RESUMEN
CDK4 and CDK6 are kinases with similar sequences that regulate cell cycle progression and are validated targets in the treatment of cancer. Glioblastoma is characterized by a high frequency of CDKN2A/CCND2/CDK4/CDK6 pathway dysregulation, making dual inhibition of CDK4 and CDK6 an attractive therapeutic approach for this disease. Abemaciclib, ribociclib, and palbociclib are approved CDK4/6 inhibitors for the treatment of HR+/HER2- breast cancer, but these drugs are not expected to show strong activity in brain tumors due to poor blood brain barrier penetration. Herein, we report the identification of a brain-penetrant CDK4/6 inhibitor derived from a literature molecule with low molecular weight and topological polar surface area (MWâ¯=â¯285 and TPSAâ¯=â¯66â¯Å2), but lacking the CDK2/1 selectivity profile due to the absence of a basic amine. Removal of a hydrogen bond donor via cyclization of the pyrazole allowed for the introduction of basic and semi-basic amines, while maintaining in many cases efflux ratios reasonable for a CNS program. Ultimately, a basic spiroazetidine (cpKaâ¯=â¯8.8) was identified that afforded acceptable selectivity over anti-target CDK1 while maintaining brain-penetration in vivo (mouse Kp,uuâ¯=â¯0.20-0.59). To probe the potency and selectivity, our lead compound was evaluated in a panel of glioblastoma cell lines. Potency comparable to abemaciclib was observed in Rb-wild type lines U87MG, DBTRG-05MG, A172, and T98G, while Rb-deficient cell lines SF539 and M059J exhibited a lack of sensitivity.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Diseño de Fármacos , Glioblastoma/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/metabolismo , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Células MCF-7 , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Relación Estructura-ActividadRESUMEN
BI 224436 is an HIV-1 integrase inhibitor with effective antiviral activity that acts through a mechanism that is distinct from that of integrase strand transfer inhibitors (INSTIs). This 3-quinolineacetic acid derivative series was identified using an enzymatic integrase long terminal repeat (LTR) DNA 3'-processing assay. A combination of medicinal chemistry, parallel synthesis, and structure-guided drug design led to the identification of BI 224436 as a candidate for preclinical profiling. It has antiviral 50% effective concentrations (EC50s) of <15 nM against different HIV-1 laboratory strains and cellular cytotoxicity of >90 µM. BI 224436 also has a low, â¼2.1-fold decrease in antiviral potency in the presence of 50% human serum and, by virtue of a steep dose-response curve slope, exhibits serum-shifted EC95 values ranging between 22 and 75 nM. Passage of virus in the presence of inhibitor selected for either A128T, A128N, or L102F primary resistance substitutions, all mapping to a conserved allosteric pocket on the catalytic core of integrase. BI 224436 also retains full antiviral activity against recombinant viruses encoding INSTI resistance substitutions N155S, Q148H, and E92Q. In drug combination studies performed in cellular antiviral assays, BI 224436 displays an additive effect in combination with most approved antiretrovirals, including INSTIs. BI 224436 has drug-like in vitro absorption, distribution, metabolism, and excretion (ADME) properties, including Caco-2 cell permeability, solubility, and low cytochrome P450 inhibition. It exhibited excellent pharmacokinetic profiles in rat (clearance as a percentage of hepatic flow [CL], 0.7%; bioavailability [F], 54%), monkey (CL, 23%; F, 82%), and dog (CL, 8%; F, 81%). Based on the excellent biological and pharmacokinetic profile, BI 224436 was advanced into phase 1 clinical trials.
Asunto(s)
Inhibidores de Integrasa VIH/farmacología , VIH-1/efectos de los fármacos , VIH-1/enzimología , Sustitución de Aminoácidos/genética , Sustitución de Aminoácidos/fisiología , Animales , Fármacos Anti-VIH/farmacología , Células CACO-2 , Clonación Molecular , Inhibidores Enzimáticos del Citocromo P-450/farmacología , ADN Viral/efectos de los fármacos , Farmacorresistencia Viral , Integrasa de VIH/biosíntesis , Integrasa de VIH/genética , Integrasa de VIH/metabolismo , Inhibidores de Integrasa VIH/metabolismo , Inhibidores de Integrasa VIH/farmacocinética , Hepatocitos/metabolismo , Humanos , Ratones , Ratas , Suero/virología , Replicación Viral/efectos de los fármacosRESUMEN
We were attracted to the therapeutic potential of inhibiting Casitas B-lineage lymphoma proto-oncogene-b (Cbl-b), a RING E3 ligase that plays a critical role in regulating the activation of T cells. However, given that only protein-protein interactions were involved, it was unclear whether inhibition by a small molecule would be a viable approach. After screening an â¼6 billion member DNA-encoded library (DEL) using activated Cbl-b, we identified compound 1 as a hit for which the cis-isomer (2) was confirmed by biochemical and surface plasmon resonance (SPR) assays. Our hit optimization effort was greatly accelerated when we obtained a cocrystal structure of 2 with Cbl-b, which demonstrated induced binding at the substrate binding site, namely, the Src homology-2 (SH2) domain. This was quite noteworthy given that there are few reports of small molecule inhibitors that bind to SH2 domains and block protein-protein interactions. Structure- and property-guided optimization led to compound 27, which demonstrated measurable cell activity, albeit only at high concentrations.
RESUMEN
A novel series of non-nucleoside thumb pocket 2 HCV NS5B polymerase inhibitors were derived from a fragment-based approach using information from X-ray crystallographic analysis of NS5B-inhibitor complexes and iterative rounds of parallel synthesis. Structure-based drug design strategies led to the discovery of potent sub-micromolar inhibitors 11a-c and 12a-c from a weak-binding fragment-like structure 1 as a starting point.
Asunto(s)
Antivirales/química , Inhibidores Enzimáticos/química , Hepacivirus/efectos de los fármacos , Proteínas no Estructurales Virales/antagonistas & inhibidores , Antivirales/síntesis química , Antivirales/farmacología , Sitios de Unión , Células CACO-2 , Permeabilidad de la Membrana Celular/efectos de los fármacos , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Hepacivirus/enzimología , Humanos , Simulación del Acoplamiento Molecular , Nucleósidos/química , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Proteínas no Estructurales Virales/metabolismo , ortoaminobenzoatos/químicaRESUMEN
We describe the structure-based design of a novel lead chemotype that binds to thumb pocket 2 of HCV NS5B polymerase and inhibits cell-based gt1 subgenomic reporter replicons at sub-micromolar concentrations (EC50<200nM). This new class of potent thumb pocket 2 inhibitors features a 1H-quinazolin-4-one scaffold derived from hybridization of a previously reported, low affinity thiazolone chemotype with our recently described anthranilic acid series. Guided by X-ray structural information, a key NS5B-ligand interaction involving the carboxylate group of anthranilic acid based inhibitors was replaced by a neutral two-point hydrogen bonding interaction between the quinazolinone scaffold and the protein backbone. The in vitro ADME and in vivo rat PK profile of representative analogs are also presented and provide areas for future optimization of this new class of HCV polymerase inhibitors.
Asunto(s)
Antivirales/química , Diseño de Fármacos , Hepacivirus/enzimología , Quinazolinonas/química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Regulación Alostérica , Animales , Antivirales/síntesis química , Antivirales/farmacocinética , Sitios de Unión , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos , Semivida , Hepacivirus/fisiología , Simulación del Acoplamiento Molecular , Estructura Terciaria de Proteína , Quinazolinonas/síntesis química , Quinazolinonas/farmacocinética , Ratas , Relación Estructura-Actividad , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/efectos de los fármacos , ortoaminobenzoatos/químicaRESUMEN
Optimization efforts on the anthranilic acid-based Thumb Pocket 2 HCV NS5B polymerase inhibitors 1 and 2 resulted in the identification of multiple structural elements that contributed to improved cell culture potency. The additive effect of these elements resulted in compound 46, an inhibitor with enzymatic (IC50) and cell culture (EC50) potencies of less than 100 nanomolar.
Asunto(s)
Antivirales/química , Inhibidores Enzimáticos/química , Hepacivirus/enzimología , Proteínas no Estructurales Virales/antagonistas & inhibidores , ortoaminobenzoatos/química , Antivirales/síntesis química , Antivirales/farmacología , Sitios de Unión , Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Simulación del Acoplamiento Molecular , Unión Proteica , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/efectos de los fármacos , ortoaminobenzoatos/síntesis química , ortoaminobenzoatos/farmacologíaRESUMEN
Significant advances have led to receptor induced-fit and conformational selection models for describing bimolecular recognition, but a more comprehensive view must evolve to also include ligand shape and conformational changes. Here, we describe an example where a ligand's "structural hinge" influences potency by inducing an "L-shape" bioactive conformation, and due to its solvent exposure in the complex, reasonable conformation-activity-relationships can be qualitatively attributed. From a ligand design perspective, this feature was exploited by successful linker hopping to an alternate "structural hinge" that led to a new and promising chemical series which matched the ligand bioactive conformation and the pocket bioactive space. Using a combination of X-ray crystallography, NMR and modeling with support from binding-site resistance mutant studies and photoaffinity labeling experiments, we were able to derive inhibitor-polymerase complexes for various chemical series.
Asunto(s)
Diamida/química , Diamida/farmacología , Descubrimiento de Drogas , Hepacivirus , Indoles/química , Conformación Molecular , Proteínas no Estructurales Virales/antagonistas & inhibidores , Regulación Alostérica , Diamida/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Concentración 50 Inhibidora , Ligandos , Modelos Moleculares , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismoRESUMEN
SAR studies at the N(1)-position of allosteric indole-based HCV NS5B inhibitors has led to the discovery of acetamide derivatives with good cellular potency in subgenomic replicons (EC(50) <200 nM). This class of inhibitors displayed improved physicochemical properties and favorable ADME-PK profiles over previously described analogs in this class.
Asunto(s)
Acetamidas/química , Antivirales/síntesis química , Ácidos Carboxílicos/química , Descubrimiento de Drogas , Hepacivirus/enzimología , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Proteínas no Estructurales Virales/antagonistas & inhibidores , Acetamidas/farmacología , Regulación Alostérica/efectos de los fármacos , Regulación Alostérica/fisiología , Antivirales/farmacología , Células CACO-2 , Ácidos Carboxílicos/farmacología , Línea Celular , ARN Polimerasas Dirigidas por ADN/antagonistas & inhibidores , ARN Polimerasas Dirigidas por ADN/metabolismo , Descubrimiento de Drogas/métodos , Hepacivirus/efectos de los fármacos , Humanos , Microsomas Hepáticos/enzimología , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas no Estructurales Virales/metabolismoRESUMEN
The role of the tetrazole moiety in the binding of aryl thiotetrazolylacetanilides with HIV-1 wild type and K103N/Y181C double mutant reverse transcriptases was explored. Different acyclic, cyclic and heterocyclic replacements were investigated in order to evaluate the conformational and electronic contribution of the tetrazole ring to the binding of the inhibitors in the NNRTI pocket. The replacement of the tetrazole by a pyrazolyl group led to reversal of selectivity, providing inhibitors with excellent potency against the double mutant reverse transcriptase.
Asunto(s)
Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/farmacología , Transcriptasa Inversa del VIH/genética , Tetrazoles/síntesis química , Tetrazoles/farmacología , Fármacos Anti-VIH/química , Técnicas Químicas Combinatorias , Diseño de Fármacos , VIH-1/efectos de los fármacos , VIH-1/genética , Modelos Moleculares , Estructura Molecular , Relación Estructura-Actividad , Tetrazoles/químicaRESUMEN
Optimization of pyridine-based noncatalytic site integrase inhibitors (NCINIs) based on compound 2 has led to the discovery of molecules capable of inhibiting virus harboring N124 variants of HIV integrase (IN) while maintaining minimal contribution of enterohepatic recirculation to clearance in rat. Structure-activity relationships at the C6 position established chemical space where the extent of enterohepatic recirculation in the rat is minimized. Desymmetrization of the C4 substituent allowed for potency optimization against virus having the N124 variant of integrase. Combination of these lessons led to the discovery of compound 20, having balanced serum-shifted antiviral potency and minimized excretion in to the biliary tract in rat, potentially representing a clinically viable starting point for a new treatment option for individuals infected with HIV.
RESUMEN
A ligand-focused strategy employed NMR, X-ray, modeling, and medicinal chemistry to expose the critical role that bioactive conformation played in the design of a variety of drugs that target the HCV protease. The bioactive conformation (bound states) were determined for key inhibitors identified along our drug discovery pathway from the hit to clinical compounds. All adopt similar bioactive conformations for the common core derived from the hit peptide DDIVPC. A carefully designed SAR analysis, based on the advanced inhibitor 1 in which the P1 to P3 side chains and the N-terminal Boc were sequentially truncated, revealed a correlation between affinity and the relative predominance of the bioactive conformation in the free state. Interestingly, synergistic conformation effects on potency were also noted. Comparisons with clinical and recently marketed drugs from the pharmaceutical industry showed that all have the same core and similar bioactive conformations. This suggested that the variety of appendages discovered for these compounds also properly satisfy the bioactive conformation requirements and allowed for a large variety of HCV protease drug candidates to be designed.
Asunto(s)
Diseño de Fármacos , Inhibidores de Proteasas/química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Cristalografía por Rayos X , Ligandos , Espectroscopía de Resonancia Magnética , Conformación Molecular , Inhibidores de Proteasas/farmacología , Relación Estructura-ActividadRESUMEN
Conformational restrictions of flexible torsion angles were used to guide the identification of new chemotypes of HCV NS5B inhibitors. Sites for rigidification were based on an acquired conformational understanding of compound binding requirements and the roles of substituents in the free and bound states. Chemical bioisosteres of amide bonds were explored to improve cell-based potency. Examples are shown, including the design concept that led to the discovery of the phase III clinical candidate deleobuvir (BI 207127). The structure-based strategies employed have general utility in drug design.
Asunto(s)
Antivirales/farmacología , Bencimidazoles/farmacología , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Hepacivirus/efectos de los fármacos , Indoles/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Antivirales/química , Bencimidazoles/química , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Hepacivirus/enzimología , Indoles/química , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación MolecularRESUMEN
An anthranilic acid series of allosteric thumb pocket 2 HCV NS5B polymerase inhibitors exhibited hindered rotation along a covalent bond axis, and the existence of atropisomer chirality was confirmed by NMR, HPLC analysis on chiral supports, and computational studies. A thorough understanding of the concerted rotational properties and the influence exerted by substituents involved in this steric phenomenon was attained through biophysical studies on a series of truncated analogues. The racemization half-life of a compound within this series was determined to be 69 min, which was consistent with a class 2 atropisomer (intermediate conformational exchange). It was further found by X-ray crystallography that one enantiomer of a compound bound to the intended HCV NS5B polymerase target whereas the mirror image atropisomer was able to bind to an unrelated HIV matrix target. Analogues were then identified that selectively inhibited the former. These studies highlight that atropisomer chirality can lead to distinct entities with specific properties, and the phenomenon of atropisomerism in drug discovery should be evaluated and appropriately managed.
Asunto(s)
Antivirales/farmacología , VIH/efectos de los fármacos , Hepacivirus/efectos de los fármacos , Proteínas no Estructurales Virales/antagonistas & inhibidores , Regulación Alostérica , Antivirales/química , Antivirales/farmacocinética , Cromatografía Líquida de Alta Presión , Cristalografía por Rayos X , Hepacivirus/enzimología , Espectroscopía de Resonancia Magnética , EstereoisomerismoRESUMEN
Future treatments for individuals infected by the hepatitis C virus (HCV) will likely involve combinations of compounds that inhibit multiple viral targets. The helicase of HCV is an attractive target with no known drug candidates in clinical trials. Herein we describe an integrated strategy for identifying fragment inhibitors using structural and biophysical techniques. Based on an X-ray structure of apo HCV helicase and in silico and bioinformatic analyses of HCV variants, we identified that one site in particular (labeled 3 + 4) was the most conserved and attractive pocket to target for a drug discovery campaign. Compounds from multiple sources were screened to identify inhibitors or binders to this site, and enzymatic and biophysical assays (NMR and SPR) were used to triage the most promising ligands for 3D structure determination by X-ray crystallography. Medicinal chemistry and biophysical evaluations focused on exploring the most promising lead series. The strategies employed here can have general utility in drug discovery.
Asunto(s)
Antivirales/farmacología , Inhibidores Enzimáticos/farmacología , Hepacivirus/efectos de los fármacos , Proteínas no Estructurales Virales/antagonistas & inhibidores , Antivirales/química , Inhibidores Enzimáticos/química , Hepacivirus/enzimología , Cinética , Espectroscopía de Resonancia Magnética , Modelos Moleculares , ARN Helicasas/antagonistas & inhibidores , Serina Endopeptidasas , Relación Estructura-Actividad , Resonancia por Plasmón de SuperficieRESUMEN
A scaffold replacement approach was used to identifying the pyridine series of noncatalytic site integrase inhibitors. These molecules bind with higher affinity to a tetrameric form compared to a dimeric form of integrase. Optimization of the C6 and C4 positions revealed that viruses harboring T124 or A124 amino acid substitutions are highly susceptible to these inhibitors, but viruses having the N124 amino acid substitution are about 100-fold less susceptible. Compound 20 had EC50 values <10 nM against viruses having T124 or A124 substitutions in IN and >800 nM in viruses having N124 substitions. Compound 20 had an excellent in vitro ADME profile and demonstrated reduced contribution of biliary excretion to in vivo clearance compared to BI 224436, the lead compound from the quinoline series of NCINIs.
RESUMEN
An assay recapitulating the 3' processing activity of HIV-1 integrase (IN) was used to screen the Boehringer Ingelheim compound collection. Hit-to-lead and lead optimization beginning with compound 1 established the importance of the C3 and C4 substituent to antiviral potency against viruses with different aa124/aa125 variants of IN. The importance of the C7 position on the serum shifted potency was established. Introduction of a quinoline substituent at the C4 position provided a balance of potency and metabolic stability. Combination of these findings ultimately led to the discovery of compound 26 (BI 224436), the first NCINI to advance into a phase Ia clinical trial.
RESUMEN
An often overlooked source of chirality is atropisomerism, which results from slow rotation along a bond axis due to steric hindrance and/or electronic factors. If undetected or not managed properly, this time-dependent chirality has the potential to lead to serious consequences, because atropisomers can be present as distinct enantiomers or diastereoisomers with their attendant different properties. Herein we introduce a strategy to reveal and classify compounds that have atropisomeric chirality. Energy barriers to axial rotation were calculated using quantum mechanics, from which predicted high barriers could be experimentally validated. A calculated rotational energy barrier of 20â kcal mol(-1) was established as a suitable threshold to distinguish between atropisomers and non-atropisomers with a prediction accuracy of 86%. This methodology was applied to subsets of drug databases in the course of which atropisomeric drugs were identified. In addition, some drugs were exposed that were not yet known to have this chiral attribute. The most valuable utility of this tool will be to predict atropisomerism along the drug discovery pathway. When used in concert with our compound classification scheme, decisions can be made during early discovery stages such as "hit-to-lead" and "lead optimization," to foresee and validate the presence of atropisomers and to exercise options of removing, further stabilizing, or rendering the chiral axis of interest more freely rotatable via SAR design, thereby decreasing this potential liability within a compound series. The strategy can also improve drug development plans, such as determining whether a drug or series should be developed as a racemic mixture or as an isolated single compound. Moreover, the work described herein can be extended to other chemical fields that require the assessment of potential chiral axes.
Asunto(s)
Preparaciones Farmacéuticas/química , Técnicas Químicas Combinatorias , Bases de Datos Factuales , Evaluación Preclínica de Medicamentos , Teoría Cuántica , Estereoisomerismo , TermodinámicaRESUMEN
The carboxylate moiety is an important pharmacophore in the medicinal chemist's arsenal and is sometimes an irreplaceable functionality in drug-target interactions. Thus, practical guidance on its use in the most optimized manner would be a welcome addition to rational drug design. Key physicochemical and ADMET-PK properties from a dataset of drugs containing a carboxylate (COOH) moiety were assembled and compared with those of a broader, general drug dataset. Our main objective was to identify features specific to COOH-containing oral drugs that could be converted into simple rules delineating the boundaries within which prospective COOH-containing chemical series and COOH-containing drug candidates would be reasonably expected to possess properties suitable for oral administration. These specific "drug-like" property rules include molecular weight, the number of rotatable bonds, the number of hydrogen bond donors and acceptors, predictions of lipophilic character (calculated log P and log D values), topological polar surface area (TPSA), and the pK(a) value of the carboxylate moiety. Similar to the various sets of criteria that have emerged over the past decade and which have significantly reshaped the way medicinal chemists think about preferred drug chemical space, we propose these specific COOH "drug-like" property rules as a guide for the design of superior COOH-containing drug candidates and as a tool to better manage the liabilities generally associated with the presence of a COOH moiety.
Asunto(s)
Ácidos Carboxílicos/química , Preparaciones Farmacéuticas/química , Administración Oral , Disponibilidad Biológica , Ácidos Carboxílicos/farmacocinética , Química Farmacéutica , Bases de Datos Factuales , Diseño de Fármacos , Preparaciones Farmacéuticas/metabolismoAsunto(s)
Bencimidazoles/química , Hepacivirus/enzimología , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , Proteínas no Estructurales Virales/antagonistas & inhibidores , Bencimidazoles/farmacología , Inhibidores Enzimáticos/química , Humanos , Concentración 50 Inhibidora , Espectroscopía de Resonancia Magnética , Estructura Molecular , Unión Proteica , Relación Estructura-ActividadRESUMEN
We present a binding free energy function that consists of force field terms supplemented by solvation terms. We used this function to calibrate the solvation model along with the binding interaction terms in a self-consistent manner. The motivation for this approach was that the solute dielectric-constant dependence of calculated hydration gas-to-water transfer free energies is markedly different from that of binding free energies (J. Comput. Chem. 2003, 24, 954). Hence, we sought to calibrate directly the solvation terms in the context of a binding calculation. The five parameters of the model were systematically scanned to best reproduce the absolute binding free energies for a set of 99 protein-ligand complexes. We obtained a mean unsigned error of 1.29 kcal/mol for the predicted absolute binding affinity in a parameter space that was fairly shallow near the optimum. The lowest errors were obtained with solute dielectric values of Din = 20 or higher and scaling of the intermolecular van der Waals interaction energy by factors ranging from 0.03 to 0.15. The high apparent Din and strong van der Waals scaling may reflect the anticorrelation of the change in solvated potential energy and configurational entropy, that is, enthalpy-entropy compensation in ligand binding (Biophys. J. 2004, 87, 3035-3049). Five variations of preparing the protein-ligand data set were explored in order to examine the effect of energy refinement and the presence of bound water on the calculated results. We find that retaining water in the final protein structure used for calculating the binding free energy is not necessary to obtain good results; that is the continuum solvation model is sufficient. Virtual screening enrichment studies on estrogen receptor and thymidine kinase showed a good ability of the binding free energy function to recover true hits in a collection of decoys.