nM). Inspection of crystal structures of the native and PS2-RNA aptamer:thrombin complexes reveals an RNA-induced fit in the latter. This leads to a close approach between PS2 and the phenyl ring edge of Phe-232 that is surrounded by pairs of lysines and arginines. To better understand the origins of the tighter binding and individual contributions to the interaction energy, we carried out QM calculations with phosphate- and dithiophosphate-benzene and dimethyl phosphate- and dimethyl dithiophosphate-benzene model systems. These calculations demonstrate that the dithiophosphate-benzene interaction is much stronger than the corresponding interaction with phosphate. QM/MM calculations with the full complexes confirmed this finding and support the hypothesis that the electric field generated by basic residues surrounding Phe-232 is key to the polarization of the PS2 moiety. Thus, disparate polarization and dispersion energies between the PO2 and PS2 complexes contribute critically to the difference in binding affinity. By comparison, easier desolvation of the dithiophosphate group compared to phosphate does not contribute decisively to the observed difference in binding affinity. Favorable polarization and dispersion energies may be a general feature of the dramatic affinity gains seen for complexes between RNAs carrying dithiophosphate groups and their binding proteins.

Prostate cancer remains a significant oncological challenge, driven by molecular factors such as KLK3 (kallikrein-related peptidase 3). Text mining of 237,357 PubMed articles identified KLK3 as the most frequently cited protein (10,477 mentions in titles; 162,619 in abstracts), with strong co-mentions of AR, TMPRSS2, and ERG (χ2, *p* < 0.001). Structural modeling of KLK3 (PDB: 2ANY) using I-TASSER yielded a high-confidence 3D structure (C-score: 0.73), validated by Ramachandran analysis, with 99.5% of residues falling in favored regions. Phytochemical profiling ofCurcuma longarevealed potent bioactive constituents, with leaf extracts showing the highest total phenolic (510.7 ± 0.07 µg/mL) and flavonoid (498.9 ± 0.05 µg/mL) content. LC-MS identified 23 bioactive compounds, which exhibited exceptional binding affinity. Virtual screening of FDA-approved drugs (-11.8 to -9.4 kcal/mol), food-derived compounds (-10.0 to -9.1 kcal/mol), and natural products (-11.4 to -8.8 kcal/mol) revealed significant differences in binding affinities. MK3207 showed the highest binding affinity (-11.7 kcal/mol), followed by MolPort-039-338-696 (-11.4 kcal/mol), with key interactions at PHE-110 and THR-167. In-silico docking shows that MK3207 exhibits the strongest binding affinity to KLK3 (- 11.7 kcal/mol), with accuracy validated by an RMSD of 0.195 Å. Pharmacokinetic and drug-likeness evaluation of MK3207 indicated moderate solubility (Log S: -4.58 to -5.02), high lipophilicity (consensus log Po/w: 3.39), favorable drug-likeness (no PAINS/Lipinski violations, bioavailability score: 0.55, synthetic accessibility: 5.21). MD simulations (100 ns) confirmed stable KLK3-ligand binding (final RMSD: 4.3 Å protein, 3.1 Å ligand; average RMSD: 3.99 Å C-α, 3.97 Å backbone, 5.41 Å sidechain). The complex exhibited moderate flexibility (RMSF peaks: 1-4 Å), 28.32% secondary structure, and persistent interactions (hydrophobic: VAL-49, PHE-110; hydrogen bonds: THR-167, SER-213). MM-GBSA analysis revealed strong binding energy (-75.57 to -66.36 kcal/mol) and consistent ligand efficiency. This study bridgescomputational drug discoveryandphytochemical analysis, nominatingCurcuma longaderivatives andMK3207as promising KLK3 inhibitors for PC therapy.

Plexin receptors regulate cell adhesion, migration, and guidance. The Rho GTPase binding domain (RBD) of plexin-A1 and -B1 can bind GTPases, including Rnd1. By contrast, plexin-C1 and -D1 reportedly bind Rnd2 but associate with Rnd1 only weakly. The structural basis of this differential Rnd1 GTPase binding to plexin RBDs remains unclear. Here, we solved the structure of the plexin-A2 RBD in complex with Rnd1 and the structures of the plexin-C1 and plexin-D1 RBDs alone, also compared with the previously determined plexin-B1 RBD.Rnd1 complex structure. The plexin-A2 RBD·Rnd1 complex is a heterodimer, whereas plexin-B1 and -A2 RBDs homodimerize at high concentration in solution, consistent with a proposed model for plexin activation. Plexin-C1 and -D1 RBDs are monomeric, consistent with major residue changes in the homodimerization loop. In plexin-A2 and -B1, the RBD β3-β4 loop adjusts its conformation to allow Rnd1 binding, whereas minimal structural changes occur in Rnd1. The plexin-C1 and -D1 RBDs lack several key non-polar residues at the corresponding GTPase binding surface and do not significantly interact with Rnd1. Isothermal titration calorimetry measurements on plexin-C1 and -D1 mutants reveal that the introduction of non-polar residues in this loop generates affinity for Rnd1. Structure and sequence comparisons suggest a similar mode of Rnd1 binding to the RBDs, whereas mutagenesis suggests that the interface with the highly homologous Rnd2 GTPase is different in detail. Our results confirm, from a structural perspective, that Rnd1 does not play a role in the activation of plexin-C1 and -D1. Plexin functions appear to be regulated by subfamily-specific mechanisms, some of which involve different Rho family GTPases.

The rational design of DNA/RNA aptamers for use as molecular probes depends on a clear understanding of their structural elements in relation to target-aptamer binding interactions. We present a simple method to create aptamer probes that can occupy two different structural states. Then, based on the difference in binding affinity between these states, target-aptamer binding interactions can be elucidated. The basis of our two-state system comes from the incorporation of azobenzene within the DNA strand. Azobenzene can be used to photoregulate the melting of DNA-duplex structures. When incorporated into aptamers, the light-regulated conformational change of azobenzene can be used to analyze how aptamer secondary structure is involved in target binding. Azobenzene-modified aptamers showed no change in target selectivity, but showed differences in binding affinity as a function of the number, position, and conformation of azobenzene modifications. Aptamer probes that can change binding affinity on demand may have future uses in targeted drug delivery and photodynamic therapy.

The thrombin binding aptamer (TBA) is a promising nucleic acid-based anticoagulant. We studied the effects of chemical modifications, such as dendrimer Trebler and NHS carboxy group, on TBA with respect to its structures and thrombin binding affinity. The two dendrimer modifications were incorporated into the TBA at the 5′ end and the NHS carboxy group was added into the thymine residues in the thrombin binding site of the TBA G-quadruplex (at T4, T13 and both T4/T13) using solid phase oligonucleotide synthesis. Circular dichroism (CD) spectroscopy confirmed that all of these modified TBA variants fold into a stable G-quadruplex. The binding affinity of TBA variants with thrombin was measured by surface plasmon resonance (SPR). The binding patterns and equilibrium dissociation constants (KD) of the modified TBAs are very similar to that of the native TBA. Molecular dynamics simulations studies indicate that the additional interactions or stability enhancement introduced by the modifications are minimized either by the disruption of TBA–thrombin interactions or destabilization elsewhere in the aptamer, providing a rational explanation for our experimental data. Overall, this study identifies potential positions on the TBA that can be modified without adversely affecting its structure and thrombin binding preference, which could be useful in the design and development of more functional TBA analogues.

We have studied the binding of recombinant human insulin-like growth factor I (hIGF-I), and hIGF-II, and rat IGF-II [rIGF-II (multiplication-stimulating activity)] to the human amniotic fluid IGF-binding protein placental protein-12 (PP12). PP12 displayed a 5- to 10-fold higher affinity for IGF-I compared to hIGF-II or rIGF-II. These differences in binding affinity were confirmed by both saturation binding analysis and competitive binding analysis using 125I-labeled IGF-I, hIGF-II, and rIGF-II and each of the unlabeled ligands. PP12 produced dose-dependent inhibition of IGF-I-stimulated [3H]thymidine incorporation in the rat thyroid follicular cell line FRTL5. Inhibition of IGF-I-stimulated thymidine incorporation paralleled the ability of PP12 to inhibit IGF-I binding to the surface of FRTL5. At a high concentration, PP12 also inhibited TSH-stimulated DNA synthesis but did not inhibit the binding of 125I-labeled TSH to FRTL5. Insulin did not inhibit the binding of 125I-labeled IGFs to PP12, and PP12 did not inhibit the ability of insulin to stimulate DNA synthesis. These data suggest that the ability of PP12 to inhibit TSH-stimulated DNA synthesis is through the inactivation of IGF produced endogenously by FRTL5. Low concentrations of PP12 produced a statistically significant enhancement of TSH-stimulated DNA synthesis; the mechanism by which this occurs remains unclear.

An in vitro Assay to Measure Targeted Drug Delivery to Bone Mineral

The Chinese horseshoe bat (Rhinolophus sinicus), reservoir host of severe acute respiratory syndrome coronavirus (SARS-CoV), carries many bat SARS-related CoVs (SARSr-CoVs) with high genetic diversity, particularly in the spike gene. Despite these variations, some bat SARSr-CoVs can utilize the orthologs of the human SARS-CoV receptor, angiotensin-converting enzyme 2 (ACE2), for entry. It is speculated that the interaction between bat ACE2 and SARSr-CoV spike proteins drives diversity. Here, we identified a series of R. sinicus ACE2 variants with some polymorphic sites involved in the interaction with the SARS-CoV spike protein. Pseudoviruses or SARSr-CoVs carrying different spike proteins showed different infection efficiencies in cells transiently expressing bat ACE2 variants. Consistent results were observed by binding affinity assays between SARS-CoV and SARSr-CoV spike proteins and receptor molecules from bats and humans. All tested bat SARSr-CoV spike proteins had a higher binding affinity to human ACE2 than to bat ACE2, although they showed a 10-fold lower binding affinity to human ACE2 compared with that of their SARS-CoV counterpart. Structure modeling revealed that the difference in binding affinity between spike and ACE2 might be caused by the alteration of some key residues in the interface of these two molecules. Molecular evolution analysis indicates that some key residues were under positive selection. These results suggest that the SARSr-CoV spike protein and R. sinicus ACE2 may have coevolved over time and experienced selection pressure from each other, triggering the evolutionary arms race dynamics.IMPORTANCE Evolutionary arms race dynamics shape the diversity of viruses and their receptors. Identification of key residues which are involved in interspecies transmission is important to predict potential pathogen spillover from wildlife to humans. Previously, we have identified genetically diverse SARSr-CoVs in Chinese horseshoe bats. Here, we show the highly polymorphic ACE2 in Chinese horseshoe bat populations. These ACE2 variants support SARS-CoV and SARSr-CoV infection but with different binding affinities to different spike proteins. The higher binding affinity of SARSr-CoV spike to human ACE2 suggests that these viruses have the capacity for spillover to humans. The positive selection of residues at the interface between ACE2 and SARSr-CoV spike protein suggests long-term and ongoing coevolutionary dynamics between them. Continued surveillance of this group of viruses in bats is necessary for the prevention of the next SARS-like disease.

Human apurinic/apyrimidinic (AP) endonuclease (hAPE) initiates the repair of an abasic site (AP site). To gain insight into the mechanisms of damage recognition of hAPE, we conducted surface plasmon resonance spectroscopy to study the thermodynamics and kinetics of its interaction with substrate DNA containing an abasic site (AP DNA). The affinity of hAPE binding toward DNA increased as much as 6-fold after replacing a single adenine (equilibrium dissociation constant, K(D), 5.3 nm) with an AP site (K(D), 0.87 nm). The enzyme-substrate complex formation appears to be thermodynamically stabilized and favored by a large change in Gibbs free energy, DeltaG degrees (-50 kJ/mol). The latter is supported by a high negative change in enthalpy, DeltaH degrees (-43 kJ/mol) and also positive change in entropy, DeltaS degrees (24 J/(K mol)), and thus the binding process is spontaneous at all temperatures. Analysis of kinetic parameters reveals small enthalpy of activation for association, DeltaH degrees++(ass) (-17 kJ/mol), and activation energy for association (E(a), -14 kJ/mol) when compared with the enthalpy of activation for dissociation, DeltaH degrees++(diss) (26 kJ/mol), and activation energy in the reverse direction (E(d), 28 kJ/mol). Furthermore, varying concentration of KCl showed an increase in binding affinity at low concentration but complete abrogation of the binding at higher concentration, implying the importance of hydrophobic, but predominantly ionic, forces in the Michaelis-Menten complex formation. Thus, low activation energy and the enthalpy of activation, which are perhaps a result of dipole-dipole interactions, play critical roles in AP site binding of APE.

This thesis presents several interlinked projects aimed at sensitive detection of chemical and biological species by the use of surface plasmon resonance-based biosensors. This optical technique takes advantage of electromagnetic field confined at the nanoscale that is generated by the coupling light to the collective oscillation of charge density at the surface of metallic films and metallic nanoparticles. In particular, the thesis utilizes surface plasmon field-enhanced fluorescence spectroscopy (SPFS) and surface plasmon resonance (SPR) biosensors for sensitive readout of heterogeneous assays on gold sensor surface. It focuses on several important aspects that are essential to unlock the potential of optical biosensors in the emerging field of biomedical sciences that require detection of species serving as biomarkers or drugs, in close contact with the human body, outside of specialized laboratories. Specially,it aims at optical biosensor systems that hold potential for continuous monitoring of compounds in complex liquid samples and the following three projects have been pursued.Firstly, fluorescence assay with weak affinity recognition elements that reversibly bind target analyte has been employed. In order to compensate for low fluorescence signal associated with the affinity binding of target analyte at the sensor surface, the SPFS was implemented. A fluorophore-labeled hairpin aptamer structure was designed for reversible real-time biosensor, which was demonstrated for a model analyte –adenosine triphosphate. The sensing concept relies on resonant fluorescence energy transfer between the fluorophore label and metal that occurs at short distances. The aptamer hairpin is opened and closed by the specific capture of target analyte which is translated to strong variations in fluorescence signal intensity amplified by the intense surface plasmon field. A fully reversible sensor was achieved with up to 23-fold increased fluorescence signal when target analyte was captured. In addition to the aptamer hairpin, split sequences were implemented in a sandwich-type assay for the same analyte. The lack of interaction of the aptamer split sequences in the absence of the analyte allowed for a highly reduced background interference. The sensor demonstrated full reversibility that allowed multiple rounds of detection on the same sensor chip, with time resolution of several minutes for ligands with equilibrium affinity binding constants at around mM concentration. Secondly, hydrogel materials were employed for the construction of a biointerface that is resistant to fouling from blood serum. It was utilized in the form of thin surface-attached layers and free-standing membranes spanning above gold-coated surface plasmon resonance-based biosensors. The hydrophilic nature of the used hydrogels provided efficient means to repel the unspecific sorption from serum, as demonstrated by optical waveguide spectroscopy method. In addition, the interaction of biomolecules with the poly-(N-isopropyl)acrylamide – based hydrogel used was thoroughly investigated by fluorescence correlation spectroscopy. These materials were tailored for filtering applications as the permeability of hydrogels can be efficiently controlled by tuning the pore size. A thermo-responsive hydrogel was used for dynamic switching of the membrane with about micrometer thickness between its closed and permeable states. Thirdly, a thin hydrogel layer was employed as an efficient 3D affinity binding matrix that takes advantage of its large surface area. This material was employed in a fluorescence assay that relies on horse-radish peroxidase label and tyramide-based enzymatic fluorescence signal amplification. On 2D surface architectures, self-quenching occurs which limits the performance characteristics of this approach. The use of 3D hydrogel architecture offers means to overcome this limitation and is demonstrated to provide 2 orders of magnitude increase in the fluorescence signal intensity compared to conventional fluorophore-labeled detection scheme.

BackgroundWater contamination with human enteric viruses has posed human health risks all over the world. Reasonable and facile methodologies for recovering and quantifying infectious enteric viruses in environmental samples are needed to address the issues of waterborne viral infectious diseases. In this study, a bacterial protein that has a binding capability with several enteric viruses is discovered, and its binding characteristics were investigated for utilizing it as a viral adsorbent in virus recovery and detection technologies.ResultsA gene of an enteric virus-binding protein (EVBP), derived from a monomer of a bacterial chaperon protein GroEL, was successfully acquired from a genomic DNA library of activated sludge microorganisms with nested PCR. Equilibrium dissociation constants between EVBP and norovirus-like particles (NoVLPs) of genotypes GI.7 and GII.4, estimated with quartz crystal microbalance method, were 240 and 210 nM, respectively. These values of equilibrium dissociation constant imply that the binding affinity between EVBP and NoVLPs is 1 to 3-log weaker than that in general antigen-antibody interactions, but about 2-log stronger than that in weak specific interactions of proteins with cations and organic polymers. The adsorptions of EVBP to norovirus, group A rotavirus and poliovirus type 1 were found to be significant in enzyme-linked immunosorbent assay. Meanwhile, the binding of native GroEL tetradecamer to viral particles was weaker than that of EVBP, presumably because of a steric hindrance. The small molecule of EVBP could have an advantage in the access to the surface of viral particles with rugged structure.ConclusionsEVBP that has a broad binding spectrum to enteric viruses was newly discovered. The broad binding characteristic of EVBP would allow us to utilize it as a novel adsorbent for detecting diverse enteric viruses in clinical and environmental samples.

The aim of this study was to develop high-affinity single-stranded DNA (ssDNA) aptamers that can selectively recognize the protein Ras and can be used as preventive and therapeutic agents for restenosis occurring after coronary surgery or angioplasty. For this purpose, we used the systematic evolution of ligands by exponential enrichment (SELEX) technique, also known as in vitro selection. Using this technique, ssDNA aptamers recognizing the Ras protein were obtained from a synthesized random ssDNA library in vitro. The binding rate and affinity of each aptamer pool, isolated in successive rounds of selection, were measured using ELISA, and the finally selected aptamer pool was cloned and sequenced. The binding affinities of each aptamer in this pool were measured. Their primary and secondary structures were analyzed using the DNAMAN 5.29 software, and the relationship between these structures and corresponding binding affinities was analyzed. The rate of aptamer pool binding to the Ras protein gradually increased from 2.4 to 34.5% along the selection process. Optical density (OD) and equilibrium dissociation constant (Kd) measurements showed that OD gradually increased from 0.220 to 1.080 and Kd decreased from 51.5 to 18.3 nM. The 11th pool of aptamers was selected based on these analyses, and cloning and sequencing of individual aptamers was performed. Secondary structure analysis revealed different conformations, but of a single type: stem‑loop. The aptamer Ra1 showed the highest affinity, with a measured OD of 1.213 and an estimated Kd of 15.3 nM. The binding affinity of the aptamer Ra1 to Ras was dose-dependent. In conclusion, high‑affinity ssDNA aptamers recognizing the Ras protein have been successfully selected. These aptamers may serve in the future as preventive and/or therapeutic agents for restenosis occurring after coronary surgery or angioplasty.

A series of pyrido- and pyrimidomorphinans (6a-h and 7a-g) were synthesized from naltrexone and evaluated for binding and biological activity at the opioid receptors. The unsubstituted pyridine 6a displayed high affinities at opioid delta, mu, and kappa receptors with K(i) values of 0.78, 1.5, and 8.8 nM, respectively. Compound 6a was devoid of agonist activity in the mouse vas deferens (MVD) and guinea pig ileum (GPI) preparations but was found to display moderate to weak antagonist activity in the MVD and GPI with K(e) values of 37 and 164 nM, respectively. The pyrimidomorphinans in general displayed lower binding potencies and delta receptor binding selectivities than their pyridine counterparts. Incorporation of aryl groups as putative delta address mimics on the pyrido- and pyrimidomorphinan framework gave ligands with significant differences in binding affinity and intrinsic activity. Attachment of a phenyl group at the 4'-position of 6a or the equivalent 6'-position of 7a led to dramatic reduction in binding potencies at all the three opioid receptors, indicating the existence of a somewhat similar steric constraint at the ligand binding sites of delta, mu, and kappa receptors. In contrast, the introduction of a phenyl group at the 5'-position of 6a did not cause any reduction in the binding affinity at the delta receptor. In comparison to the unsubstituted pyridine 6a, the 5'-phenylpyridine 6c showed improvements in mu/delta and kappa/delta binding selectivity ratios as well as in the delta antagonist potency in the MVD. Interestingly, introduction of a chlorine atom at the para position of the pendant 5'-phenyl group of 6c not only provided further improvements in delta antagonist potency in the MVD but also shifted the intrinsic activity profile of 6c from an antagonist to that of a mu agonist in the GPI. Compound 6d thus possesses the characteristics of a nonpeptide mu agonist/delta antagonist ligand with high affinity at the delta receptor (K(i) = 2.2 nM), high antagonist potency in the MVD (K(e) = 0.66 nM), and moderate agonist potency in the GPI (IC(50) = 163 nM). Antinociceptive evaluations in mice showed that intracerebroventricular (icv) injections of 6d produced a partial agonist effect in the 55 degrees C tail-flick assay and a full agonist effect in the acetic acid writhing assay (A(50) = 7.5 nmol). No signs of overt toxicity were observed with this compound in the dose ranges tested. Moreover, repeated icv injections of an A(90) dose did not induce any significant development of antinociceptive tolerance in the acetic acid writhing assay. The potent delta antagonist component of this mixed mu agonist/delta antagonist may be responsible for the diminished propensity to produce tolerance that this compound displays.

We report the binding free energy calculation and its decomposition for the complexes of alpha-lytic protease and its protein inhibitors using molecular dynamics simulation. Standard mechanism serine protease inhibitors eglin C and OMTKY3 are known to have strong binding affinity for many serine proteases. Their binding loops have significant similarities, including a common P1 Leu as the main anchor in the binding interface. However, recent experiments demonstrate that the two inhibitors have vastly different affinity towards alpha-lytic protease (ALP), a bacterial serine protease. OMTKY3 inhibits the enzyme much more weakly (by approximately 10(6) times) than eglin C. Moreover, a variant of OMTKY3 with five mutations, OMTKY3M, has been shown to inhibit 10(4) times more strongly than the wild-type inhibitor. The underlying mechanisms for the unusually large difference in binding affinities and the effect of mutation are not well understood. Here we use molecular dynamics simulation with molecular mechanics-Poisson Boltzmann/surface area method (MM-PB/SA) to investigate quantitatively the binding specificity. The calculated absolute binding free energies correctly differentiate the thermodynamic stabilities of these protein complexes, but the magnitudes of the binding affinities are systematically overestimated. Analysis of the binding free energy components provides insights into the molecular mechanism of binding specificity. The large DeltaDeltaG(bind) between eglin C and wild type OMTKY3 towards ALP is mainly attributable to the stronger nonpolar interactions in the ALP-eglin C complex, arising from a higher degree of structural complementarity. Here the electrostatic interaction contributes to a lesser extent. The enhanced inhibition in the penta-mutant OMTKY3M over its wild type is entirely due to an overall improvement in the solvent-mediated electrostatic interactions in the ALP-OMTKY3M complex. The results suggest that for these protein-complexes and similar enzyme-inhibitor systems (1) the binding is driven by nonpolar interactions, opposed by overall electrostatic and solute entropy contributions; (2) binding specificity can be tuned by improving the complementarity in electrostatics between two associating proteins. Binding free energy decomposition into contributions from individual protein residues provides additional detailed information on the structural determinants and subtle conformational changes responsible for the binding specificity.

Glutamate dehydrogenase (GDH), which is present in most bacteria and eukaryotes’ mitochondria, plays an important role in amino acid metabolism. In general, GDH converts 2-oxoglutarate to l-glutamate using NAD(P)H as a cofactor, and vice versa. Acquiring more structural information about the GDH of Synechocystis sp. PCC 6803 could be helpful in many studies related to amino acid metabolism in cyanobacteria. In this study, homology modeling studies were conducted to achieve an acceptable structure of the GDH using recognized templates. To this end, a computational approach was used to demonstrate the coenzyme specificity of GDH for NADPH and NADH. The present study involved homology modeling of GDH and docking analyses of NADPH, NADH, 2-oxoglutarate, and l-glutamate into the predictive model of GDH. The results of this study suggest that GDH has similar coenzyme specificity for NADH and NADPH, while NADH has a better binding affinity than NADPH. Furthermore, the binding sites of 2-oxoglutarate and l-glutamate are similar to each other with differences in binding affinity.