Niche filling slows the diversification of Himalayan songbirds.

Speciation generally involves a three-step process--range expansion, range fragmentation and the development of reproductive isolation between spatially separated populations. Speciation relies on cycling through these three steps and each may limit the rate at which new species form. We estimate phylogenetic relationships among all Himalayan songbirds to ask whether the development of reproductive isolation and ecological competition, both factors that limit range expansions, set an ultimate limit on speciation. Based on a phylogeny for all 358 species distributed along the eastern elevational gradient, here we show that body size and shape differences evolved early in the radiation, with the elevational band occupied by a species evolving later. These results are consistent with competition for niche space limiting species accumulation. Even the elevation dimension seems to be approaching ecological saturation, because the closest relatives both inside the assemblage and elsewhere in the Himalayas are on average separated by more than five million years, which is longer than it generally takes for reproductive isolation to be completed; also, elevational distributions are well explained by resource availability, notably the abundance of arthropods, and not by differences in diversification rates in different elevational zones. Our results imply that speciation rate is ultimately set by niche filling (that is, ecological competition for resources), rather than by the rate of acquisition of reproductive isolation.

Clinically Important Misclassification of Aortic Valve Stenosis Severity Using Non-Invasive Techniques: Simultaneous Echocardiography and Cardiac Catheterization During Transcatheter Aortic Valve Implantation in Awake Patients.

BACKGROUND: Suitability for transcatheter aortic valve (AV) implantation (TAVI) is determined by using transthoracic echocardiography (TTE), although left-sided cardiac catheterization (LCC) provides directly measured pressure data. TAVI in awake patients permits simultaneous comparison of TTE and LCC under physiologically relevant left ventricular loading conditions. We hypothesized that clinically important discrepancies between TTE and LCC would be identified. METHODS AND RESULTS: TAVI was performed in 108 awake patients undergoing intra-procedural TTE and LCC between January 1, 2016 and December 31, 2016, based upon pre-procedure TTE data. Intra-procedural assessments simultaneously were performed before and after prosthesis implantation. Based upon mean trans-AV systolic ejection pressure gradient (MSEPG), AS was graded as: mild (<20 mm Hg; grade 1), moderate (20 - <40 mm Hg; grade 2), or severe (≥40 mm Hg; grade 3). In 79 of the 108 (73.1%) patients, intra-procedural TTE and LCC assessments were concordant. In 2 of the 108 (1.9%) patients, TTE overestimated AS severity by ≥1 grade. In 27 of the 108 (25.0%) patients, TTE underestimated AS severity by ≥1 grade. In total, AS severity reclassification occurred in 29 (26.9%) patients. Overall, TTE underestimated MSEPG by 8.9 ± 1.2 mm Hg (TTE MSEPG versus LCC MSEPG; P < .001). CONCLUSION: Current TTE criteria appear to frequently and importantly underestimate AS severity. Because decision-making regarding TAVI often exclusively is based upon TTE data, these findings suggest either a continued role for LCC in the diagnostic assessment of AS in patients who do not meet standard TTE criteria or lowering TTE cutoffs for TAVI.

Diffuse alveolar hemorrhage secondary to plastic fume exposure: A case report.

A 31-year non-smoker man, working in plastic making industry for 12 years presented with cough and streaking hemoptysis for 2 days. Computed tomography (CT) of chest showed patchy ground glass opacities with interlobular septal thickening in bilateral lung parenchyma. Fiber optic bronchoscopy (FOB) was done. Sequential lavage was taken which showed progressively increasing hemorrhagic fluid. His diffusion capacity for carbon monoxide (DLCO) was 38.08 mL/mmHg/Mi (126%) predicted on day 2 of admission, 32.36 ml/mmHg/Mi (106%) predicted on discharge and 39.63 mL/mmHg/Mi (130%) predicted on going back to work. He was diagnosed with plastic fume exposure related pulmonary alveolar hemorrhage.

Simulations of site-specific target-mediated pharmacokinetic models for guiding the development of bispecific antibodies.

Bispecific antibodies (BAbs) are novel constructs that are under development and show promise as new therapeutic modalities for cancer and autoimmune disorders. The aim of this study is to develop a semi-mechanistic modeling approach to elucidate the disposition of BAbs in plasma and possible sites of action in humans. Here we present two case studies that showcase the use of modeling to guide BAb development. In case one, a BAb is directed against a soluble and a membrane-bound ligand for treating systemic lupus erythematosus, and in case two, a BAb targets two soluble ligands as a potential treatment for ulcerative colitis and asthma. Model simulations revealed important differences between plasma and tissues, when evaluated for drug disposition and target suppression. Target concentrations at tissue sites and type (soluble vs membrane-bound), tissue-site binding, and binding affinity are all major determinants of BAb disposition and subsequently target suppression. For the presented case studies, higher doses and/or frequent dosing regimens are required to achieve 80 % target suppression in site specific tissue (the more relevant matrix) as compared to plasma. Site-specific target-mediated models may serve to guide the selection of first-in-human doses for new BAbs.

Isolation of genes conferring salt tolerance from Piriformospora indica by random overexpression in Escherichia coli.

Piriformospora indica, a root endophytic fungus identified in the Indian Thar desert, colonizes the roots of plants and provides resistance towards biotic stress as well as tolerance to abiotic stress in the plants. Despite its positive impact on the host, little is known about the P. indica genes that are involved in salt stress tolerance. Therefore this study was conducted to identify and isolate high salinity-tolerance genes from P. indica. Thirty-six salinity-tolerance genes were obtained by functional screening, based on random over expression of a P. indica cDNA library in Escherichia coli grown on medium supplemented with 0.6 M NaCl. The salinity tolerance conferred by these 36 genes in bacteria was further confirmed by using another strain of E. coli (DH5α) transformants. However when the expression of these 36 genes was analysed in P. indica using quantitative RT-PCR, we found only six genes were up-regulated by salt stress. These six genes are involved in different cellular processes, such as metabolism, energy and biosynthetic processes, DNA repair, regulation of protein turnover, transport and salt stress tolerance. This work presents the basis for further molecular analyses of the mechanisms of salt tolerance in P. indica and for the use of this endophyte to confer salt tolerance to plants.

Quantitative analysis of target coverage and germinal center response by a CXCL13 neutralizing antibody in a T-dependent mouse immunization model.

PURPOSE: Study the impact of CXCL13 neutralization on germinal center (GC) response in vivo, and build quantitative relationship between target coverage and pharmacological effects at the target tissue. METHODS: An anti-CXCL13 neutralizing monoclonal antibody was dosed in vivo in a T-dependent mouse immunization (TDI) model. A quantitative site-of-action (SoA) model was developed to integrate antibody PK and total CXCL13 levels in serum and spleen towards estimating target coverage as a function of dose. To aid in the SoA model development, a radio-labeled study using [I(125)] CXCL13 was conducted in mice. Model estimated target coverage was linked to germinal center response using a sigmoidal inhibitory effect model. RESULTS: In vivo studies demonstrated that CXCL13 inhibition led to an architectural change in B-cell follicles, dislocation of GCs and a significant reduction in the GC absolute numbers per square area (GC/mm(2)). The SoA modeling analysis indicated that ~79% coverage in spleen was required to achieve 50% suppression of GC/mm(2). The 3 mg/kg dose with 52% spleen coverage resulted in no PD suppression, whereas 30 mg/kg with 93% coverage achieved close to maximum PD suppression, highlighting the steepness of PD response. CONCLUSIONS: This study showcases an application of SoA modeling towards a quantitative understanding of CXCL13 pharmacology.

Evolution of species recognition when ecology and sexual selection favor signal stasis.

The process of reproductive character displacement involves divergence and/or the narrowing of variance in traits involved in species recognition, driven by interactions between taxa. However, stabilizing sexual selection may favor stasis and species similarity in these same traits if signals are optimized for transmission through the prevailing environment. Further, sexual selection may promote increased variability within species to facilitate individual recognition. Here we ask how the conflicting selection pressures of species recognition and sexual selection are resolved in a genus of Himalayan birds that sing exceptionally similar songs. We experimentally show that small differences in two traits (note shape and peak frequency) are both necessary and sufficient for species recognition. Song frequency shows remarkable clinal variation along the Himalayan elevational gradient, being most divergent where species co-occur, the classic signature of reproductive character displacement. Note shape shows no such clinal variation but varies more between individuals of an allopatric species than it does among individuals within species which co-occur. We argue that the different note shapes experience similar transmission constraints, and differences produced through species interactions spread back through the entire species range. Our results imply that reproductive character displacement is likely to be common.

Population Pharmacokinetics of Sacituzumab Govitecan in Patients with Metastatic Triple-Negative Breast Cancer and Other Solid Tumors.

BACKGROUND AND OBJECTIVE: Sacituzumab govitecan (SG) is an antibody-drug conjugate composed of an antibody with affinity for Trop-2 coupled to SN-38 via hydrolyzable linker. SG is approved for patients with metastatic triple-negative breast cancer (mTNBC) who have received two or more prior chemotherapies (at least one in a metastatic setting) and for patients with pretreated hormone receptor positive (HR+)/human epidermal growth factor receptor 2 negative (HER2-) metastatic breast cancer. METHODS: In these analyses, the pharmacokinetics of SG, free SN-38, and total antibody (tAB) were characterized using data from 529 patients with mTNBC or other solid tumors across two large clinical trials (NCT01631552; ASCENT, NCT02574455). Three population pharmacokinetic models were constructed using non-linear mixed-effects modeling; clinically relevant covariates were evaluated to assess their impact on exposure. Models for SG and tAB were developed independently whereas free SN-38 was sequentially generated via a first-order release process from SG. RESULTS: Pharmacokinetics of the three analytes were each described by a two-compartment model with estimated body weight-based scaling exponents for clearance and volume. Typical parameter estimates for clearance and steady-state volume of distribution were 0.133 L/h and 3.68 L for SG and 0.0164 L/h and 4.26 L for tAB, respectively. Mild-to-moderate renal impairment, mild hepatic impairment, age, sex, baseline albumin level, tumor type, UGT1A1 genotype, or Trop-2 expression did not have a clinically relevant impact on exposure for any of the three analytes. CONCLUSIONS: These analyses support the approved SG dosing regimen of 10 mg/kg as intravenous infusion on days 1 and 8 of 21-day cycles and did not identify a need for dose adjustment based on evaluated covariates or disease characteristics.

Comparative analysis of soil quality and enzymatic activities under different tillage based nutrient management practices in soybean-wheat cropping sequence in Vertisols.

In the modern era, intensive agricultural practices such as agrochemicals are applied in excessive amounts to enhance agricultural production. However, imbalanced adoption of these chemicals has arisen in the dwindling of agriculture factor productivity and soil quality. To maintain soil fertility and production, these chemical fertilizers must be supplemented with organic inputs. Keeping this in the backdrop, a research trail was established during 2018-19 and 2019-20 years at Research Farm of Agriculture University, Kota, India. The treatment setup was comprised of 5 treatment modules viz., conservation tillage + organic management (CAOM), conservation tillage + chemical management (CACM), conventional tillage + chemical management (CTCM), conventional tillage + organic management (CTOM) and the package of practices (PoPs) with four replications. Results indicated that the highest organic carbon (0.68%), bacterial (29.11 × 107 cfu g-1), fungal (4.77 × 104 cfu g-1), actinomycetes populations (5.67 × 104 cfu g-1), acid phosphatase (44.1 µg g-1 h-1), urease (45.3 µg g-1 h-1) and dehydrogenase (23.3 µg triphenylformazan [TPF] g-1 h-1) activity in soil were found in the treatment of conservation organic system during both the years of study at each soil depth. In contrast to other parameters, the highest system productivity was observed with conservation chemical crop management approaches, with a soybean equivalent yield of 4615 kg ha-1 in a soybean-wheat system of production. Furthermore, the soil quality index (SQI) significantly varied from the lowest score (0.30) at 45-60 cm layer of soil in the package of practices to the highest score (0.92) at 0-15 cm layer of soil with regards to the conservation organic which shows, 206.67 percent enhancement through the soil profile of various crop management practices. The SQI variation from 0-15 to 45-60 cm soil depth was 130.0, 81.08, 60.0, 175.0 and 83.33 percent, respectively, for CAOM, CACM, CTCM, CTOM and PoPs. Amongst, different systems, the highest mean performance was noticed under the conservation organic systems for physical and biological properties. Hence, in line with the salient outcome, we may propose that the conservation chemical system needs to be followed to improve crop productivity, whereas, conservation organic seems a good option for soil health with long-term viability.

Optimizing outcomes and managing adverse events in locally advanced or metastatic urothelial cancer: a clinical pharmacology perspective.

INTRODUCTION: Bladder cancer (BC) is the sixth most common type of cancer with epithelial/urothelial and non-urothelial origins. Urothelial carcinoma (UC) involves neoplastic cells of epithelial origin and accounts for 90% of all BC cases. Current review aims to discuss the latest advances and challenges in the treatment of UC with an emphasis on clinical pharmacology considerations. AREAS COVERED: Data including clinical efficacy and safety outcomes as well as precautions reported in published clinical studies obtained from PubMed and package inserts were collected and summarized in the review. Recent decade saw the approval of multiple drugs for the treatment of BC in both adjuvant/neoadjuvant setting as well as for unresectable tumors. Checkpoint blockers (pembrolizumab, nivolumab, atezolizumab, and avelumab), antibody drug conjugates (enfortumab vedotin and sacituzumab govitecan) and targeted therapies (erdafitinib) are now available in first-line (cisplatin-ineligible), second-line and third-line settings along with conventional platinum-based chemotherapy. While the survival outcomes have improved especially in refractory and unresponsive patients, the response rates are relatively low and patient safety needs further optimization. EXPERT OPINION: Additional studies on combination therapies, dose adjustments in special populations and impact of anti-drug antibodies on drug exposure are needed to further improve clinical outcomes.

End fire linear antenna array synthesis using differential evolution inspired Adaptive Naked Mole Rat Algorithm.

Linear antenna arrays (LAAs) play a critical role in smart system communication applications such as the Internet of Things (IoT), mobile communication and beamforming. However, minimizing secondary lobes while maintaining a low beamwidth remains challenging. This study presents an enhanced synthesis methodology for LAAs using the Adaptive Naked Mole Rat Algorithm (ANMRA). ANMRA, inspired by mole-rat mating habits, improves exploration and exploitation capabilities for directive LAA applications. The performance of ANMRA is assessed using the CEC 2019 benchmark test functions, a widely adopted standard for statistical evaluation in optimization algorithms. The proposed methodology results are also benchmarked against state-of-the-art algorithms, including the Salp Swarm Algorithm (SSA), Cuckoo Search (CS), Artificial Hummingbird Algorithm (AHOA), Chimp Optimization Algorithm (ChOA), and Naked Mole Rat Algorithm (NMRA). The results demonstrate that ANMRA achieves superior performance among the benchmarked algorithms by successfully minimizing secondary lobes and obtaining a narrow beamwidth. The ANMRA controlled design achieves the lowest Side Lobe Level (SLL) of - 37.08 dB and the smallest beamwidth of 74.68°. The statistical assessment using the benchmark test functions further confirms the effectiveness of ANMRA. By optimizing antenna element magnitude and placement control, ANMRA enables precise primary lobe placement, grating lobe elimination, and high directivity in LAAs. This research contributes to advancing smart system communication technologies, particularly in the context of IoT and beamforming applications, by providing an enhanced synthesis methodology for LAAs that offers improved performance in terms of secondary lobe reduction and beamwidth optimization.

Nutritional Diversity in Native Germplasm of Maize Collected From Three Different Fragile Ecosystems of India.

Native germplasm resources are adapted to specific ecological niches. They have sustained over generations owing to the preference of local communities for their unique taste, the utility to particular dishes, and the low cost of cultivation. They may help eradicate malnutrition and act as a source for trait-linked genes. The present dataset comprises thirty-three native germplasm of maize collected from Rajasthan, Himachal Pradesh, and Andhra Pradesh states of India with an altitudinal variation of 386-2,028 m. They were evaluated for proximate composition, minerals, nutritional attributes, and antioxidant activity and compared with the standard values reported in the Indian Food Composition Table 2017 (IFCT2017). The nutritional profile showed moisture content in the range of 7.16-10.9%, ash 0.73-1.93%, crude protein 8.68-12.0%, crude fat 3.72-8.03%, dietary fiber 5.21-11.2%, and available carbohydrates 60.6-69.8%. Three accessions, namely, Malan 11 (7.06%), Malan 24 (7.20%), and Yellow Chamba Local 02 (8.03%) exhibited almost double the crude fat content as compared with the values notified in IFCT2017 (3.77). Total sugar content obtained was in the range of 5.00-11.3%, whereas the starch content was found between 50.9 and 64.9%. All the germplasm except Yellow Chamba Local reflected a higher protein content than reported values in IFCT2017 (8.80). Sathi, Safed Chamba Local, and Ragal Makka had nearly 12% protein content. Mineral malnutrition, mainly due to iron (Fe) deficiency, is a worldwide issue to science, humanity, and society. The mineral profile revealed that most germplasm had a higher iron content. Accessions with the iron content of nearly three times of IFCT2017 reported value were identified in germplasm belonging to three states. A negative relationship was observed between the altitude of the sample collection site and available carbohydrate content. In contrast, available carbohydrate showed inverse correlations with dietary fiber, protein, and fat content. The information generated in this study can be utilized to promote these germplasm as nutrifood, nutritional surveillance, labeling, and crop improvement programs.

Eculizumab Pharmacokinetics and Pharmacodynamics in Patients With Neuromyelitis Optica Spectrum Disorder.

Objective: To investigate the pharmacokinetics and pharmacodynamics of the approved 900/1,200 mg dosing regimen for the terminal complement component 5 (C5) inhibitor eculizumab in patients with neuromyelitis optica spectrum disorder (NMOSD). Methods: Data were analyzed from 95 patients with aquaporin-4-IgG-positive NMOSD who received eculizumab during the PREVENT study (ClinicalTrials.gov: NCT01892345). Relationships were explored between eculizumab exposure and free complement C5 concentrations, terminal complement activity, and clinical outcomes. Results: Pharmacokinetic data were well-described by a two-compartment model with first-order elimination, and time-variant body-weight and plasmapheresis/plasma exchange effects. Steady-state serum eculizumab concentrations were achieved by Week 4 and were sustained, with serum trough eculizumab concentrations maintained above the 116 µg/ml threshold for complete complement inhibition throughout 168 weeks of treatment in all post-baseline samples from 89% of patients. Complete inhibition of terminal complement was achieved at Day 1 peak and pre-dosing trough eculizumab concentration in nearly all post-baseline samples assessed (free C5 <0.5 µg/ml in all post-baseline samples from 96% of patients; in vitro hemolysis <20% in all post-baseline samples from 93% of patients). Kaplan-Meier survival analysis of time to first relapse showed separation of eculizumab-treated patients from those receiving placebo, but no separation based on eculizumab exposure quartile, indicating an optimized dose regimen with maximized efficacy. Conclusions: The approved eculizumab dosing regimen (900/1,200 mg) for adults with aquaporin-4-IgG-positive NMOSD is confirmed by rigorous quantitative model-based analysis of exposure-response. The data demonstrate that eculizumab's mechanism of action translates into clinical effect by achieving rapid, complete, and sustained terminal complement inhibition.

Improved extrapolation of hepatobiliary clearance from in vitro sandwich cultured rat hepatocytes through absolute quantification of hepatobiliary transporters.

Previously we have reported that hepatobiliary transporter expressions in sandwich cultured hepatocytes (SCH) are altered 2- to 5-fold. This change could limit the model's predictive power for in vivo biliary clearance. The present study was designed to better establish in vitro to in vivo correlation (IVIVC) of biliary clearance. Eleven compounds representing the substrates of Mrp2/Abcc2, Bcrp/Abcg2 and Bsep/Abcb11 were tested in the sandwich cultured rat hepatocyte (SCRH) model. Simultaneously, the absolute difference of hepatobiliary transporters between rat livers and SCRH at day 5 post culture was determined by LC-MS/MS. This difference was integrated into the well-stirred hepatic prediction model. A correction factor named "g_factor" was mathematically defined to reflect the difference in hepatobiliary transporter expressions between the SCRH model and in vivo models, as well as the contribution of multiple transporters. When the g_factor correction was applied, the in vivo biliary clearance prediction was significantly improved. In addition, for those compounds which are poorly permeable and/or undergo transporter-dependent active uptake, the known intracellular concentrations of substrates were used to estimate intrinsic bile clearance. This led to further improvement in the prediction of in vivo bile secretion. While the rate-limiting processes of uptake transporters in the SCRH model remain to be further determined, we showed that integration of the absolute difference of hepatobiliary transporter proteins and transport contributions could improve the predictability of SCRH model. This integration is fundamental for increased confidence in the IVIVC of human biliary clearance.

Dorsal onlay lingual mucosal graft urethroplasty for urethral strictures in women.

OBJECTIVE: To describe the technique and results of dorsal onlay lingual mucosal graft (LMG) urethroplasty for the definitive management of urethral strictures in women. PATIENTS AND METHODS: In all, 15 women (mean age 42 years) with a history suggestive of urethral stricture who had undergone multiple urethral dilatations and/or urethrotomy were selected for dorsal onlay LMG urethroplasty after thorough evaluation, from October 2006 to March 2008. After a suprameatal inverted-U incision, the dorsal aspect of the urethra was dissected and urethrotomy was done at the 12 o'clock position across the strictured segment. Tailored LMG harvested from the ventrolateral aspect of the tongue was then sutured to the urethrotomy wound over an 18 F silicone catheter. RESULTS: The preoperative mean maximum urinary flow rate of 7.2 mL/s increased to 29.87 mL/s, 26.95 mL/s and 26.86 mL/s with a 'normal' flow rate curve at 3, 6 and 12 months follow-up, respectively. One patient at the 3-month follow-up had submeatal stenosis and required urethral dilatation thrice at monthly intervals. At the 1-year follow-up, none of the present patients had any neurosensory complications, urinary incontinence, or long-term functional/aesthetic complication at the donor site. CONCLUSION: LMG urethroplasty using the dorsal onlay technique should be offered for correction of persistent female urethral stricture as it provides a simple, safe and effective approach with durable results.

Substitution urethroplasty for anterior urethral strictures: buccal versus lingual mucosal graft.

AIM: To compare the results of substitution urethroplasty and donor site morbidity between buccal mucosal graft (BMG) and lingual mucosal graft (LMG). PATIENTS AND METHODS: Patients who underwent single-stage dorsal onlay free oral mucosal graft substitution urethroplasty by Barbagli's technique between January 2004 and August 2008 were included in this study. Patients who underwent buccal (cheek, lip) mucosal graft urethroplasty were included in group I and those who underwent LMG urethroplasty (tongue) were included in group II. All patients underwent complete evaluation of the stricture including inspection of the oral cavity. Exclusion criteria were stricture length <3 cm and complex strictures which required a multistage procedure. RESULTS: The results of urethroplasty were similar in both groups in terms of blood loss, duration of postoperative hospitalization, complications encountered at urethroplasty site, mean postoperative Q(max) and mean postoperative AUA symptom score. Early slurring of speech complications was seen in group II, but not in group I. The long-term complications of persistent oral discomfort, perioral numbness and tightness of the mouth were seen only in group I. CONCLUSION: LMG urethroplasty is a good substitute for BMG urethroplasty with equally good results of urethroplasty with lower donor site morbidity.

Multivitamin and micronutrient treatment improves semen parameters of azoospermic patients with maturation arrest.

The study was undertaken to evaluate the efficacy of multivitamin and micronutrient supplementation in azoospermic patients with maturation arrest. A total of 35 azoospermic patients showing maturation arrest on testicular biopsy were recruited in this study. The patients were divided into two groups. Untreated group (n=11) without any treatment and treated group (n=24) who received multivitamins, micronutrients and co-enzyme Q10. The sperm concentration, motility and morphology were evaluated at monthly interval. The results showed reduction in liquefaction time and relative viscosity of the semen in the treated group. Further, in treated group there was appearance of spermatozoa (4.0 million/ml) exhibiting progressive motility (7%) and normal morphology (6%), even in the first follow up visit. The sperm count, motility and normal morphology increased significantly on subsequent visits. Within 3 months (3 visits) 2 pregnancies were reported. These observations indicate that multivitamin and micronutrient supplementation improve the qualitative and quantitative parameters of seminogram in patients with azoospermia of maturation arrest.

Prostate-specific antigen is a "chymotrypsin-like" serine protease with unique P1 substrate specificity.

Prostate-specific antigen (PSA), a serine protease belonging to the human kallikrein family, is best known as a prostate cancer biomarker. Emerging evidence suggests that PSA may also play a salient role in prostate cancer development and progression. With large amounts of enzymatically active PSA continuously and selectively produced by all stages of prostate cancer, PSA is an attractive target. PSA inhibitors, therefore, may represent a promising class of therapeutics and/or imaging agents. PSA displays chymotrypsin-like specificity, cleaving after hydrophobic residues, in addition to possessing a unique ability to cleave after glutamine in the P1 position. In this study, we investigated the structural motifs of the PSA S1 pocket that give it a distinct architecture and specificity when compared to the S1 pocket of chymotrypsin. Using the previously described PSA substrate Ser-Ser-Lys-Leu-Gln (SSKLQ) as a template, peptide aldehyde based inhibitors containing novel P1 aldehydes were made and tested against both proteases. Glutamine derivative aldehydes were highly specific for PSA while inhibitors with hydrophobic P1 aldehydes were potent inhibitors of both proteases with K(i) values <500 nM. The crystal structure of PSA was used to generate a model that allowed GOLD docking studies to be performed to further understand the critical interactions required for inhibitor binding to the S1 pockets of PSA and chymotrypsin. In conclusion, these results provide experimental and structural evidence that the S1 specificity pocket of PSA is distinctly different from that of chymotrypsin and that the development of highly specific PSA inhibitors is feasible.

Molecular insights into substrate specificity of prostate specific antigen through structural modeling.

Prostate Specific Antigen's (PSA) role as a biomarker for prostate cancer is well established but the physiological role of its serine protease activity in the pathobiology of normal prostate and prostate carcinogenesis remains largely unknown. In light of recent studies that implicate PSA's enzymatic activity in the initiation and/or progression of prostate cancer, we performed a molecular modeling study of substrate binding at the catalytic site of PSA wherein a PSA-selective substrate (HSSKLQ) was docked in an acyl-enzyme conformation to a three-dimensional homology model of PSA. Additionally, virtual positional scanning studies were conducted to gain mechanistic insights into substrate recognition of PSA. Subsequently, 13 novel peptide substrates of 6-aa length and four peptide substrates with varying length were synthesized and assayed for PSA hydrolysis to evaluate the experimental validity of docking insights. Additionally, six novel aldehyde-containing transition state analog inhibitors were synthesized and tested for their inhibitory potencies. The experimental data on the hydrolysis rates of the newly synthesized substrates and inhibitory potencies of the aldehyde peptides agreed with the docking predictions, providing validation of the docking methodology and demonstrating its utility towards the design of substrate-mimetic inhibitors that can be used to explore PSA's role in the pathobiology of prostate cancer.

Potent and selective peptidyl boronic acid inhibitors of the serine protease prostate-specific antigen.

Prostate cancer cells produce high (microgram to milligram/milliliter) levels of the serine protease Prostate-Specific Antigen (PSA). PSA is enzymatically active in the extracellular fluid surrounding prostate cancers but is found at 1,000- to 10,000-fold lower concentrations in the circulation, where it is inactivated due to binding to abundant serum protease inhibitors. The exclusive presence of high levels of active PSA within prostate cancer sites makes PSA an attractive candidate for targeted imaging and therapeutics. A synthetic approach based on a peptide substrate identified first peptide aldehyde and then boronic acid inhibitors of PSA. The best of these had the sequence Cbz-Ser-Ser-Lys-Leu-(boro)Leu, with a K(i) for PSA of 65 nM. The inhibitor had a 60-fold higher K(i) for chymotrypsin. A validated model of PSA's catalytic site confirmed the critical interactions between the inhibitor and residues within the PSA enzyme.