The discovery and maturation of peptide biologics targeting the small G-protein Cdc42: A bioblockade for Ras-driven signaling.

Aberrant Ras signaling drives 30% of cancers, and inhibition of the Rho family small GTPase signaling has been shown to combat Ras-driven cancers. Here, we present the discovery of a 16-mer cyclic peptide that binds to Cdc42 with nanomolar affinity. Affinity maturation of this sequence has produced a panel of derived candidates with increased affinity and modulated specificity for other closely-related small GTPases. The structure of the tightest binding peptide was solved by NMR, and its binding site on Cdc42 was determined. Addition of a cell-penetrating sequence allowed the peptides to access the cell interior and engage with their target(s), modulating signaling pathways. In Ras-driven cancer cell models, the peptides have an inhibitory effect on proliferation and show suppression of both invasion and motility. As such, they represent promising candidates for Rho-family small GTPase inhibitors and therapeutics targeting Ras-driven cancers. Our data add to the growing literature demonstrating that peptides are establishing their place in the biologics arm of drug discovery.

Bond swapping from a charge cloud allows flexible coordination of upstream signals through WASP: Multiple regulatory roles for the WASP basic region.

Wiskott-Aldrich syndrome protein (WASP) activates the actin-related protein 2/3 homolog (Arp2/3) complex and regulates actin polymerization in a physiological setting. Cell division cycle 42 (Cdc42) is a key activator of WASP, which binds Cdc42 through a Cdc42/Rac-interactive binding (CRIB)-containing region that defines a subset of Cdc42 effectors. Here, using site-directed mutagenesis and binding affinity determination and kinetic assays, we report the results of an investigation into the energetic contributions of individual WASP residues to both the Cdc42-WASP binding interface and the kinetics of complex formation. Our results support the previously proposed dock-and-coalesce binding mechanism, initiated by electrostatic steering driven by WASP's basic region and followed by a coalescence phase likely driven by the conserved CRIB motif. The WASP basic region, however, appears also to play a role in the final complex, as its mutation affected both on- and off-rates, suggesting a more comprehensive physiological role for this region centered on the C-terminal triad of positive residues. These results highlight the expanding roles of the basic region in WASP and other CRIB-containing effector proteins in regulating complex cellular processes and coordinating multiple input signals. The data presented improve our understanding of the Cdc42-WASP interface and also add to the body of information available for Cdc42-effector complex formation, therapeutic targeting of which has promise for Ras-driven cancers. Our findings suggest that combining high-affinity peptide-binding sequences with short electrostatic steering sequences could increase the efficacy of peptidomimetic candidates designed to interfere with Cdc42 signaling in cancer.

A dock and coalesce mechanism driven by hydrophobic interactions governs Cdc42 binding with its effector protein ACK.

Cdc42 is a Rho-family small G protein that has been widely studied for its role in controlling the actin cytoskeleton and plays a part in several potentially oncogenic signaling networks. Similar to most other small G proteins, Cdc42 binds to many downstream effector proteins to elicit its cellular effects. These effector proteins all engage the same face of Cdc42, the conformation of which is governed by the activation state of the G protein. Previously, the importance of individual residues in conferring binding affinity has been explored for residues within Cdc42 for three of its Cdc42/Rac interactive binding (CRIB) effectors, activated Cdc42 kinase (ACK), p21-activated kinase (PAK), and Wiskott-Aldrich syndrome protein (WASP). Here, in a complementary study, we have used our structure of Cdc42 bound to ACK via an intrinsically disordered ACK region to guide an analysis of the Cdc42 interface on ACK, creating a panel of mutant proteins with which we can now describe the complete energetic landscape of the Cdc42-binding site on ACK. Our data suggest that the binding affinity of ACK relies on several conserved residues that are critical for stabilizing the quaternary structure. These residues are centered on the CRIB region, with the complete binding region anchored at each end by hydrophobic interactions. These findings suggest that ACK adopts a dock and coalesce binding mechanism with Cdc42. In contrast to other CRIB-family effectors and indeed other intrinsically disordered proteins, hydrophobic residues likely drive Cdc42-ACK binding.

Hereditary thrombotic thrombocytopenic purpura and the hereditary TTP registry.

Hereditary thrombotic thrombocytopenic purpura, Upshaw-Schulman syndrome, ADAMTS13 Hereditary thrombotic thrombocytopenic purpura (TTP), also known as Upshaw-Schulman syndrome, is a rare recessively inherited disease. Underlying is a severe constitutional deficiency of the von Willebrand factor-cleaving protease, ADAMTS13, due to compound heterozygous or homozygous mutations in the ADAMTS13 gene. The clinical picture is variable and more and more patients with an adult-onset are diagnosed. In the majority of countries the only available treatment is plasma, which when administered regularly can efficiently prevent acute disease bouts. The decision to initiate regular prophylaxis is often not easy, as evidence based guidelines and long term outcome data are lacking. Through the hereditary TTP registry (www.ttpregistry.net, ClinicalTrials.gov identifier: NCT01257269), which was initiated in 2006 and is open to all patients diagnosed with Upshaw-Schulman syndrome and their family members, we aim to gain further information and insights into this rare disease, which eventually will help to improve clinical management of affected patients.

The Oklahoma Thrombotic Thrombocytopenic Purpura-haemolytic Uraemic Syndrome Registry. A model for clinical research, education and patient care.

The Oklahoma Thrombotic Thrombocytopenic Purpura-Haemolytic Uraemic Syndrome (TTP-HUS) Registry has a 24 year record of success for collaborative clinical research, education, and patient care. This article tells the story of how the Registry began and it describes the Registry's structure and function. The Registry provides a model for using a cohort of consecutive patients to investigate a rare disorder. Collaboration between Oklahoma, United States and Bern, Switzerland has been the basis for successful interpretation of Registry data. Registry data have provided new insights into the evaluation and management of TTP. Because recovery from acute episodes of TTP has been assumed to be complete, the increased prevalence of hypertension, diabetes, depression, and death documented by long-term follow-up was unexpected. Registry data have provided opportunities for projects for students and trainees, education of physicians and nurses, and also for patients themselves. During our follow-up, patients have also educated Registry investigators about problems that persist after recovery from an acute episode of TTP. Most important, Registry data have resulted in important improvements for patient care.

Evaluation of bleeding and thrombotic events during long-term use of romiplostim in patients with chronic immune thrombocytopenia (ITP).

BACKGROUND: Romiplostim is a peptibody protein that raises platelet counts during long-term treatment of patients with chronic immune thrombocytopenia (ITP). Clinical outcomes related to increased platelet counts include a reduced risk of bleeding and a potential risk of thrombosis. OBJECTIVE: To evaluate bleeding and thrombotic events occurring in chronic ITP patients during two phase 3, randomized, placebo-controlled, 24-week studies of romiplostim and during subsequent treatment in an open-label extension study. PATIENTS/METHODS: In the phase 3 trials, 125 patients were randomized to romiplostim or placebo; romiplostim dose was adjusted to maintain platelet counts of 50-200 x 10(9) L(-1). Patients who completed the phase 3 trials could enroll in the extension study in which all patients received romiplostim. RESULTS: In the phase 3 trials, a significantly greater percentage of patients treated with placebo (34%) had bleeding adverse events of moderate or greater severity than did patients treated with romiplostim (15%, P = 0.018). In the extension study, the incidence of bleeding adverse events of moderate or greater severity decreased from 23% of patients in the first 24 weeks to 12% after 24-48 weeks, remaining < or = 6% thereafter. The exposure-adjusted incidence of thrombotic events was 0.1 per 100 patient-weeks in the phase 3 studies, and 0.08 per 100 patient-weeks in the extension study where patients received romiplostim for up to 144 additional weeks. CONCLUSIONS: The incidence and severity of bleeding was decreased in chronic ITP patients treated with romiplostim compared with placebo, and the incidence of thrombotic events was not different between the two groups.

Management of patients with refractory immune thrombocytopenic purpura.

In immune thrombocytopenic purpura (ITP), thrombocytopenia is a result of both increased platelet destruction and insufficient platelet production. In adults, the course is commonly chronic, but most patients never experience serious bleeding even with severe thrombocytopenia. In case series of consecutive adult patients identified at the time of diagnosis, the frequency of death from bleeding is low, < 1%. The goal of treatment is only to prevent bleeding, not to correct the platelet count to normal. All current treatments are designed to diminish the increased platelet destruction, either by immunosuppression or splenectomy. The frequency of death from complications of treatment is similar to the frequency of death from bleeding. Perhaps because of increasing recognition of both the infrequent occurrence of serious bleeding and the risks of immunosuppressive treatment and splenectomy, data from case series across the past 30 years suggest a trend toward less therapy and fewer splenectomies among patients with ITP. However treatment is necessary for patients with severe and symptomatic thrombocytopenia. Splenectomy remains the most effective treatment for ITP, with two-thirds of patients achieving durable complete remissions. Immunosuppressive agents, including rituximab and combinations of agents, may be less effective than splenectomy in achieving complete remissions and the remissions may also be less durable. New agents for patients with ITP are currently in development that enhance platelet production, rather than diminish platelet destruction. In preliminary reports, these agents have been effective in maintaining safe platelet counts in patients with chronic ITP that was refractory to splenectomy and other treatments.

The incidence of thrombotic thrombocytopenic purpura-hemolytic uremic syndrome: all patients, idiopathic patients, and patients with severe ADAMTS-13 deficiency.

BACKGROUND: Accurate estimates of the incidence of thrombotic thrombocytopenic purpura (TTP) are important to assess the resources required for current treatments as well as to anticipate the need to develop new treatments. Previous estimates have been indirect and have not reported data on patients with ADAMTS-13 deficiency. OBJECTIVE: To determine the incidence of patients with TTP-hemolytic uremic syndrome (HUS) in three categories: all patients with clinically suspected TTP-HUS, patients with idiopathic TTP-HUS, and patients with severe ADAMTS-13 deficiency. METHODS: Incidence rates were estimated from the Oklahoma TTP-HUS Registry, analyzing all 206 consecutive patients from January 1, 1996 to June 30, 2004 who were treated with plasma exchange for their initial episode of clinically suspected TTP-HUS. ADAMTS-13 activity was measured in 186 (90%) of the 206 patients. RESULTS: The age-sex-race standardized annual incidence rates were 11.29 x 10(6) (95% CI: 9.70-12.88) for all patients with clinically suspected TTP-HUS; 4.46 x 10(6) (95% CI: 3.43-5.50) for patients with idiopathic TTP-HUS; and 1.74 x 10(6) (95% CI: 1.06-2.41) for patients with severe ADAMTS-13 deficiency (<5% activity). In all three categories, the incidence rates were greater for women and for blacks. For patients with severe ADAMTS-13 deficiency, the age-sex standardized incidence rate ratio of blacks to non-blacks was 9.29 (95% CI: 4.33-19.93). CONCLUSIONS: Accurate incidence rate estimates for all patients with clinically suspected TTP-HUS, idiopathic TTP-HUS, and TTP associated with severe ADAMTS-13 deficiency have been determined. The greater incidence among women and blacks is comparable with their increased risk for other autoimmune disorders.

Initial management of adults with idiopathic (immune) thrombocytopenic purpura.

Since idiopathic (immune) thrombocytopenic purpura (ITP) in adults is usually a chronic condition with few spontaneous remissions, the goal of treatment is not cure, but to maintain a hemostatically safe platelet level. The indication for treatment should be based not merely on platelet counts, but also clinical indices of bleeding. Although most patients show good initial response to prednisone, the side effects of steroids limit this treatment. Currently, long-term management usually involves splenectomy. Since splenectomy has surgical risks and may also predispose the patient to sepsis, a clinical trial using anti-D (WinRho-SDR) has been performed to determine whether this treatment can safely delay or avoid the need for surgery. The use of WinRho may also reveal the occurrence of spontaneous remissions, a previously unrecognized subgroup of adults with chronic ITP.

Unintentional platelet removal by plasmapheresis.

Therapeutic plasmapheresis may remove platelets as well as plasma. Unintentional platelet loss, if not recognized, may lead to inappropriate patient assessment and treatment. A patient with thrombotic thrombocytopenic purpura-hemolytic uremic syndrome (TTP-HUS) is reported in whom persistent thrombocytopenia was interpreted as continuing active disease; thrombocytopenia resolved only after plasma exchange treatments were stopped. This observation prompted a systematic study of platelet loss with plasmapheresis. Data are reported on platelet loss during 432 apheresis procedures in 71 patients with six disease categories using three different instruments. Comparing the first procedure recorded for each patient, there was a significant difference among instrument types (P<0.001); platelet loss was greater with the Fresenius AS 104 (17.5%, N = 21) than with the COBE Spectra (1.6%, N = 26) or the Haemonetics LN9000 (2.6%, N = 24). With all procedures, platelet loss ranged from 0 to 71%. Among disease categories, platelet loss was greater in patients with dysproteinemias who were treated for hyperviscosity symptoms. Absolute platelet loss with the first recorded apheresis procedure, in the 34 patients who had a normal platelet count before the procedure, was also greater with the AS 104 (2.23 x 10(11) platelets) than with the Spectra (0.29 x 10(11) platelets) or the LN9000 (0.37 x 10(11) platelets). In 39 patients in whom data were collected on consecutive days, platelet removal by plasmapheresis correlated with a decreased patient platelet count (r = 0.40, P = 0.011). In these 39 patients, the platelet counts were significantly decreased at 24 hours (P = 0.002).

Quinine-associated thrombotic thrombocytopenic purpura-hemolytic uremic syndrome: frequency, clinical features, and long-term outcomes.

BACKGROUND: Quinine-associated thrombotic thrombocytopenic purpura-hemolytic uremic syndrome (TTP-HUS) is thought to be uncommon and to have a good prognosis. OBJECTIVE: To describe the frequency, clinical features, and long-term outcomes of quinine-associated TTP-HUS. DESIGN: Case series. SETTING: Hospitals in central-western Oklahoma. PATIENTS: 225 consecutive patients with TTP-HUS, 1989-2000. MEASUREMENTS: Presenting features and clinical outcomes. RESULTS: Thrombotic thrombocytopenic purpura-hemolytic uremic syndrome was associated with quinine in 17 patients. Four patients died, and 7 survivors currently have chronic renal failure. Since 1 July 1995, 132 patients with clinically suspected TTP-HUS were explicitly asked about drug exposure. Fourteen (11%) had taken quinine, and 7 had taken other drugs associated with TTP-HUS. Neurologic abnormalities were as severe in patients with quinine-associated TTP-HUS as in the 118 patients who had not taken quinine. CONCLUSIONS: Quinine is a common cause of drug-associated TTP-HUS and can cause death and chronic renal failure. When the disorder is described as TTP-HUS rather than only as HUS, the severity of neurologic abnormalities and the occasional absence of renal failure are emphasized. If recurrent disease is to be prevented, clinicians must recognize quinine as a possible cause.

Thrombotic thrombocytopenic purpura-hemolytic uremic syndrome: diagnosis and treatment.

Prompt recognition of thrombotic thrombocytopenic purpura-hemolytic uremic syndrome (TTP-HUS) and initiation of plasma exchange treatment is critical as it substantially decreases mortality. Nevertheless, death and long-term complications remain common. The recent relaxation of diagnostic criteria has dramatically increased the number of patients treated for clinically suspected TTP-HUS.

Drug-associated thrombotic thrombocytopenic purpura-hemolytic uremic syndrome.

Thrombotic thrombocytopenic purpura-hemolytic uremic syndrome (TTP-HUS) is an inclusive term describing diverse syndromes of multiple etiologies with the common features of thrombocytopenia and microangiopathic hemolytic anemia. Other organ involvement, including renal failure, neurologic abnormalities, and gastrointestinal symptoms, is common. Adverse reactions to drugs increasingly are reported as a potential cause of TTP-HUS. More than 50 drugs and other substances have been associated with the development of TTP-HUS, but many case reports are difficult to interpret because there is uncertainty regarding the diagnosis of TTP-HUS and because there is uncertainty regarding the relation of drug exposure to the onset of TTP-HUS. A systematic analysis of reports of drug-associated TTP-HUS will be required to better understand the strength of clinical evidence linking drugs to the etiology of TTP-HUS. In this review, five drugs that have been the subject of the most and the most recent reports of drug-associated TTP-HUS are discussed: mitomycin C, cyclosporine, quinine, ticlopidine, and clopidogrel. The clinical features of TTP-HUS associated with these drugs are different, suggesting two principal mechanisms by which drugs may cause TTP-HUS: dose-related toxicity (mitomycin C, cyclosporine), and immune-mediated reaction (quinine, ticlopidine, clopidogrel). The role of plasma exchange is uncertain, but this treatment is appropriate because of the high mortality and morbidity of drug-associated TTP-HUS. Recognition of a drug-associated etiology in a patient with TTP-HUS is critical to avoid re-exposure and recurrent illness.

Thrombotic thrombocytopenic purpura-like syndromes following bone marrow transplantation: an analysis of associated conditions and clinical outcomes.

The diagnosis and treatment of thrombotic thrombocytopenic purpura (TTP) in patients following BMT are often uncertain and unsuccessful. To better understand the evaluation and management of these patients, we describe 17 patients treated with plasma exchange for a presumptive diagnosis of TTP following BMT during a 10 year period, 1989-1998. Because of the uncertainty of the diagnosis, these patients are described as having a 'TTP-like syndrome'. All 17 patients had received an allogeneic BMT. Comparison with the other 245 patients who had an allogeneic BMT during the same period demonstrated that patients with a TTP-like syndrome more frequently had unrelated and/or HLA-mismatched donors, and had also experienced more serious complications: grade III-IV acute GVHD and systemic bacterial, fungal, and viral infections. Three months after the diagnosis of the TTP-like syndrome, only four of 17 patients (24%) were alive; currently only one patient survives. These data emphasize: (1) the diagnosis of TTP following BMT is uncertain because of the presence of multiple BMT-associated complications. (2) The outcome of patients with TTP-like syndromes following BMT is poor. (3) Urgent intervention with plasma exchange when TTP is suspected following BMT may not always be appropriate. Alternative explanations for the signs and symptoms should be considered and treated aggressively.

Cryoprecipitate poor plasma does not improve early response in primary adult thrombotic thrombocytopenic purpura (TTP).

Thrombotic thrombocytopenic purpura (TTP) is a potentially fatal disease that is treated with plasma exchange and typically with replacement with fresh frozen plasma (FFP). This approach results in an approximate 50% response rate following 1 week of therapy and 80% survival. Cryoprecipitate poor plasma (CPP) is plasma from which the cryoprecipitate fraction is removed. CPP has been reported to be successful as salvage therapy in refractory TTP and has been suggested to be superior to FFP in retrospective studies. The present report compares initial therapy of TTP with exchange using replacement with either FFP or CPP in a multi-institutional prospective randomized study performed by the North American TTP Group (NATG Group) from 1993 to 1995. Initial therapy also included corticosteroids. Antiplatelet drugs or vinca alkaloids were not employed. A severity score index, response score, and individual clinical parameters (platelet count, LDH x upper limit of normal, hemoglobin level, and creatinine) were compared at their nadir or peak values, baseline, and days +6 and +13 of therapy. Thirteen patients were randomized to FFP exchange and 14 to CPP exchange. Results were equivalent for all parameters. Survival was equal with three deaths in each group. These data indicate that the efficacy of FFP and CPP are the same in the initial treatment of TTP in adults.