Urinary and fecal incontinence in patients with advanced ovarian cancer treated with CRS + HIPEC.

PURPOSE: The objective of this work was to analyze the long-term prevalence of urinary and fecal incontinence and their impact on quality of life in patients with advanced and recurrent ovarian cancer treated with cytoreductive surgery and hyperthermic intraoperative intraperitoneal chemotherapy (CRS + HIPEC). METHODS: This cross-sectional study included a series of patients with advanced and recurrent ovarian cancer treated by CRS + HIPEC, with a disease-free period of at least 12 months after the procedure. Urinary incontinence was evaluated using the International Consultation on Incontinence Questionnaire - Short Form (ICIQ-SF), fecal incontinence using the Wexner test and the Fecal Incontinence Quality of Life (FIQL) questionnaire and global quality of life using the Short Form 36 (SF-36) survey. RESULTS: A total of 64 patients were included in the study, with a median age of 55 years (range 28-78). The urinary incontinence rate was 45% and the fecal incontinence rate was 20%. Up to 14% of the patients presented both types of incontinence. The presence of urinary or fecal incontinence generated a significant negative impact on quality of life in relation to patients without incontinence. DISCUSSION: Urinary and fecal incontinence is frequent in the follow-up of ovarian cancer patients treated with CRS + HIPEC. Reconsidering the approach to the pelvis without peritoneal metastases in the peritoneum could modify the incidence of these pelvic floor dysfunctions.

Risk factors and management of incisional hernia after cytoreduction and hyperthermic intraperitoneal chemotherapy (HIPEC) in patients with peritoneal surface malignancies.

BACKGROUND: The incidence of incisional hernia in patients with peritoneal surface malignancies treated by cytoreduction plus hyperthermic intraperitoneal chemotherapy (HIPEC) remains unclear, and the criteria commonly used to indicate their repair cannot be applied in these patients. The objective of this work was to analyze the incidence of incisional hernias in these patients, identify the risk factors associated with their appearance, and propose an algorithm for their management. METHODS: We analyzed a series of patients with malignant pathologies of the peritoneal surface treated by cytoreduction with peritonectomy and HIPEC procedures between January 2008 and June 2017. Only patients with a minimum postoperative follow-up period of 12 months were included. RESULTS: Our series included 282 patients, 28 (10%) of whom developed an incisional hernia during the follow-up period. Fifty-one patients, all with ovarian cancer with peritoneal dissemination, did not receive HIPEC after cytoreduction as they were part of the control arm of the CARCINOHIPEC clinical trial (NCT02328716) or because they did not provide specific informed consent. In the multivariate analysis, treatment with HIPEC (OR 2.56, 95% CI [1.57, 4.31], p = 0.032) and the administration of preoperative systemic chemotherapy (OR = 1.59, 95% CI [1.26, 3.58], p = 0.041) were found to be independent factors related to the appearance of an incisional hernia. CONCLUSIONS: The incidence of incisional hernia after cytoreduction and HIPEC is within the ranges described in the literature for other abdominal surgery procedures. The use of systemic chemotherapy and treatment with HIPEC, in particular, were identified as factors related to their occurrence.

2'-Deoxyribosyltransferase from Leishmania mexicana, an efficient biocatalyst for one-pot, one-step synthesis of nucleosides from poorly soluble purine bases.

Processes catalyzed by enzymes offer numerous advantages over chemical methods although in many occasions the stability of the biocatalysts becomes a serious concern. Traditionally, synthesis of nucleosides using poorly water-soluble purine bases, such as guanine, xanthine, or hypoxanthine, requires alkaline pH and/or high temperatures in order to solubilize the substrate. In this work, we demonstrate that the 2'-deoxyribosyltransferase from Leishmania mexicana (LmPDT) exhibits an unusually high activity and stability under alkaline conditions (pH 8-10) across a broad range of temperatures (30-70 °C) and ionic strengths (0-500 mM NaCl). Conversely, analysis of the crystal structure of LmPDT together with comparisons with hexameric, bacterial homologues revealed the importance of the relationships between the oligomeric state and the active site architecture within this family of enzymes. Moreover, molecular dynamics and docking approaches provided structural insights into the substrate-binding mode. Biochemical characterization of LmPDT identifies the enzyme as a type I NDT (PDT), exhibiting excellent activity, with specific activity values 100- and 4000-fold higher than the ones reported for other PDTs. Interestingly, LmPDT remained stable during 36 h at different pH values at 40 °C. In order to explore the potential of LmPDT as an industrial biocatalyst, enzymatic production of several natural and non-natural therapeutic nucleosides, such as vidarabine (ara A), didanosine (ddI), ddG, or 2'-fluoro-2'-deoxyguanosine, was carried out using poorly water-soluble purines. Noteworthy, this is the first time that the enzymatic synthesis of 2'-fluoro-2'-deoxyguanosine, ara G, and ara H by a 2'-deoxyribosyltransferase is reported.

A functional BH3 domain in an aquaporin from Leishmania infantum.

Despite the absence of sequences showing significant similarity to any of the members of the Bcl-2 family of proteins in protozoa, experiments carried out in yeast or trypanosomatids have demonstrated that ectopic expression of some of these members alters their response to different death stimuli. Because the BH3 domain is the smallest common signature in all the proteins of this family of apoptosis regulators and also because they are essential for molecular interactions between antagonistic members, we looked for sequences with significant similarity to the BH3 motif in the Leishmania infantum genome. Among the top scoring ones, we found the MYLALQNLGDEV amino-acid stretch at the C terminus of a previously described aquaporin, now renamed as Li-BH3AQP. This motif is highly conserved in homologous proteins from other species of the Leishmania genus. The association of Li-BH3AQP with human Bcl-XL was demonstrated by both co-immunoprecipitation and yeast two-hybrid experiments. Ectopic expression of Li-BH3AQP reduced viability of HeLa cells and this deleterious effect was abrogated by the simultaneous overexpression of Bcl-XL. Although we were not able to demonstrate a reduction in parasite viability when the protein was overexpressed in Leishmania promastigotes, a prodeath effect could be observed when the parasites overexpressing Li-BH3AQP were treated with staurosporine or antimycin A. Surprisingly, these parasites were more resistant, compared with wild-type parasites, to hypotonic stress or nutrient deprivation. The prodeath activity was abolished upon replacement of two highly conserved amino acids in this BH3 domain. Taken together, these results point to Li-BH3AQP as the first non-enzymatic protein ever described in trypanosomatids that is involved in cell death.

Epigenetic regulation of ID4 in the determination of the BRCAness phenotype in breast cancer.

BRCAness breast tumors represent a group of sporadic tumors characterized by a reduction in BRCA1 gene expression. As BRCA1 is involved in double-strand breaks (DSBs) repair, dysfunctional BRCA pathway could make a tumor sensitive to DNA damaging drugs (e.g., platinum agents). Thus, accurately identifying BRCAness could contribute to therapeutic decision making in patients harboring these tumors. The purpose of this study was to identify if BRCAness tumors present a characteristic methylation profile and/or were related to specific clinico-pathological features. BRCAness was measured by MLPA in 63 breast tumors; methylation status of 98 CpG sites within 84 cancer-related genes was analyzed by MS-MLPA. Protein and mRNA expressions of the selected genes were measured by quantitative real-time PCR and Western Blot. BRCAness was associated with younger age, higher nuclear pleomorphism, and triple-negative (TN) status. Epigenetically, we found that the strongest predictors for BRCAness tumors were the methylations of MLH1 and PAX5 plus the unmethylations of CCND2 and ID4. We determined that ID4 unmethylation correlated with the expression levels of both its mRNA and protein. We observed an inverse relation between the expressions of ID4 and BRCA1. To the best of our knowledge, this is the first report suggesting an epigenetic regulation of ID4 in BRCAness tumors. Our findings give new information of BRCAness etiology and encourage future studies on potential drug targets for BRCAness breast tumors.

Synthesis and evaluation of hybrid molecules targeting the vinca domain of tubulin.

Some hybrids of vinca alkaloids and phomopsin A, linked by a glycine pattern, have been synthesized in one or two steps, by an insertion reaction and shown to inhibit microtubule assembly. These compounds have been elaborated in order to interact with both the "vinca site" and the "peptide site" of the vinca domain in tubulin. Two out of three hybrids are potent inhibitors of microtubules assembly and they present good cytotoxicity against different cell lines. Molecular modelling studies show that they could bind, within the vinca domain, in similar spatial regions as those of vinca and phomopsin thanks to the flexibility provided by the glycine linker used to elaborate these hybrids.

The pyrimidine nucleoside phosphorylase of Mycoplasma hyorhinis and how it may affect nucleoside-based therapy.

Mycoplasmas are opportunistic parasites and some species are suggested to preferentially colonize tumor tissue in cancer patients. We could demonstrate that the annotated thymidine phosphorylase (TP) gene in the genome of Mycoplasma hyorhinis encodes a pyrimidine nucleoside phosphorylase (PyNPHyor) that not only efficiently catalyzes thymidine but also uridine phosphorolysis. The kinetic characteristics of PyNPHyor-catalyzed nucleoside and nucleoside analogue (NA) phosphorolysis were determined. We demonstrated that the expression of such an enzyme in mycoplasma-infected cell cultures dramatically alters the activity of various anticancer/antiviral NAs such as 5-halogenated pyrimidine nucleosides, including 5-trifluorothymidine (TFT). Due to their close association with human cancers, the presence of mycoplasmas may markedly influence the therapeutic efficiency of nucleoside-based drugs.

Methylation profile of triple-negative breast carcinomas.

Breast cancer is a group of clinically, histopathologically and molecularly heterogeneous diseases, with different outcomes and responses to treatment. Triple-negative (TN) breast cancers are defined as tumors that lack the expression of estrogen receptor, progesterone receptor and epidermal growth factor receptor 2. This subgroup accounts for 15% of all types of breast cancer and its prevalence is higher among young African, African-American and Latino women. The hypermethylation of CpG islands (CpGI) is a common epigenetic alteration for suppressing gene expression in breast cancer and has been shown to be a key factor in breast carcinogenesis. In this study we analyzed the hypermethylation of 110 CpGI within 69 cancer-related genes in TN tumors. For the methylation analysis, we used the methyl-specific multiplex-ligation probe amplification assay. We found that the number of methylated CpGI is similar between TN and non-TN tumors, but the methylated genes between the groups are different. The methylation profile of TN tumors is defined by the methylation of five genes (that is, CDKN2B, CD44, MGMT, RB and p73) plus the non-methylation of 11 genes (that is, GSTP1, PMS2, MSH2, MLH1, MSH3, MSH6, DLC1, CACNA1A, CACNA1G, TWIST1 and ID4). We conclude that TN tumors have a specific methylation profile. Our findings give new information for better understanding tumor etiology and encourage future studies on potential drug targets for triple-negative breast tumors, which now lack a specific treatment.

PM01183, a new DNA minor groove covalent binder with potent in vitro and in vivo anti-tumour activity.

BACKGROUND AND PURPOSE: PM01183 is a new synthetic tetrahydroisoquinoline alkaloid that is currently in phase I clinical development for the treatment of solid tumours. In this study we have characterized the interactions of PM01183 with selected DNA molecules of defined sequence and its in vitro and in vivo cytotoxicity. EXPERIMENTAL APPROACH: DNA binding characteristics of PM01183 were studied using electrophoretic mobility shift assays, fluorescence-based melting kinetic experiments and computational modelling methods. Its mechanism of action was investigated using flow cytometry, Western blot analysis and fluorescent microscopy. In vitro anti-tumour activity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and the in vivo activity utilized several human cancer models. KEY RESULTS: Electrophoretic mobility shift assays demonstrated that PM01183 bound to DNA. Fluorescence-based thermal denaturation experiments showed that the most favourable DNA triplets providing a central guanine for covalent adduct formation are AGC, CGG, AGG and TGG. These binding preferences could be rationalized using molecular modelling. PM01183-DNA adducts in living cells give rise to double-strand breaks, triggering S-phase accumulation and apoptosis. The potent cytotoxic activity of PM01183 was ascertained in a 23-cell line panel with a mean GI(50) value of 2.7 nM. In four murine xenograft models of human cancer, PM01183 inhibited tumour growth significantly with no weight loss of treated animals. CONCLUSIONS AND IMPLICATIONS: PM01183 is shown to bind to selected DNA sequences and promoted apoptosis by inducing double-strand breaks at nanomolar concentrations. The potent anti-tumour activity of PM01183 in several murine models of human cancer supports its development as a novel anti-neoplastic agent.

Identification of aspartic acid-203 in human thymidine phosphorylase as an important residue for both catalysis and non-competitive inhibition by the small molecule "crystallization chaperone" 5'-O-tritylinosine (KIN59).

Thymidine phosphorylase (TP) is a catabolic enzyme in thymidine metabolism that is frequently upregulated in many solid tumors. Elevated TP levels are associated with tumor angiogenesis, metastasis and poor prognosis. Therefore, the use of TP inhibitors might offer a promising strategy for cancer treatment. The tritylated inosine derivative 5'-O-tritylinosine (previously designated KIN59) is a non-competitive inhibitor of TP which was previously found to be instrumental for the crystallization of human TP. A combination of computational studies including normal mode analysis, automated ligand docking and molecular dynamics simulations were performed to define a plausible binding site for 5'-O-tritylinosine on human TP. A cavity in which 5'-O-tritylinosine could fit was identified in the vicinity of the Gly405-Val419 loop at a distance of about 11A from the substrate-binding site. In the X-ray crystal structure, this pocket is characterized by an intricate hydrogen-bonding network in which Asp203 was found to play an important role to afford the loop stabilization that is required for efficient enzyme catalysis. Site-directed mutagenesis of this amino acid residue afforded a mutant enzyme with a severely compromised catalytic efficiency (V(max)/K(m) of mutant enzyme approximately 50-fold lower than for wild-type TP) and pronounced resistance to the inhibitory effect of 5'-O-tritylinosine. In contrast, the D203A mutant enzyme kept full sensitivity to the competitive inhibitors 6-aminothymine and 6-amino-5-bromouracil, which is in line with the kinetic properties of these inhibitors. Our findings reveal the existence of a previously unrecognized site in TP that can be targeted by small molecules to inhibit the catalytic activity of TP.

Primary tumour expression of the cysteine cathepsin inhibitor Stefin A inhibits distant metastasis in breast cancer.

Using the clinically relevant 4T1-derived syngeneic murine model of spontaneous mammary metastasis to bone, we have identified the cysteine cathepsin inhibitor Stefin A as a gene differentially expressed in primary and metastatic mammary tumours. In primary tumours, Stefin A expression correlated inversely with metastatic potential in 4T1-derived lines and was not detected in tumour cells in culture, indicating induction only within the tumour microenvironment. Enforced expression of Stefin A in the highly metastatic 4T1.2 cell line significantly reduced spontaneous bone metastasis following orthotopic injection into the mammary gland. Consistent with the mouse data, Stefin A expression correlated with disease-free survival (absence of distant metastasis) in a cohort of 142 primary tumours from breast cancer patients. This was most significant for patients with invasive ductal carcinoma expressing Stefin A, who were less likely to develop distant metastases (log rank test, p = 0.0075). In a multivariate disease-free survival analysis (Cox proportional hazards model), Stefin A expression remained a significant independent prognostic factor in patients with invasive ductal carcinoma (p = 0.0014), along with grade and progesterone receptor (PR) status. In human lung and bone metastases, we detected irregular Stefin A staining patterns, with expression often localizing to micrometastases (<0.2 mm) in direct contact with the stroma. We propose that Stefin A, as a cysteine cathepsin inhibitor, may be a marker of increased cathepsin activity in metastases. Using immunohistology, the cathepsin inhibitor was detected co-expressed with cathepsin B in lung and bone metastases in both the murine model and human tissues. We conclude that Stefin A expression reduces distant metastasis in breast cancer and propose that this may be due to the inhibition of cysteine cathepsins, such as cathepsin B.

Predictive value of apoptosis, proliferation, HER-2, and topoisomerase IIalpha for anthracycline chemotherapy in locally advanced breast cancer.

PURPOSE: Laboratory evidence indicates that tumor growth depends on the balance between cell proliferation and cell death, and many anticancer agents may exert their therapeutic effect by decreasing proliferation and increasing apoptosis. Additionally, clinical observations indicate that overexpression of HER-2 or topoisomerase IIalpha (topo IIalpha) may be predictors of better response to anthracyclines in breast cancer. The objective of this study was to determine if proliferation (Ki-67), apoptosis (TUNEL), and expression of HER-2 and topo IIalpha are affected by anthracycline treatment, and if these molecular markers predict anthracycline responsiveness. EXPERIMENTAL DESIGN: Thirty-three women with primary breast tumors > or =3 cm received either doxorubicin (75 mg/m(2)) or epirubicin (120 mg/m(2)) for 4 cycles before surgery. Clinical response was evaluated after 4 cycles of treatment. Changes in molecular markers were assessed from core needle taken before treatment (D0), at 24-48 h (Dl) and on day 7 (D7) while on treatment, and from the surgical specimen excised on day 84 (D84) after the fourth cycle of chemotherapy. RESULTS: The overall response rate was 51% (17 of 33 patients), with a 12% complete clinical response rate (4 of 33 patients). There were trends for tumors with higher apoptosis and topo IIalpha at baseline (D0) to be more responsive to anthracyclines, p = 0.1 and p = 0.08, respectively. Median apoptosis increased from D0 to Dl (p = 0.06) while median Ki-67 decreased (p = 0.07). Overall, expression of HER-2 remained stable throughout the chemotherapy administration. By Day 84, topo IIalpha had significantly decreased from baseline in responders, while it increased in non-responders, p = 0.03. CONCLUSIONS: In human primary breast cancer, anthracycline treatment causes an early increase in apoptosis and a decrease in proliferation. In this pilot study, higher apoptosis and topo IIalphaa levels in primary tumors were associated with greater responsiveness to anthracyclines, and topo IIalpha levels declined in responsive tumors.

Towards new thymidine phosphorylase/PD-ECGF inhibitors based on the transition state of the enzyme reaction.

Computational studies have been conducted to built a closed form of TPase and to characterize the transition state of the phosphorylisis reaction catalyzed by TPase. The results obtained point to a crucial role of His-85 and the O2 of thymine in the catalysis. This modelled transition state forms the basis for the design of new TPase inhibitors.

Molecular dynamics simulations of the conformational changes of the glutamate receptor ligand-binding core in the presence of glutamate and kainate.

Excitatory synaptic transmission is mediated by ionotropic glutamate receptors (iGluRs) through the induced transient opening of transmembrane ion channels. The three-dimensional structure of the extracellular ligand-binding core of iGluRs shares the overall features of bacterial periplasmic binding proteins (PBPs). In both families of proteins, the ligand-binding site is arranged in two domains separated by a cleft and connected by two peptide stretches. PBPs undergo a typical hinge motion of the two domains associated with ligand binding that leads to a conformational change from an open to a closed form. The common architecture suggests a similar closing mechanism in the ligand-binding core of iGluRs induced by the binding of specific agonists. Starting from the experimentally determined kainate-bound closed form of the S1S2 GluR2 construct, we have studied by means of molecular dynamics simulations the opening motion of the ligand-binding core in the presence and in the absence of both glutamate and kainate. Our results suggest that the opening/closing interdomain hinge motions are coupled to conformational changes in the insertion region of the transmembrane segments. These changes are triggered by the interaction of the agonists with the essential Glu 209 residue. A plausible mechanism for the coupling of agonist binding to channel gating is discussed.

Comparative binding energy analysis of the substrate specificity of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10.

Comparative binding energy (COMBINE) analysis was conducted for 18 substrates of the haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (DhlA): 1-chlorobutane, 1-chlorohexane, dichloromethane, 1,2-dichloroethane, 1,2-dichloropropane, 2-chloroethanol, epichlorohydrine, 2-chloroacetonitrile, 2-chloroacetamide, and their brominated analogues. The purpose of the COMBINE analysis was to identify the amino acid residues determining the substrate specificity of the haloalkane dehalogenase. This knowledge is essential for the tailoring of this enzyme for biotechnological applications. Complexes of the enzyme with these substrates were modeled and then refined by molecular mechanics energy minimization. The intermolecular enzyme-substrate energy was decomposed into residue-wise van der Waals and electrostatic contributions and complemented by surface area dependent and electrostatic desolvation terms. Partial least-squares projection to latent structures analysis was then used to establish relationships between the energy contributions and the experimental apparent dissociation constants. A model containing van der Waals and electrostatic intermolecular interaction energy contributions calculated using the AMBER force field explained 91% (73% cross-validated) of the quantitative variance in the apparent dissociation constants. A model based on van der Waals intermolecular contributions from AMBER and electrostatic interactions derived from the Poisson-Boltzmann equation explained 93% (74% cross-validated) of the quantitative variance. COMBINE models predicted correctly the change in apparent dissociation constants upon single-point mutation of DhlA for six enzyme-substrate complexes. The amino acid residues contributing most significantly to the substrate specificity of DhlA were identified; they include Asp124, Trp125, Phe164, Phe172, Trp175, Phe222, Pro223, and Leu263. These residues are suitable targets for modification by site-directed mutagenesis.

Identification of a putative binding site for [2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3'-spiro-5''-(4''-amino-1'',2''-oxathiole-2'',2''-dioxide)thymine (TSAO) derivatives at the p51-p66 interface of HIV-1 reverse transcriptase.

A binding site for TSAO-m(3)T at the interface between the p66 and p51 subunits of HIV-1 reverse transcriptase (RT) and distinct from that of "classical" HIV-1 non-nucleoside inhibitors is proposed. The feasibility of the binding mode was assessed by carrying out nanosecond molecular dynamics simulations for the complexes of TSAO-m(3)T with reduced models of both the wild-type enzyme and a more sensitive R172A mutant. The molecular model is in agreement with a previous proposal, with known structure-activity and mutagenesis data for this unique class of inhibitors, and also with recent biochemical evidence indicating that TSAO analogues can affect enzyme dimerization. The relative importance of residues involved in dimer formation and TSAO-RT complex stabilization was assessed by a combination of surface area accessibility, molecular mechanics, and continuum electrostatics calculations. A structure-based modification introduced into the lead compound yielded a new derivative with improved antiviral activity.

Exploring the role of the 5'-position of TSAO-T. Synthesis and anti-HIV evaluation of novel TSAO-T derivatives.

Various analogues of the anti-HIV-1 agent TSAO-T, [1-[2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]thymine]-3'-spiro-5"-(4"-amino-1",2"-oxathiole-2",2"-dioxide) have been synthesized in which the 5'-TBDMS group has been replaced by alkyl-, alkenyl- or aromatic ether groups, substituted amines, carbamoyl or (thio)acyl groups. The compounds synthesized were evaluated for their inhibitory effect on HIV-1 and HIV-2 replication in cell culture. Replacement of the 5'-TBDMS group by an acyl, aromatic or a cyclic moiety markedly diminish or even eliminate the anti-HIV activity. However, the presence at that position of an alkyl or alkenyl chain, partially retain antiviral activity. These observations suggest that the 5'-TBDMS group of the TSAO molecule plays a crucial role.

Advances in the chemistry and pharmacology of ecteinascidins, a promising new class of anti-cancer agents.

Ecteinascidins are marine natural products consisting of two or three linked tetrahydroisoquinoline subunits and an active carbinolamine functional group. Their potent antiproliferative activity against a variety of tumor cells has made them attractive candidates for development as anticancer agents. The lead compound, ecteinascidin 743 (ET 743), is currently in phase II clinical trials but the low amounts present in its natural source, the tunicate Ecteinascidia turbinata, made it necessary to develop efficient synthetic procedures. Recent improvements on the original synthesis are reviewed as well as new strategies starting from readily available cyanosafracin B. ET 743 is known to bind to the minor groove of DNA giving rise to a covalent adduct with the exocyclic amino group at position 2 of a guanine in a fashion similar to saframycin antibiotics. Some of the resulting complexes have been studied by a variety of biochemical and spectroscopic methods and also by computer simulations. The rules for sequence specificity have been well established (preferred targets are RGC and YGG, where R and Y stand for purine and pyrimidine, respectively), and it has been shown that binding of ET 743 to DNA is accompanied by minor groove widening and DNA bending towards the major groove. Although the precise target for antitumor action remains to be unambiguously defined, a role in affecting the transcriptional regulation of some inducible genes is rapidly emerging.

Comparative binding energy (COMBINE) analysis of human neutrophil elastase inhibition by pyridone-containing trifluoromethylketones.

The complexes of human neutrophil elastase with a series of 40 N3-substituted trifluoromethylketone-based pyridone inhibitors have been modelled. The series spans three orders of magnitude in inhibition constants despite the fact that it was originally developed in an attempt to improve the oral activity of a lead compound. Ligand-receptor interaction energies calculated using molecular mechanics did not correlate well with the experimental activities. A good correlation with activity was found, however, when a COMBINE analysis of the same data was carried out, which allowed a quantitative interpretation of the modelled complexes. The essence of this method is to partition the ligand-receptor interaction energies into individual residue-based van der Waals and electrostatic contributions, and to subject the resulting energy matrix to partial least squares analysis. Incorporation of two additional descriptors representing the electrostatic energy contributions to the partial desolvation of both the receptor and the ligands improved the QSAR model, as did the replacement of the distance-dependent electrostatic contributions with solvent-screened electrostatic interactions calculated by numerically solving the Poisson-Boltzmann equation. The model was validated both internally (cross-validation) and externally, using a set of twelve 6-phenyl-pyridopyrimidine analogs. The analysis reveals the subtle interplay of binding forces which occurs within the enzyme active site and provides objective information that can be interpreted in the light of the receptor structure. This information, gained from a series of real compounds, can be easily translated into 3D real or virtual database queries in the search for more active derivatives.