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The CDK4/6 inhibitor palbociclib reduces tumor growth by decreasing retinoblastoma (RB) protein phosphorylation and inducing cell cycle arrest at the G1/S phase transition. Palbociclib in combination with anti-hormonal therapy brings significant benefit to breast cancer patients. In this study, novel combination approaches and underlying molecular/cellular mechanisms for palbociclib were explored in squamous cell lung cancer (SqCLC), the second most common subtype of non-small cell lung cancer. While approximate 20% lung patients benefit from immunotherapy, most SqCLC patients who receive platinum-doublet chemotherapy as first-line treatment, which often includes a taxane, are still in need of more effective combination therapies. Our results demonstrated enhanced cytotoxicity and anti-tumor effect with palbociclib plus taxanes at clinically achievable doses in multiple SqCLC models with diverse cancer genetic backgrounds. Comprehensive gene expression analysis revealed a sustained disruption of pRB-E2F signaling by combination that was accompanied with enhanced regulation of pleiotropic biological effects. These included several novel mechanisms such as abrogation of G2/M and mitotic spindle assembly checkpoints, as well as impaired induction of hypoxia-inducible factor 1 alpha (HIF-1α). The decrease in HIF-1α modulated a couple key angiogenic and anti-angiogenic factors, resulting in an enhanced anti-angiogenic effect. This preclinical work suggests a new therapeutic opportunity for palbociclib in lung and other cancers currently treated with taxane based chemotherapy as standard of care.

Effective treatment options for advanced colorectal cancer (CRC) are limited, survival rates are poor and this disease continues to be a leading cause of cancer-related deaths worldwide. Despite being a highly heterogeneous disease, a large subset of individuals with sporadic CRC typically harbor relatively few established ‘driver’ lesions. Here, we describe a collection of genetically engineered mouse models (GEMMs) of sporadic CRC that combine lesions frequently altered in human patients, including well-characterized tumor suppressors and activators of MAPK signaling. Primary tumors from these models were profiled, and individual GEMM tumors segregated into groups based on their genotypes. Unique allelic and genotypic expression signatures were generated from these GEMMs and applied to clinically annotated human CRC patient samples. We provide evidence that a Kras signature derived from these GEMMs is capable of distinguishing human tumors harboring KRAS mutation, and tracks with poor prognosis in two independent human patient cohorts. Furthermore, the analysis of a panel of human CRC cell lines suggests that high expression of the GEMM Kras signature correlates with sensitivity to targeted pathway inhibitors. Together, these findings implicate GEMMs as powerful preclinical tools with the capacity to recapitulate relevant human disease biology, and support the use of genetic signatures generated in these models to facilitate future drug discovery and validation efforts.

P-cadherin is a calcium-dependent cell–cell adhesion glycoprotein. P-cadherin expression is restricted to the myoepithelial cells in normal breast tissue, and aberrant staining has also been described in invasive tumors. Several small studies have reported P-cadherin as a marker of poor outcome in breast cancer patients but its prognostic significance in relation to other variables has not been established in a large series of breast cancers. A tissue microarray was constructed from 3992 cases of invasive breast carcinoma, and P-cadherin expression was evaluated using immunohistochemistry. Median follow-up was 12.5 years. The immunohistochemistry-based definitions of cancer subtypes were luminal (ER+ or PR+/HER2−), luminal/HER2+ (ER+ or PR+/HER2+), HER2+ (ER−/PR−/HER2+), and basal (ER−/PR−/HER2−/CK5/6+ or EGFR+). Clinical covariate and biomarker associations were assessed using contingency tables, and Pearson's χ2or Fisher's exact test. Survival associations were assessed using Kaplan–Meier plots, logrank and Breslow tests, and Cox proportional hazards regression analysis. P-cadherin was expressed in 34.8% (1290/3710, 50% cut point) of cases. P-cadherin staining was strongly associated with HER2+ and basal carcinoma subtypes (P<0.0005). P-cadherin-positive patients showed significantly poorer short-term (0–10 years) overall survival, disease-specific survival, distant relapse-free interval, and locoregional relapse-free interval in univariable models (P<0.05). In multivariable Cox models containing standard clinical covariates and cancer subtypes, P-cadherin did not show independent prognostic value. P-cadherin expression was positively associated with histological grade, chemotherapy, Ki-67, EGFR, CK5/6, p53, YB-1, and HER2 expression (P<0.002), and negatively associated with age at diagnosis, ER, PR, and Bcl-2 expression (P<0.0005). This study shows the value of P-cadherin as a marker of poor prognosis. The large sample size of this series clarifies contradictory findings of many smaller studies. P-cadherin positivity is associated with high-grade tumor subtypes and well-established markers of poor prognosis, and may represent a promising antibody therapeutic target.

P-cadherin is a calcium-dependent cell–cell adhesion glycoprotein. P-cadherin expression is restricted to the myoepithelial cells in normal breast tissue, and aberrant staining has also been described in invasive tumors. Several small studies have reported P-cadherin as a marker of poor outcome in breast cancer patients but its prognostic significance in relation to other variables has not been established in a large series of breast cancers. A tissue microarray was constructed from 3992 cases of invasive breast carcinoma, and P-cadherin expression was evaluated using immunohistochemistry. Median follow-up was 12.5 years. The immunohistochemistry-based definitions of cancer subtypes were luminal (ER+ or PR+/HER2−), luminal/HER2+ (ER+ or PR+/HER2+), HER2+ (ER−/PR−/HER2+), and basal (ER−/PR−/HER2−/CK5/6+ or EGFR+). Clinical covariate and biomarker associations were assessed using contingency tables, and Pearson's χ2or Fisher's exact test. Survival associations were assessed using Kaplan–Meier plots, logrank and Breslow tests, and Cox proportional hazards regression analysis. P-cadherin was expressed in 34.8% (1290/3710, 50% cut point) of cases. P-cadherin staining was strongly associated with HER2+ and basal carcinoma subtypes (P<0.0005). P-cadherin-positive patients showed significantly poorer short-term (0–10 years) overall survival, disease-specific survival, distant relapse-free interval, and locoregional relapse-free interval in univariable models (P<0.05). In multivariable Cox models containing standard clinical covariates and cancer subtypes, P-cadherin did not show independent prognostic value. P-cadherin expression was positively associated with histological grade, chemotherapy, Ki-67, EGFR, CK5/6, p53, YB-1, and HER2 expression (P<0.002), and negatively associated with age at diagnosis, ER, PR, and Bcl-2 expression (P<0.0005). This study shows the value of P-cadherin as a marker of poor prognosis. The large sample size of this series clarifies contradictory findings of many smaller studies. P-cadherin positivity is associated with high-grade tumor subtypes and well-established markers of poor prognosis, and may represent a promising antibody therapeutic target.

Glucocorticoids, through activation of the glucocorticoid receptor (GR), regulate hepatic gluconeogenesis. Elevated hepatic expression and activity of 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) play a key role in ligand-induced activation of the GR through the production of cortisol. Evidence from genetically modified mice suggests that inhibition of 11betaHSD1 might be a therapeutic approach to treat the metabolic syndrome. We have identified a potent 11betaHSD1 inhibitor, 4'-cyano-biphenyl-4-sulfonic acid (6-amino-pyridin-2-yl)-amide (PF-915275), that is selective for the primate and human enzymes. The objective of this study was to demonstrate target inhibition with PF-915275 and to quantify the relationship between target inhibition and drug exposure in monkeys. We characterized the ability of PF-915275 to inhibit the conversion of prednisone, a synthetic cortisone analog that can be distinguished from the endogenous substrate cortisone, enabling a direct measure of substrate to product conversion without the complication of feedback. Adult cynomolgus monkeys were administered either vehicle or various doses of PF-915275 followed by a 10-mg/kg dose of prednisone. Prednisone conversion to prednisolone and the concentrations of PF-915275 were measured by liquid chromatography/tandem mass spectrometry. PF-915275 dose-dependently inhibited 11betaHSD1-mediated conversion of prednisone to prednisolone, with a maximum of 87% inhibition at a 3-mg/kg dose. An exposure-response relationship was demonstrated, with an estimated EC(50) of 391 nM (total) and 17 nM (free). Insulin levels were also reduced in a dose-related manner. These results should enable the development of a biomarker for evaluating target modulation in humans that will aid in identifying 11betaHSD1 inhibitors to treat diabetes and other related metabolic diseases.

CONTEXT: The pathophysiological importance of glucocorticoids (GCs) is exemplified by patients with Cushing’s syndrome who develop hypertension, obesity, and insulin resistance. At a cellular level, availability of GCs to the glucocorticoid and mineralocorticoid receptors is controlled by the isoforms of 11{szligbeta}-hydroxysteroid dehydrogenase (11{szligbeta}-HSD). In liver and adipose tissue, 11{szligbeta}-HSD1 converts endogenous, inactive cortisone to active cortisol but also catalyzes the bioactivation of the synthetic prednisone to prednisolone. OBJECTIVE: The objective of the study was to compare markers of 11{szligbeta}-HSD1 activity and demonstrate that inhibition of 11{szligbeta}-HSD1 activity limits glucocorticoid availability to adipose tissue. DESIGN AND SETTING: This was a clinical study. PATIENTS: Seven healthy male volunteers participated in the study. INTERVENTION: Intervention included carbenoxolone (CBX) single dose (100 mg) and 72 hr of continuous treatment (300 mg/d). MAIN OUTCOME MEASURES: Inhibition of 11{szligbeta}-HSD1 was monitored using five different mechanistic biomarkers (serum cortisol and prednisolone generation, urinary corticosteroid metabolite analysis by gas chromatography/mass spectrometry, and adipose tissue microdialysis examining cortisol generation and glucocorticoid-mediated glycerol release). RESULTS: Each biomarker demonstrated reduced 11{szligbeta}-HSD1 activity after CBX administration. After both a single dose and 72 hr of treatment with CBX, cortisol and prednisolone generation decreased as did the urinary tetrahydrocortisol+5-tetrahydrocortisol to tetrahydrocortisone ratio. Using adipose tissue microdialysis, we observed decreased interstitial fluid cortisol availability with CBX treatment. Furthermore, a functional consequence of 11{szligbeta}-HSD1 inhibition was observed, namely decreased prednisone-induced glycerol release into adipose tissue interstitial fluid indicative of inhibition of GC-mediated lipolysis. CONCLUSION: CBX is able to inhibit rapidly the generation of active GC in human adipose tissue. Importantly, limiting GC availability in vivo has functional consequences including decreased glycerol release.

Monte Carlo simulations of molecular recognition at the consensus binding site of the constant fragment (Fc) of human immunoglobulin G (Ig) protein have been performed to analyze structural and thermodynamic aspects of binding for the 13‐residue cyclic peptide DCAWHLGELVWCT. The energy landscape analysis of a hot spot at the intermolecular interface using alanine scanning and equilibrium‐simulated tempering dynamics with the simplified, knowledge‐based energy function has enabled the role of the protein hot spot residues in providing the thermodynamic stability of the native structure to be determined. We have found that hydrophobic interactions between the peptide and the Met‐252, Ile‐253, His‐433, and His‐435 protein residues are critical to guarantee the thermodynamic stability of the crystallographic binding mode of the complex. Binding free energy calculations, using a molecular mechanics force field and a solvation energy model, combined with alanine scanning have been conducted to determine the energetic contribution of the protein hot spot residues in binding affinity. The conserved Asn‐434, Ser‐254, and Tyr‐436 protein residues contribute significantly to the binding affinity of the peptide–protein complex, serving as an energetic hot spot at the intermolecular interface. The results suggest that evolutionary conserved hot spot protein residues at the intermolecular interface may be partitioned in fulfilling thermodynamic stability of the native binding mode and contributing to the binding affinity of the complex. Proteins 2002;48:539–557. © 2002 Wiley‐Liss, Inc.

Monte Carlo simulations of molecular recognition at the consensus binding site of the constant fragment (Fc) of human immunoglobulin G (Ig) protein have been performed to analyze structural and thermodynamic aspects of binding for the 13-residue cyclic peptide DCAWHLGELVWCT. The energy landscape analysis of a hot spot at the intermolecular interface using alanine scanning and equilibrium-simulated tempering dynamics with the simplified, knowledge-based energy function has enabled the role of the protein hot spot residues in providing the thermodynamic stability of the native structure to be determined. We have found that hydrophobic interactions between the peptide and the Met-252, Ile-253, His-433, and His-435 protein residues are critical to guarantee the thermodynamic stability of the crystallographic binding mode of the complex. Binding free energy calculations, using a molecular mechanics force field and a solvation energy model, combined with alanine scanning have been conducted to determine the energetic contribution of the protein hot spot residues in binding affinity. The conserved Asn-434, Ser-254, and Tyr-436 protein residues contribute significantly to the binding affinity of the peptide–protein complex, serving as an energetic hot spot at the intermolecular interface. The results suggest that evolutionary conserved hot spot protein residues at the intermolecular interface may be partitioned in fulfilling thermodynamic stability of the native binding mode and contributing to the binding affinity of the complex. Proteins 2002;48:539–557. © 2002 Wiley-Liss, Inc.

Computer simulations using the simplified energy function and simulated tempering dynamics have accurately determined the native structure of the pYVPML, SVLpYTAVQPNE, and SPGEpYVNIEF peptides in the complexes with SH2 domains. Structural and equilibrium aspects of the peptide binding with SH2 domains have been studied by generating temperature‐dependent binding free energy landscapes. Once some native peptide–SH2 domain contacts are constrained, the underlying binding free energy profile has the funnel‐like shape that leads to a rapid and consistent acquisition of the native structure. The dominant native topology of the peptide–SH2 domain complexes represents an extended peptide conformation with strong specific interactions in the phosphotyrosine pocket and hydrophobic interactions of the peptide residues C‐terminal to the pTyr group. The topological features of the peptide–protein interface are primarily determined by the thermodynamically stable phosphotyrosyl group. A diversity of structurally different binding orientations has been observed for the amino‐terminal residues to the phosphotyrosine. The dominant native topology for the peptide residues carboxy‐terminal to the phosphotyrosine is tolerant to flexibility in this region of the peptide–SH2 domain interface observed in equilibrium simulations. The energy landscape analysis has revealed a broad, entropically favorable topology of the native binding mode for the bound peptides, which is robust to structural perturbations. This could provide an additional positive mechanism underlying tolerance of the SH2 domains to hydrophobic conservative substitutions in the peptide specificity region. Proteins 2001;45:456–470. © 2001 Wiley‐Liss, Inc.

Computer simulations using the simplified energy function and simulated tempering dynamics have accurately determined the native structure of the pYVPML, SVLpYTAVQPNE, and SPGEpYVNIEF peptides in the complexes with SH2 domains. Structural and equilibrium aspects of the peptide binding with SH2 domains have been studied by generating temperature-dependent binding free energy landscapes. Once some native peptide–SH2 domain contacts are constrained, the underlying binding free energy profile has the funnel-like shape that leads to a rapid and consistent acquisition of the native structure. The dominant native topology of the peptide–SH2 domain complexes represents an extended peptide conformation with strong specific interactions in the phosphotyrosine pocket and hydrophobic interactions of the peptide residues C-terminal to the pTyr group. The topological features of the peptide–protein interface are primarily determined by the thermodynamically stable phosphotyrosyl group. A diversity of structurally different binding orientations has been observed for the amino-terminal residues to the phosphotyrosine. The dominant native topology for the peptide residues carboxy-terminal to the phosphotyrosine is tolerant to flexibility in this region of the peptide–SH2 domain interface observed in equilibrium simulations. The energy landscape analysis has revealed a broad, entropically favorable topology of the native binding mode for the bound peptides, which is robust to structural perturbations. This could provide an additional positive mechanism underlying tolerance of the SH2 domains to hydrophobic conservative substitutions in the peptide specificity region. Proteins 2001;45:456–470. © 2001 Wiley-Liss, Inc.

Common failures in predicting crystal structures of ligand-protein complexes are investigated for three ligand-protein systems by a combined thermodynamic and kinetic analysis of the binding energy landscapes. Misdocked predictions in ligand-protein docking are classified as `soft' and `hard' failures. While a soft failure arises when the search algorithm is unable to find the global energy minimum corresponding to the crystal structure, a hard failure results from a flaw of the energy function to qualify the crystal structure as the predicted lowest energy conformation in docking simulations. We find that neither the determination of a single structure with the lowest energy nor finding the most common binding mode is sufficient to predict crystal structures of the complexes, which belong to the category of hard failures. In a proposed hierarchical approach, structural similarity clustering of the conformations, generated from equilibrium simulations with the simplified energy function, is followed by energy refinement with the AMBER force field. This protocol, that involves a hierarchy of energy functions, resolves some common failures in ligand-protein docking and detects crystallographic binding modes that were not found during docking simulations.

The thermodynamics of molecular recognition is investigated by a statistical energy landscape approach, where the temperature profile of the ligand–protein binding process is determined using the weighted histogram analysis method. The analysis reveals differences in the binding energy landscapes of two molecular fragments with the FKBP12 protein, which are reflected in their characteristic transition temperatures. The approach provides insight into the nature of transitions between unbound and bound phases of ligand–protein complexes. One molecular fragment proceeds from the unbound phase to the native complex via a short-lived intermediate at relatively high temperature. The second fragment has a significantly more rugged binding energy landscape and goes from unbound to a long-lived nonspecific bound species consisting of isoenergetic yet structurally different binding modes, and later via a second-order-like transition to the native complex. Emerging universalities in molecular recognition and protein folding mechanisms are highlighted in the context of the kinetic partitioning mechanism. ©1999 John Wiley & Sons, Inc. Int J Quant Chem 73: 113–121, 1999

We present the results of molecular docking simulations with HIV-1 protease for the sb203386 and skf107457 inhibitors by Monte Carlo simulated annealing. A simplified piecewise linear energy function, the standard AMBER force field, and the AMBER force field with solvation and a soft-core smoothing component are employed in simulations with a single-protein conformation to determine the relationship between docking simulations with a simple energy function and more realistic force fields. The temperature-dependent binding free energy profiles of the inhibitors interacting with a single protein conformation provide a detailed picture of relative thermodynamic stability and a distribution of ligand binding modes in agreement with experimental crystallographic data. Using the simplified piecewise linear energy function, we also performed Monte Carlo docking simulations with an ensemble of protein conformations employing preferential biased sampling of low-energy protein conformations, and the results are analyzed in connection with the free energy profiles. ©1999 John Wiley & Sons, Inc. Int J Quant Chem 72: 73–84, 1999

Abstract.: Binding-energy landscapes are used to investigate the thermodynamics of molecular recognition for the pteridine ring, a recognition anchor in binding with dihydrofolate reductase, and two molecules with the same shape but different heteroatom substitutions. The relative importance of hydrogen bonding and hydrophobic interactions in this system is analyzed by comparing these three different decorations of the pteridine scaffold.

The thermodynamic and kinetic aspects of molecular recognition for the methotrexate (MTX)–dihydrofolate reductase (DHFR) ligand–protein system are investigated by the binding energy landscape approach. The impact of ‘hot’ and ‘cold’ errors in ligand mutations on the thermodynamic stability of the native MTX–DHFR complex is analyzed, and relationships between the molecular recognition mechanism and the degree of ligand optimization are discussed. The nature and relative stability of intermediates and thermodynamic phases on the ligand–protein association pathway are studied, providing new insights into connections between protein folding and molecular recognition mechanisms, and cooperativity of ligand–protein binding. The results of kinetic docking simulations are rationalized based on the thermodynamic properties determined from equilibrium simulations and the shape of the underlying binding energy landscape. We show how evolutionary ligand selection for a receptor active site can produce well‐optimized ligand–protein systems such as MTX–DHFR complex with the thermodynamically stable native structure and a direct transition mechanism of binding from unbound conformations to the unique native structure. Copyright © 1999 John Wiley & Sons, Ltd.

The thermodynamic and kinetic aspects of molecular recognition for the methotrexate (MTX)–dihydrofolate reductase (DHFR) ligand–protein system are investigated by the binding energy landscape approach. The impact of ‘hot’ and ‘cold’ errors in ligand mutations on the thermodynamic stability of the native MTX–DHFR complex is analyzed, and relationships between the molecular recognition mechanism and the degree of ligand optimization are discussed. The nature and relative stability of intermediates and thermodynamic phases on the ligand–protein association pathway are studied, providing new insights into connections between protein folding and molecular recognition mechanisms, and cooperativity of ligand–protein binding. The results of kinetic docking simulations are rationalized based on the thermodynamic properties determined from equilibrium simulations and the shape of the underlying binding energy landscape. We show how evolutionary ligand selection for a receptor active site can produce well-optimized ligand–protein systems such as MTX–DHFR complex with the thermodynamically stable native structure and a direct transition mechanism of binding from unbound conformations to the unique native structure. Copyright © 1999 John Wiley & Sons, Ltd.

Mean field analysis of FKBP12 complexes with FK506 and rapamycin has been performed by using structures obtained from molecular docking simulations on a simple, yet robust molecular recognition energy landscape. When crystallographic water molecules are included in the simulations as an extension of the FKBP12 protein surface, there is an appreciable stability gap between the energy of the native FKBP12–FK506 complex and energies of conformations with the “native-like” binding mode. By contrast, the energy spectrum of the FKBP12–rapamycin complex is dense regardless of the presence of the water molecules. The stability gap in the FKBP12–FK506 system is determined by two critical water molecules from the effector region that participate in a network of specific hydrogen bond interactions. This interaction pattern protects the integrity and precision of the composite ligand-protein effector surface in the binary FKBP12–FK506 complex and is preserved in the crystal structure of the FKBP12–FK506–calcineurin ternary complex. These features of the binding energy landscapes provide useful insights into specific and nonspecific aspects of FK506 and rapamycin recognition. Proteins 28:313–324, 1997. © 1997 Wiley-Liss, Inc.

Computational structure prediction of streptavidin-peptide complexes for known recognition sequences and a number of random di-, tri-, and tetrapeptides has been conducted, and mechanisms of peptide recognition with streptavidin have been investigated by a new computational protocol. The structural consensus criterion, which is computed from multiple docking simulations and measures the accessibility of the dominant binding mode, identifies recognition motifs from a set of random peptide sequences, whereas energetic analysis is less discriminatory. The predicted conformations of recognition tripeptide and tetrapeptide sequences are also in structural harmony and composed of peptide fragments that are individually unfrustrated in their bound conformation, resulting in a minimally frustrated energy landscape for recognition peptides. Proteins 28:421–433, 1997. © 1997 Wiley-Liss, Inc.

Mean field analysis of FKBP12 complexes with FK506 and rapamycin has been performed by using structures obtained from molecular docking simulations on a simple, yet robust molecular recognition energy landscape. When crystallographic water molecules are included in the simulations as an extension of the FKBP12 protein surface, there is an appreciable stability gap between the energy of the native FKBP12–FK506 complex and energies of conformations with the “native‐like” binding mode. By contrast, the energy spectrum of the FKBP12–rapamycin complex is dense regardless of the presence of the water molecules. The stability gap in the FKBP12–FK506 system is determined by two critical water molecules from the effector region that participate in a network of specific hydrogen bond interactions. This interaction pattern protects the integrity and precision of the composite ligand‐protein effector surface in the binary FKBP12–FK506 complex and is preserved in the crystal structure of the FKBP12–FK506–calcineurin ternary complex. These features of the binding energy landscapes provide useful insights into specific and nonspecific aspects of FK506 and rapamycin recognition. Proteins 28:313–324, 1997. © 1997 Wiley‐Liss, Inc.

Computational structure prediction of streptavidin‐peptide complexes for known recognition sequences and a number of random di‐, tri‐, and tetrapeptides has been conducted, and mechanisms of peptide recognition with streptavidin have been investigated by a new computational protocol. The structural consensuscriterion, which is computed from multiple docking simulations and measures the accessibility of the dominant binding mode, identifies recognition motifs from a set of random peptide sequences, whereas energetic analysis is less discriminatory. The predicted conformations of recognition tripeptide and tetrapeptide sequences are also in structural harmonyand composed of peptide fragments that are individually unfrustrated in their bound conformation, resulting in a minimally frustrated energy landscape for recognition peptides. Proteins 28:421–433, 1997. © 1997 Wiley‐Liss, Inc.

Combination therapy with EGFR inhibitors may overcome acquired resistance to PI3K pathway inhibitors in some colorectal cancer patients.