Renal and Multiorgan Safety of 177Lu-PSMA-617 in Patients with Metastatic Castration-Resistant Prostate Cancer in the VISION Dosimetry Substudy.

In the VISION trial, [177Lu]Lu-PSMA-617 (177Lu-PSMA-617) plus protocol-permitted standard of care significantly improved overall survival and radiographic progression-free survival compared with standard of care alone in patients with prostate-specific membrane antigen-positive metastatic castration-resistant prostate cancer. This VISION dosimetry substudy quantified absorbed doses of 177Lu-PSMA-617 in the kidneys and other organs. Methods: Participants were a separate cohort of 30 nonrandomized patients receiving standard of care plus 177Lu-PSMA-617 at 7.4 GBq per cycle for up to 6 cycles. Blood samples, whole-body conjugate planar image scintigraphy, and abdominal SPECT/CT images were collected. SPECT/CT images were collected at 2, 24, 48, and 168 h after administration in cycle 1 and at a single time point 48 h after administration in cycles 2-6. Outcomes were absorbed dose per unit activity per cycle and cumulative absorbed dose over all cycles. Cumulative absorbed doses were predicted by extrapolation from cycle 1, and calculation of observed values was based on measurements of cycle 1 and cycles 2-6. Safety was also assessed. Results: Mean (±SD) absorbed doses per cycle in the kidneys were 0.43 ± 0.16 Gy/GBq in cycle 1 and 0.44 ± 0.21 Gy/GBq in cycles 2-6. The observed and predicted 6-cycle cumulative absorbed doses in the kidneys were 15 ± 6 and 19 ± 7 Gy, respectively. Observed and predicted cumulative absorbed doses were similar in other at-risk organs. Safety findings were consistent with those in the VISION study; no patients experienced renal treatment-emergent adverse events of a grade higher than 3. Conclusion: The renal cumulative absorbed 177Lu-PSMA-617 dose was below the established limit. 177Lu-PSMA-617 had a good overall safety profile, and low renal radiotoxicity was not a safety concern. Cumulative absorbed doses in at-risk organs over multiple cycles can be predicted by extrapolation from cycle 1 data in patients with metastatic castration-resistant prostate cancer receiving 177Lu-PSMA-617.

Synthesis and biological evaluation of new cytotoxic indazolo[4,3-gh]isoquinolinone derivatives.

A series of indazolo[4,3-gh]isoquinolinones derivatives have been synthesized to decrease cardiotoxic side effects in comparison to Mitoxantrone. The antiproliferative effects of different side chains were investigated and tested on at least four different cell lines of cervix, ovarian, CNS, NSCLC (non-small-cell lung cancer) and colon carcinoma. In addition to antiproliferative activities, influence on cell cycle and intercalation behavior have been tested.

Pharmacokinetics and Safety of Multiple-Dose Alpelisib in Participants with Moderate or Severe Hepatic Impairment: A Phase 1, Open-Label, Parallel Group Study.

The pharmacokinetics (PK) and safety of single-dose alpelisib (300 mg) were assessed in participants with moderate to severe hepatic impairment (n = 6 each) compared with their matching healthy controls (n = 11). Blood samples were collected upto 144 hours post-dose and evaluated by liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay. The primary PK parameters (maximum plasma concentration [Cmax], area under the curve [AUC]inf and AUClast) and secondary PK parameters (AUC0-t, apparent total body clearance [CL/F], apparent volume of distribution [Vz/F], time of maximum observed concentration [Tmax], and half-life [T1/2]) of oral alpelisib 300 mg were determined from individual plasma concentration-time profiles using non­compartmental analysis. Cmax of alpelisib decreased by approximately 17% in the moderate hepatic impairment group vs. the healthy control group (geometric mean ratio; GMR [90% confidence interval; CI], 0.833 [0.530, 1.31]). Cmax in the severe hepatic impairment group was comparable to that of the healthy control group (GMR [90% CI], 1.00 [0.636, 1.58]). AUClast for alpelisib decreased by approximately 27% in the moderate hepatic impairment group vs. the healthy control group (GMR [90% CI], 0.726 [0.487, 1.08]). AUClast was 26% higher in the severe hepatic impairment group compared with the healthy control group (GMR [90% CI], 1.26 [0.845, 1.87]). Overall, 3 participants (13.0%) experienced at least 1 adverse event which were either grade 1 or 2. Adverse events did not lead to study drug discontinuation. No grade 3 or 4 adverse events, serious adverse events or deaths were reported. The results indicate that a single dose of alpelisib was well tolerated in this study population. There was no significant impact of moderate or severe hepatic impairment on the exposure of alpelisib.

A Phase 1b/2 Study of Alpelisib in Combination with Cetuximab in Patients with Recurrent or Metastatic Head and Neck Squamous Cell Carcinoma.

BACKGROUND: Alpelisib in combination with cetuximab showed synergistic anti-tumour activity in head and neck squamous cell carcinoma (HNSCC) models. OBJECTIVES: The recommended phase 2 dose (RP2D) was determined in a phase 1b dose-escalation study. Phase 2 evaluated anti-tumour activity with a randomised part in cetuximab-naïve patients and a non-randomised part in cetuximab-resistant patients. PATIENTS AND METHODS:  Alpelisib was administered in 28 d cycles as whole tablets, suspension from crushed tablets or suspension from dispersible tablets in patients with platinum-resistant, recurrent/metastatic HNSCC. RESULTS: The RP2D determined for alpelisib was 300 mg/d. Alpelisib-cetuximab achieved an overall response rate of 25% and 9.9% and disease control rate of 75% and 43.7% in phase 1b and phase 2 studies, respectively. Median progression-free survival (PFS) per central review was 86 d for combination treatment and 87 d for cetuximab monotherapy (unadjusted HR 1.12; 95% CI 0.69-1.82; P > 0.05). When adjusted for baseline covariates [sum of longest diameters from central data, haemoglobin and white blood cell (WBC), the results favoured combination treatment (adjusted HR 0.54; 95% CI 0.30-0.97; P = 0.039). PFS per investigator assessment resulted in an unadjusted HR of 0.76 (95% CI 0.49-1.19; P > 0.05) favouring combination treatment. The median PFS in cetuximab-resistant patients was 3.9 months. CONCLUSIONS: The addition of alpelisib to cetuximab did not demonstrate a PFS benefit in cetuximab-naïve patients with advanced HNSCC. The alpelisib-cetuximab combination showed moderate activity in cetuximab-resistant patients, with a consistent safety profile. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov NCT01602315; EudraCT 2011-006017-34.

Physiologically Based Biopharmaceutics Modeling to Demonstrate Virtual Bioequivalence and Bioequivalence Safe-space for Ribociclib which has Permeation Rate-controlled Absorption.

A physiologically based biopharmaceutics model (PBBM) was developed to support formulation development of ribociclib, an orally bioavailable selective CDK4/6 inhibitor. Ribociclib is a weak base with moderate permeability and complete in vitro dissolution under stomach pH. GastroPlus™ was used to simulate the pharmacokinetics (PK) in healthy volunteers after capsule dosing. Simulations showed rapid, complete dissolution in human stomach without intestinal precipitation and with permeation-controlled absorption. Permeability was identified as controlling the systemic exposure. PBBM predicted bioequivalence (BE) between capsule and tablet in healthy volunteers, despite non-similarity between in vitro dissolution kinetics (f2<50). BE was verified in a clinical study. Then virtual bioequivalence (VBE) simulations predicted comparable PK in cancer patients between capsule and tablet of commercial batch, which was also confirmed in a clinical study. Finally, virtual trial simulations using virtual batches with slower dissolution were used to define an in vitro BE safe-space for tablets, where BE is expected. PBBM can identify drugs with permeability-controlled absorption for which formulation optimization can focus more on manufacturability rather than dissolution. PBBM can be used to predict BE study outcomes, define clinically relevant specification and BE safe-space, superseding dissolution similarity f2 criteria.

Alpelisib in combination with everolimus ± exemestane in solid tumours: Phase Ib randomised, open-label, multicentre study.

BACKGROUND AND PURPOSE: Combined mTORC1 inhibition with everolimus (EVE) and phosphatidylinositol 3-kinase catalytic subunit p110α blockade with alpelisib (ALP) has demonstrated synergistic efficacy in preclinical models and supports testing the combination of ALP and EVE in the clinical setting. The primary objective was to determine the maximum tolerated dose (MTD)/recommended dose for expansion (RDE) of ALP in combination with EVE and in combination with EVE and exemestane (EXE) and subsequently assess safety, preliminary efficacy and effect of ALP on the pharmacokinetics of EVE and determine the magnitude of the drug-drug interaction. PATIENTS AND METHODS: Dose escalation phases were conducted in patients with advanced solid tumours and in postmenopausal women with hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) advanced breast cancer (ABC). The dose expansion phase was conducted in patients with pancreatic neuroendocrine tumour and renal cell carcinoma (RCC) (both mechanistic target of rapamycin inhibitor [mTORi]-naive), in patients with mTORi-pretreated solid tumours and in postmenopausal women with HR+, HER2- ABC. RESULTS: During the doublet escalation phase, dose-limiting toxicities (DLTs) were reported in 5 of 10 (50%) patients: one patient had grade (Gr) 2 hyperglycemia and one patient had Gr 3 diarrhoea in the 300 mg dose group, one patient had Gr 2 hyperglycemia and one patient had Gr 4 hypocalcaemia in the 250 mg dose group, and one patient in the 200 mg dose group had Gr 3 diarrhoea and Gr 3 stomatitis. The combination of ALP 250 mg + EVE 2.5 mg was declared as the MTD/RDE in subjects with advanced solid tumours. In the triplet escalation phase, one patient who received ALP 200 mg + EVE 2.5 mg + EXE 25 mg had a DLT of Gr 3 acute kidney injury. This dose combination was declared as the MTD and RDE in subjects with advanced HR-positive HER2-negative BC. The common adverse events (≥30% patients), occurring across all phases, were hyperglycaemia, stomatitis, diarrhoea, nausea, asthenia, decreased appetite and fatigue. The sixteen-week progression-free survival rate was 52.4% (90% confidence interval [CI]: 32.8, 71.4) in the RCC cohort, 35.3% (90% CI: 16.6, 58.0) in the prior pNET cohort and 30.0% (90% CI: 8.7, 60.7) in the prior mTORi cohort. The pharmacokinetics of 2.5 mg of EVE was largely unchanged in the presence of ALP, independent of the dose (250 mg or 300 mg). There were no clinically relevant drug-drug interactions observed between ALP and EVE. CONCLUSION: The overall safety profile of ALP with EVE and EXE is manageable and reversible; no unexpected safety signals were noted compared with the individual safety profiles. Pharmacokinetics of ALP, EVE and EXE was largely unchanged in combination with each other.

Physiologically Based Pharmacokinetic Modeling of Oral Absorption, pH, and Food Effect in Healthy Volunteers to Drive Alpelisib Formulation Selection.

A physiologically based pharmacokinetic (PBPK) human model for alpelisib, an oral α-specific class I phosphatidylinositol-3-kinase (PI3K) inhibitor, was established to simulate oral absorption and plasma pharmacokinetics of healthy subjects to allow model-informed drug development. The GastroPlus™ model consisted of an advanced absorption gut model, which was linked to a 2-compartmental model. Systemic clearance and volume of distribution were estimated using population pharmacokinetics (popPK). Various food effect and pH-mediated absorption drug-drug interaction (DDI) scenarios were modeled. In fasted healthy subjects, simulated absorption was lower (ca. 70% for a 300-mg dose) due to pH and bile acid concentration-dependent solubility. Ranitidine showed a significant pH-mediated DDI effect only in the fasted but not fed state. The PBPK model identified that more drug is absorbed in the fed state, and alpelisib intestinal permeability is rate limiting to systemic exposure. Simulations for healthy subject showed a positive food effect with ca. 2-fold increase in plasma Cmax and 1.5-fold increase in AUC0-inf with a meal compared with fasted conditions. The PBPK model was verified using clinical food effect data with pivotal clinical formulation (PCF) and then applied to predict the performance of a commercial formulation (CF) in healthy volunteers. The model successfully predicted the outcome of a clinical bioequivalence study for PCF and CF with included in vitro dissolution data, both fasted and fed state. Estimated predictive errors (based on plasma Cmax, AUC0-t) were equal or below 30%. The alpelisib model for healthy subjects enables future bioequivalence formulation assessments, in fasted, fed, or altered pH conditions. Graphical Abstract.

Alpelisib Plus Fulvestrant in PIK3CA-Altered and PIK3CA-Wild-Type Estrogen Receptor-Positive Advanced Breast Cancer: A Phase 1b Clinical Trial.

Importance: The phosphatidylinositol 3-kinase (PI3K) pathway is frequently activated in patients with estrogen receptor-positive (ER+), endocrine therapy-resistant breast cancers. Objective: To assess the maximum tolerated dose (MTD), safety, and activity of alpelisib, an oral, PI3Kα-specific inhibitor, plus fulvestrant in patients with ER+ advanced breast cancer (ABC). Design, Setting, and Participants: An open-label, single-arm, phase 1b study of alpelisib plus fulvestrant was conducted at 10 centers in 5 countries. Participants were 87 postmenopausal women with PIK3CA-altered or PIK3CA-wild-type ER+ ABC, whose cancer progressed during or after antiestrogen therapy. The study began enrolling patients October 5, 2010, and the data cutoff was March 22, 2017. Interventions: Escalating doses of alpelisib were administered once daily, starting at 300 mg, plus fixed-dose fulvestrant, 500 mg, in the dose-escalation phase; alpelisib at the recommended phase 2 dose plus fulvestrant in the dose-expansion phase. Main Outcomes and Measures: The primary end point was determination of the MTD of once-daily alpelisib plus fulvestrant. Secondary end points included safety and preliminary activity. Results: From October 5, 2010, to March 22, 2017, 87 women (median age: 58 years [range, 37-79 years]; median of 5 prior lines of antineoplastic therapy) received escalating once-daily doses of alpelisib (300 mg, n = 9; 350 mg, n = 8; 400 mg, n = 70) plus fixed-dose fulvestrant (500 mg). During dose escalation, dose-limiting toxic effects were reported in 1 patient (alpelisib, 400 mg): diarrhea (grade 2), vomiting, fatigue, and decreased appetite (all grade 3). The MTD of alpelisib when combined with fulvestrant was 400 mg once daily, and the recommended phase 2 dose was 300 mg once daily. Overall, the most frequent grade 3/4 adverse events with alpelisib, 400 mg, once daily (≥10% of patients), regardless of causality, were hyperglycemia (19 [22%]) and maculopapular rash (11 [13%]); 9 patients permanently discontinued therapy owing to adverse events. Median progression-free survival at the MTD was 5.4 months (95% CI, 4.6-9.0 months). Median progression-free survival with alpelisib, 300 to 400 mg, once daily plus fulvestrant was longer in patients with PIK3CA-altered tumors (9.1 months; 95% CI, 6.6-14.6 months) vs wild-type tumors (4.7 months; 95% CI, 1.9-5.6 months). Overall response rate in the PIK3CA-altered group was 29% (95% CI, 17%-43%), with no objective tumor responses in the wild-type group. Conclusions and Relevance: Alpelisib plus fulvestrant has a manageable safety profile in patients with ER+ ABC, and data suggest that this combination may have greater clinical activity in PIK3CA-altered vs wild-type tumors. Trial Registration: ClinicalTrials.gov identifier: NCT01219699.

Phosphatidylinositol 3-Kinase α-Selective Inhibition With Alpelisib (BYL719) in PIK3CA-Altered Solid Tumors: Results From the First-in-Human Study.

Purpose We report the first-in-human phase Ia study to our knowledge ( ClinicalTrials.gov identifier: NCT01219699) identifying the maximum tolerated dose and assessing safety and preliminary efficacy of single-agent alpelisib (BYL719), an oral phosphatidylinositol 3-kinase α (PI3Kα)-selective inhibitor. Patients and Methods In the dose-escalation phase, patients with PIK3CA-altered advanced solid tumors received once-daily or twice-daily oral alpelisib on a continuous schedule. In the dose-expansion phase, patients with PIK3CA-altered solid tumors and PIK3CA-wild-type, estrogen receptor-positive/human epidermal growth factor receptor 2-negative breast cancer received alpelisib 400 mg once daily. Results One hundred thirty-four patients received treatment. Alpelisib maximum tolerated doses were established as 400 mg once daily and 150 mg twice daily. Nine patients (13.2%) in the dose-escalation phase had dose-limiting toxicities of hyperglycemia (n = 6), nausea (n = 2), and both hyperglycemia and hypophosphatemia (n = 1). Frequent all-grade, treatment-related adverse events included hyperglycemia (51.5%), nausea (50.0%), decreased appetite (41.8%), diarrhea (40.3%), and vomiting (31.3%). Alpelisib was rapidly absorbed; half-life was 7.6 hours at 400 mg once daily with minimal accumulation. Objective tumor responses were observed at doses ≥ 270 mg once daily; overall response rate was 6.0% (n = 8; one patient with endometrial cancer had a complete response, and seven patients with cervical, breast, endometrial, colon, and rectal cancers had partial responses). Stable disease was achieved in 70 (52.2%) patients and was maintained > 24 weeks in 13 (9.7%) patients; disease control rate (complete and partial responses and stable disease) was 58.2%. In patients with estrogen receptor-positive/human epidermal growth factor receptor 2-negative breast cancer, median progression-free survival was 5.5 months. Frequently mutated genes (≥ 10% tumors) included TP53 (51.3%), APC (23.7%), KRAS (22.4%), ARID1A (13.2%), and FBXW7 (10.5%). Conclusion Alpelisib demonstrated a tolerable safety profile and encouraging preliminary activity in patients with PIK3CA-altered solid tumors, supporting the rationale for selective PI3Kα inhibition in combination with other agents for the treatment of PIK3CA-mutant tumors.

Purification and biochemical properties of Rac1, 2, 3 and the splice variant Rac1b.

Rac proteins (Rac1, 1b, 2, 3) belong to the GTP-binding proteins (or GTPases) of the Ras superfamily and thus act as molecular switches cycling between an active GTP-bound and an inactive GDP-bound form through nucleotide exchange and hydrolysis. Like most other GTPases, these proteins adopt different conformations depending on the bound nucleotide, the main differences lying in the conformation of two short and flexible loop structures designated as the switch I and switch II region. The three distinct mammalian Rac isoforms, Rac1, 2 and 3, share a very high sequence identity (up to 90%), with Rac1b being an alternative splice variant of Rac1 with a 19 amino acid insertion in vicinity to the switch II region. We have demonstrated that Rac1 and Rac3 are very closely related with respect to their biochemical properties, such as effector interaction, nucleotide binding, and hydrolysis. In contrast, Rac2 displays a slower nucleotide association and is more efficiently activated by the Rac-GEF Tiam1. Modeling and normal mode analysis corroborate the hypothesis that the altered molecular dynamics of Rac2, in particular at the switch I region, may be responsible for different biochemical properties. On the other hand, our structural and biochemical analysis of Rac1b has shown that, compared with Rac1, Rac1b has an accelerated GEF-independent GDP/GTP-exchange and an impaired GTP-hydrolysis, accounting for a self-activating GTPase. This chapter discusses the use of fluorescence spectroscopic methods, allowing real-time monitoring of the interaction of nucleotides, regulators, and effectors with the Rac proteins at submicromolar concentrations and quantification of the kinetic and equilibrium constants.

Absorption, distribution, metabolism, and excretion of [(14)C]BYL719 (alpelisib) in healthy male volunteers.

PURPOSE: To determine the pharmacokinetics of the p110α-selective inhibitor alpelisib (BYL719) in humans, to identify metabolites in plasma and excreta, and to characterize pathways of biotransformation. METHODS: Four healthy male volunteers received a single oral dose of [(14)C]-labeled alpelisib (400 mg, 2.78 MBq). Blood, urine, and feces samples were collected throughout the study. Total radioactivity was measured by liquid scintillation counting, and metabolites were quantified and identified by radiometry and mass spectrometry. Complementary in vitro experiments characterized the hydrolytic, oxidative, and conjugative enzymes involved in metabolite formation. RESULTS: Over 50 % of [(14)C] alpelisib was absorbed, with a T(max) of 2 h and an elimination half-life from plasma of 13.7 h. Over the first 12 h, exposure to alpelisib and the primary metabolite M4 was 67.9 and 26.7 % of total drug-related material in circulation, respectively. Mass balance was achieved, with 94.5 % of administered radioactivity recovered in excreta. In total, 38.2 % of alpelisib was excreted unchanged, while 39.5 % was excreted as M4. Based on the excreta pools analyzed, excretion occurred mainly via feces (79.8 % of administered dose); 13.1 % was excreted via urine. In vitro experiments showed that spontaneous and enzymatic hydrolysis contributed to M4 formation, while CYP3A4-mediated oxidation and UGT1A9-mediated glucuronidation formed minor metabolites. Alpelisib was well tolerated, and no new safety concerns were raised during this study. CONCLUSIONS: Alpelisib was rapidly absorbed and cleared by multiple metabolic pathways; the primary metabolite M4 is pharmacologically inactive. Alpelisib has limited potential for drug-drug interactions and is therefore a promising candidate for combination therapy.

Crystal structure of Rnd3/RhoE: functional implications.

The Rnd proteins constitute an exceptional subfamily within the Rho GTPase family. They possess extended chains at both termini and four prominent amino acid deviations causing GTPase deficiency. Herein, we report the crystal structure of the Rnd3/RhoE G-domain (amino acids 19-200) at 2.0 A resolution. This is the first GTP-structure of a Rho family member which reveals a similar fold but striking differences from RhoA concerning (i) GTPase center, (ii) charge distribution at several surface areas, (iii) C3-transferase binding site and (iv) interacting interfaces towards RhoA regulators and effectors.

Comparative functional analysis of the Rac GTPases.

Small GTPases of the Rho family including Rac, Rho and Cdc42 regulate different cellular processes like reorganization of the actin cytoskeleton by acting as molecular switches. The three distinct mammalian Rac proteins share very high sequence identity but how their specificity is achieved is hitherto unknown. Here we show that Rac1 and Rac3 are very closely related concerning their biochemical properties, such as effector interaction, nucleotide binding and hydrolysis. In contrast, Rac2 displays a slower nucleotide association and is more efficiently activated by the Rac-GEF Tiam1. Modeling and normal mode analysis support the idea that altered dynamics of Rac2 at the switch I region may be responsible for different biochemical properties. These results provide insight into the individual functionalities of the Rac isoforms.

BetaQ114N and betaT110V mutations reveal a critically important role of the substrate alpha-carboxylate site in the reaction specificity of tryptophan synthase.

In the PLP-requiring alpha2beta2 tryptophan synthase complex, recognition of the substrate l-Ser at the beta-site includes a loop structure (residues beta110-115) extensively H-bonded to the substrate alpha-carboxylate. To investigate the relationship of this subsite to catalytic function and to the regulation of substrate channeling, two loop mutants were constructed: betaThr110 --> Val, and betaGln114 --> Asn. The betaT110V mutation greatly impairs both catalytic activity in the beta-reaction, and allosteric communication between the alpha- and beta-sites. The crystal structure of the betaT110V mutant shows that the modified l-Ser carboxylate subsite has altered protein interactions that impair beta-site catalysis and the communication of allosteric signals between the alpha- and beta-sites. Purified betaQ114N consists of two species of mutant protein, one with a reddish color (lambdamax = 506 nm). The reddish species is unable to react with l-Ser. The second betaQ114N species displays significant catalytic activities; however, intermediates obtained on reaction with substrate l-Ser and substrate analogues exhibit perturbed UV/vis absorption spectra. Incubation with l-Ser results in the formation of an inactive species during the first 15 min with lambdamax approximately 320 nm, followed by a slower conversion over 24 h to the species with lambdamax = 506 nm. The 320 and 506 nm species originate from conversion of the alpha-aminoacrylate external aldimine to the internal aldimine and alpha-aminoacrylate, followed by the nucleophilic attack of alpha-aminoacrylate on C-4' of the internal aldimine to give a covalent adduct with PLP. Subsequent treatment with sodium hydroxide releases a modified coenzyme consisting of a vinylglyoxylic acid moiety linked through C-4' to the 4-position of the pyridine ring. We conclude that the shortening of the side chain accompanying the replacement of beta114-Gln by Asn relaxes the steric constraints that prevent this reaction in the wild-type enzyme. This study reveals a new layer of structure-function interactions essential for reaction specificity in tryptophan synthase.

Allosteric regulation of tryptophan synthase channeling: the internal aldimine probed by trans-3-indole-3'-acrylate binding.

Substrate channeling in the tryptophan synthase bienzyme complex from Salmonella typhimurium is regulated by allosteric interactions triggered by binding of ligand to the alpha-site and covalent reaction at the beta-site. These interactions switch the enzyme between low-activity forms with open conformations and high-activity forms with closed conformations. Previously, allosteric interactions have been demonstrated between the alpha-site and the external aldimine, alpha-aminoacrylate, and quinonoid forms of the beta-site. Here we employ the chromophoric l-Trp analogue, trans-3-indole-3'-acrylate (IA), and noncleavable alpha-site ligands (ASLs) to probe the allosteric properties of the internal aldimine, E(Ain). The ASLs studied are alpha-d,l-glycerol phosphate (GP) and d-glyceraldehyde 3-phosphate (G3P), and examples of two new classes of high-affinity alpha-site ligands, N-(4'-trifluoromethoxybenzoyl)-2-aminoethyl phosphate (F6) and N-(4'-trifluoromethoxybenzenesulfonyl)-2-aminoethyl phosphate (F9), that were previously shown to bind to the alpha-site by optical spectroscopy and X-ray crystal structures [Ngo, H., Harris, R., Kimmich, N., Casino, P., Niks, D., Blumenstein, L., Barends, T. R., Kulik, V., Weyand, M., Schlichting, I., and Dunn, M. F. (2007) Synthesis and characterization of allosteric probes of substrate channeling in the tryptophan synthase bienzyme complex, Biochemistry 46, 7713-7727]. The binding of IA to the beta-site is stimulated by the binding of GP, G3P, F6, or F9 to the alpha-site. The binding of ASLs was found to increase the affinity of the beta-site of E(Ain) for IA by 4-5-fold, demonstrating for the first time that the beta-subunit of the E(Ain) species undergoes a switching between low- and high-affinity states in response to the binding of ASLs.

Allosteric regulation of substrate channeling in tryptophan synthase: modulation of the L-serine reaction in stage I of the beta-reaction by alpha-site ligands.

In the tryptophan synthase bienzyme complex, indole produced by substrate cleavage at the alpha-site is channeled to the beta-site via a 25 A long tunnel. Within the beta-site, indole and l-Ser react with pyridoxal 5'-phosphate in a two-stage reaction to give l-Trp. In stage I, l-Ser forms an external aldimine, E(Aex1), which converts to the alpha-aminoacrylate aldimine, E(A-A). Formation of E(A-A) at the beta-site activates the alpha-site >30-fold. In stage II, indole reacts with E(A-A) to give l-Trp. The binding of alpha-site ligands (ASLs) exerts strong allosteric effects on the reaction of substrates at the beta-site: the distribution of intermediates formed in stage I is shifted in favor of E(A-A), and the binding of ASLs triggers a conformational change in the beta-site to a state with an increased affinity for l-Ser. Here, we compare the behavior of new ASLs as allosteric effectors of stage I with the behavior of the natural product, d-glyceraldehyde 3-phosphate. Rapid kinetics and kinetic isotope effects show these ASLs bind with affinities ranging from micro- to millimolar, and the rate-determining step for conversion of E(Aex1) to E(A-A) is increased by 8-10-fold. To derive a structure-based mechanism for stage I, X-ray structures of both the E(Aex1) and E(A-A) states complexed with the different ASLs were determined and compared with structures of the ASL complexes with the internal aldimine [Ngo, H., Harris, R., Kimmich, N., Casino, P., Niks, D., Blumenstein, L., Barends, T. R., Kulik, V., Weyand, M., Schlichting, I., and Dunn, M. F. (2007) Biochemistry 46, 7713-7727].

Synthesis and characterization of allosteric probes of substrate channeling in the tryptophan synthase bienzyme complex.

Allosteric interactions regulate substrate channeling in Salmonella typhimurium tryptophan synthase. The channeling of indole between the alpha- and beta-sites via the interconnecting 25 A tunnel is regulated by allosteric signaling arising from binding of ligand to the alpha-site, and covalent reaction of l-Ser at the beta-site. This signaling switches the alpha- and beta-subunits between open conformations of low activity and closed conformations of high activity. Our objective is to synthesize and characterize new classes of alpha-site ligands (ASLs) that mimic the binding of substrates, 3-indole-d-glycerol 3'-phosphate (IGP) or d-glyceraldehyde 3-phosphate (G3P), for use in the investigation of alpha-site-beta-site interactions. The new synthesized IGP analogues contain an aryl group linked to an O-phosphoethanolamine moiety through amide, sulfonamide, or thiourea groups. The G3P analogue, thiophosphoglycolohydroxamate, contains a hydroxamic acid group linked to a thiophosphate moiety. Crystal structures of the internal aldimine complexed with G3P and with three of the new ASLs are presented. These structural and solution studies of the ASL complexes with the internal aldimine form of the enzyme establish the following. (1) ASL binding occurs with high specificity and relatively high affinities at the alpha-site. (2) Binding of the new ASLs slows the entry of indole analogues into the beta-site by blocking the tunnel opening at the alpha-site. (3) ASL binding stabilizes the closed conformations of the beta-subunit for the alpha-aminoacrylate and quinonoid forms of the enzyme. (4) The new ASLs exhibit allosteric properties that parallel the behaviors of IGP and G3P.

Models of the cooperative mechanism for Rho effector recognition: implications for RhoA-mediated effector activation.

Activated GTPases of the Rho family regulate a spectrum of functionally diverse downstream effectors, initiating a network of signal transduction pathways by interaction and activation of effector proteins. Although effectors are defined as proteins that selectively bind the GTP-bound state of the small GTPases, there have been also several indications for a nucleotide-independent binding mode. By characterizing the molecular mechanism of RhoA interaction with its effectors, we have determined the equilibrium dissociation constants of several Rho-binding domains of three different effector proteins (Rhotekin, ROCKI/ROK beta/p160ROCK, PRK1/PKNalpha where ROK is RhoA-binding kinase) for both RhoA.GDP and RhoA.GTP using fluorescence spectroscopy. In addition, we have identified two novel Rho-interacting domains in ROCKI, which bind RhoA with high affinity but not Cdc42 or Rac1. Our results, together with recent structural data, support the notion of multiple effector-binding sites in RhoA and strongly indicate a cooperative binding mechanism for PRK1 and ROCKI that may be the molecular basis of Rho-mediated effector activation.

Inactivation and activation of Ras by the neurotrophin receptor p75.

The neurotrophin receptor p75 induces neurotrophic and/or apoptotic signalling pathways and can also cooperate with the neurotrophic Trk receptor tyrosine kinases. Its intracellular part encloses a so-called 'death domain' with a segment similar to the wasp venom mastoparan which binds small GTPases such as Rho. To study possible interactions of p75 and Ras (and Rho) we used wild-type and mutant genes of p75 stably expressed by MDCK cells which normally have neither Trk nor p75. We found that p75 can directly bind the GTPases Ras and Rho and that the unstimulated p75 inactivates total cellular Ras through a differential influence on the dissociation of GDP and GTP from Ras and an exchange of bound Ras.GDP for free Ras.GTP. These properties of p75 could also be demonstrated in vitro and should therefore be cell type-independent. Stimulation of p75 with nerve growth factor causes Ras activation via adapter proteins known from Trk signalling and induces rapid outgrowth of cellular processes. Both inactivation and activation of Ras by p75 are controlled by the phosphorylation state of the receptor's two intracellular tyrosines. p75 also influences Rho activation and inactivation, and the combined interactions of the receptor with the two GTPases Ras and Rho can regulate neurite formation in an efficient, synergistic way.