Antibodies have proved to be a valuable mode of therapy for numerous diseases, mainly owing to their high target binding affinity and specificity. Unfortunately, antibodies are also limited in several respects, chief amongst those being the extremely high cost of manufacture. Therefore, non-antibody binding proteins have long been sought after as alternative therapies. New binding protein scaffolds are constantly being designed or discovered with some already approved for human use by the FDA. This review focuses on protein scaffolds that are either already being used in humans or are currently being evaluated in clinical trials. Although not all are expected to be approved, the significant benefits ensure that these molecules will continue to be investigated and developed as therapeutic alternatives to antibodies. Based on the location of the amino acids that mediate ligand binding, we place all the protein scaffolds under clinical development into two general categories: scaffolds with ligand-binding residues located in exposed flexible loops, and those with the binding residues located in protein secondary structures, such as α-helices. Scaffolds that fall under the first category include adnectins, anticalins, avimers, Fynomers, Kunitz domains, and knottins, while those belonging to the second category include affibodies, β-hairpin mimetics, and designed ankyrin repeat proteins (DARPins). Most of these scaffolds are thermostable and can be easily produced in microorganisms or completely synthesized chemically. In addition, many of these scaffolds derive from human proteins and thus possess very low immunogenic potential. Additional advantages and limitations of these protein scaffolds as therapeutics compared to antibodies will be discussed.
Amino Acids ; metabolism ; Animals ; Antibodies ; therapeutic use ; Humans ; Ligands ; Protein Engineering ; methods ; Protein Structure, Secondary ; Recombinant Proteins ; chemistry ; therapeutic use

Objective: To investigate the clinical significance of endothelin A receptor (ETAR) expression in high-grade serous ovarian carcinoma (HGSOC). To design ETAR carboxyl terminal (ETAR-C) amino acids derived polypeptide and to study the inhibitory effect on ovarian epithelial carcinoma cells in vitro. Methods: (1) A total of 126 patients who received surgical treatment and were diagnosed with HGSOC by postoperative pathological examination in Central Hospital of Xuzhou from January 1, 2007 to December 31, 2017 were selected. All patients had completed clinicopathological data and follow-up data. Cancer tissue samples were collected and ETAR mRNA expression in HGSOC tissues was detected by reverse transcript-PCR. The clinical significance was analyzed. (2) ETAR-C fusion polypeptide was designed based on the sequence of carboxyl terminal amino acids of ETAR, expressed and purified in vitro. The effects of ETAR-C fusion polypeptide on migration and invasion ability of ovarian cancer SKOV3 and CAOV3 cells were detected by scratch test and invasion test, respectively. The effect of ETAR-C fusion polypeptide on chemosensitivity of cisplatin-resistant ovarian cancer SKOV3/cDDP and CAOV3/cDDP cells was determined by methyl thiazolyl tetrazolium (MTT) colorimetric assay. The effect of ETAR-C fusion polypeptide on β-arrestin-1 expression in ovarian cancer SKOV3 and CAOV3 cells was detected by western blot. Results: (1) The relative expression level of ETAR mRNA in HGSOC tissues was 18.6±5.1. Patients with HGSOC were divided into high ETAR mRNA expression (n=76) and low ETAR mRNA expression (n=50) with 61.7% as cut-off value analyzed by X-Tile software. High expression of ETAR mRNA was significantly correlated with abdominal water volume, platinum drug resistance, and cancer antigen 125 (CA₁₂₅) value in HGSOC patients (all P<0.05), but was not related to the age of patients with HGSOC and the size of postoperative residual lesions (all P>0.05). The 5-year progression free survival rates were 18.4% and 28.0%, and the 5-year overall survival rates were 38.2% and 52.0% in HGSOC patients with high and low ETAR mRNA expression respectively, there were statistically significant differences (P=0.046, P=0.034). (2) The results of scratch test and invasion test showed that the scratch healing rate and cell invasion rate of SKOV3 or CAOV3 cells treated with endothelin-1 (ET-1) and ET-1+ETAR-C were respectively compared, and the differences were statistically significant (all P<0.05). MTT assay showed that the inhibition rates of ETAR-C fusion polypeptide treated in SKOV3/cDDP and CAOV3/cDDP cells were significantly higher than those of control cells after the addition of 4, 6, 8, 10, 12, and 24 μg/ml cisplatin (all P<0.05). Western blot analysis showed that the relative expression levels of β-arrestin-1 in SKOV3 or CAOV3 cells treated with ET-1 and ET-1+ETAR-C were 1.85±0.09 and 1.13±0.09 (SKOV3 cells), 2.14±0.15 and 1.66±0.12 (CAOV3 cells), respectively. The differences were statistically significant (all P<0.05). Conclusions: The prognosis of HGSOC patients with high expression of ETAR mRNA is significantly worse than those with low expression of ETAR mRNA. ETAR might be a new target for HGSOC treatment. The ETAR-C fusion polypeptide that interferes with the interaction of ETAR and β-arrestin-1 has good inhibitory effect on ovarian cancer cells in vitro, and might have clinical application potential.
Female ; Humans ; Amino Acids/therapeutic use* ; beta-Arrestins/therapeutic use* ; Cell Line, Tumor ; Cisplatin/pharmacology* ; Clinical Relevance ; Ovarian Neoplasms/pathology* ; Receptor, Endothelin A/therapeutic use* ; RNA, Messenger/metabolism*

OBJECTIVETo evaluate the effect of compound branch chain amino acid(BCAA) injection on nutritional support in patients after radical resection for colorectal cancer.

METHODSFifty patients with radical resection for colorectal cancer were randomly divided into two groups, and received compound branch chain amino acid (n=25) or compound amino acid(n=25) as control per day from postoperative day(POD)1 to POD 7. The levels of total protein, albumin, pre-albumin, transferring, nitrogen balance and complications were compared between the two groups.

RESULTSOn POD 8, the levels of albumin, pre- albumin, transferring were (36.12+/-3.16)g/L, (237.10+/-37.29)mg/L, and (2.18+/- 1.34)g/L in study group, and (30.61+/-3.55)g/L, (191.73+/-27.60)mg/L, and (1.71+/-0.84)g/L respectively in the control group(all P< 0.05). Nitrogen balance increased significantly from POD5, and increased to normal on POD 6 in study group, significantly higher than that in the control group (9.91+/-6.53 vs - 9.73+/-11.21, P=0.024). The complication rate of incision infection and delayed healing was 8.3% in study group, significantly lower than 38.1% in the control group(P< 0.05).

CONCLUSIONCompared with compound amino acid, compound branch chain amino acid injection can reduce proteolysis, correct negative nitrogen balance and promote wound healing.

Amino Acids ; therapeutic use ; Colorectal Neoplasms ; therapy ; Female ; Humans ; Male ; Middle Aged ; Nutritional Support ; methods ; Postoperative Care

OBJECTIVETo explore the influence of L-arginine supplementation on the plasma amino acid spectrum in burn patients.

METHODSTen burn patients were randomly divided into burn control (n = 5, with compound 14 amino acid injection accounting for 2% of the total caloric value), and experimental (n = 5, with intravenous injection of L-arginine which accounted for 2% of total caloric value) groups. The intake of other nutrients for these two groups of patients was the same. The nutrient regimen was begun on the 3 PBD, with one quarter of the daily supply. On 4 and 5 PBD, one half of the daily supply was given, and from 6 to 21 PBD full supplementation was given. Venous blood samples were collected on 3, 7, 14, 21 and 28 PBD for the determination of plasma levels of amino acids. Ten normal volunteers served as normal control.

RESULTSThe plasma level of citrulline in both groups was significantly lower than normal value (P < 0.05) on 3 PBD before L-arginine supplementation. There was no obvious difference in plasma levels of ornithine and arginine in the two groups on 3 PBD compared with normal value (P > 0.05). The plasma level of ornithine, citrulline and arginine in burn control group declined on 3 PBD. The plasma level of arginine in experimental group on 14, 21 and 28 PBD were 280 +/- 121 micromol/L, 223 +/- 106 micromol/L and 110 +/- 44 micromol/L, respectively, which were significantly higher than those in burn control group (124 +/- 21 micromol/L, 59 +/- 15 micromol/L, 50 +/- 26 micromol/L). The plasma level of ornithine (30 +/- 5 micromol/L) and citrulline (162 +/- 44 micromol/L) on 21 PBD in experimental group were markedly higher than those in burn control group (8 +/- 7 micromol/L, 66 +/- 4 micromol/L, P < 0.05 or 0.01). There was no difference in the plasma levels of other amino acids at all postburn time points between the two groups (P > 0.05).

CONCLUSIONThe production process of L-arginine from citrulline was accelerated after burns. The plasma levels of L-arginine, ornithine and citrulline were increased markedly after L-arginine supplementation, while that of other amino acids was not influenced. The pharmacological effects of L-arginine may be related to the promotion of ornithine cycle.

Adolescent ; Adult ; Amino Acids ; blood ; Arginine ; therapeutic use ; Burns ; blood ; drug therapy ; Female ; Humans ; Male ; Parenteral Nutrition ; Wound Healing

OBJECTIVETo observe the changes of urinary deoxypyridinoline crosslink/creatinine (UDpd/Cr) in rats after OVX and intervention by estrogen and bisphosphonate and investigate the possible application of deoxypyridinoline in osteoporosis diagnosis and treatment.

METHODS40 female 6-month-old virginal Wistar rats were divided into 5 groups, ovariectomized or sham ovariectomized. (1) Ovxb (n = 8): sacrificed at 6 weeks after OVX; (2) Sham (n = 8): sham ovariectomized; (3) Ovxe (n = 8): sacrificed at 14 weeks after OVX; (4) O + E (n = 9):OVX + 17 beta estradiol [20 micrograms/(kg.d) ih]; (5) O + C (n = 7):OVX + cimadronate [0.2 mg/(kg.d)]; Treatment started 6 weeks after OVX and lasted 8 weeks. Rats in group 2-5 were sacrificed at 14 weeks after OVX. Urinary and serum biochemical parameters were measured, pQCT scanning of femur, bone biomechanical test in femur were determined.

RESULTSOVX resulted in increasing of UDpd/Cr 133.3% (P < 0.01). The ratio of UCa/Cr also increased in OVX groups but without any significant compared with Sham (P > 0.05). UDpd/Cr were reduced by 54.6% and 51.8% (P < 0.01) in O + E, O + C group respectively compared with Ovxe. The significant negative correlationships were found between UDpd/Cr and bone mass, BMD and biomechanic characteristics.

CONCLUSIONSUDpd/Cr ratio is a sensitive bone resorption marker, a marked changes were observed when the rats ovariectomized or treated with estradiol and cimadronate. There were best correlation between UDpd/Cr and bone mineral density and bone biomechanic characteristics. It is fair to apply UDpd/Cr ratio for osteoporosis diagnosis and treatment.

Amino Acids ; urine ; Animals ; Bone Density ; Creatinine ; urine ; Diphosphonates ; therapeutic use ; Estradiol ; therapeutic use ; Female ; Osteoporosis ; drug therapy ; urine ; Ovariectomy ; Rats ; Rats, Wistar

Amino Acids ; blood ; Amino Acids, Branched-Chain ; blood ; Capsules ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Hepatitis B, Chronic ; blood ; drug therapy ; Humans ; Liver Cirrhosis ; blood ; drug therapy ; Male ; Phytotherapy

OBJECTIVETo observe the effects of polydatin on dynamic changes of excitatory amino acids in cerebrospinal fluid and water content of brain tissue of cerebral hemorrhage rats. And to discuss the therapeutic action and mechanisms of polydatin on brain hemorrhagic injured rats.

METHODA quantitative determination method of Asp and Glu was established by microdialysis-HPLC. The cerebral hemorrhage model in rats was induced by local injection of type VII collagenase. The dynamic changes of Asp and Glu in cerebrospinal fluid were observed on 0, 6, 12, 24, 36, 48, 60, 72, 84, 96, 108 h of cerebral hemorrhage rats, and then the water content of brain tissue was detected.

RESULTThe content of Asp and Glu increased rapidly within 24 h after cerebral hemorrhage, and to the highest in 24 h, then decreased gradually. Compared with the cerebral hemorrhage model group, the content of Asp and Glu increased slowly in polydatin group, and there were significant differences in 12-72 h and 6-84 h (P < 0.01, P < 0.05), but there was no significant difference after 84 h and 96 h. Compared with sham group, water content of brain tissue significantly higher in model group, while significantly lower (P < 0.01) in polydatin group.

CONCLUSIONPolydatin can inhibit increasing content of Asp and Glu in cerebrospinal fluid dynamics, and significantly inhibit cerebral edema of cerebral hemorrhage rats. It shows that the mechanisms of anti-cerebral hemorrhage injury of polydatin may be related to increasing of excitatory amino acids after cerebral hemorrhage.

Animals ; Aspartic Acid ; cerebrospinal fluid ; Cerebral Hemorrhage ; cerebrospinal fluid ; drug therapy ; Disease Models, Animal ; Drugs, Chinese Herbal ; therapeutic use ; Excitatory Amino Acids ; cerebrospinal fluid ; Glucosides ; therapeutic use ; Glutamic Acid ; cerebrospinal fluid ; Humans ; Male ; Rats ; Rats, Sprague-Dawley ; Stilbenes ; therapeutic use

OBJECTIVESTo observe the effect of recombinant human growth hormone on metabolism in severely burned patients.

METHODSFrom January 1999 to January 2001, 50 patients, aged 12 to 50 years, with over 30% total body surface area (TBSA) and 10% full-thickness burns, were randomized in a double-blind study. In the control group normal saline was used as a placebo (control group), while 0.3 IU/kg(-1) /d(-1) recombinant human growth hormone was given from postoperative day 1 to day 10 in the rhGH group. The excised burn wounds were closed with microautograft and allograft skin. Blood samples were collected at 6:00 am for assaying of growth hormone, blood glucose, blood insulin, anti-insulin antibody, glucagon, cortisol, serum amino acid profile, transferring, proalbumin, total protein, dielectric, and resting energy expenditure (REE) was also measured.

RESULTSThe concentration of blood GH in both groups was lower (t = 2.806, P < 0.05) than that of physiological values before surgery. However, the concentration of GH on POD 3 in the rhGH group was significantly higher than that of normal values, but a higher level was observed on POD 7 in the rhGH group than that of the control group (t = 3.142, P < 0.05). Although the concentration of anti-insulin antibody was slightly increased, there was no significant difference between the two groups. The concentration of glucagons tended to decrease with an increase in the concentration of blood glucose, and it was marked in the rhGH group. There was no significant difference between the two groups. The concentration of cortisol was higher than normal values, but no significant difference was observed between the two groups. With the administration of rhGH, the plasma concentration of amino acids was lower than that of the control group (t = 2.714, P < 0.05), and the urinary output of 3-MH in the rhGH group was lower than that of the control group (t = 2.207, P < 0.05).

CONCLUSIONSAdministration of rhGH in patients with major burn after surgery could improve their metabolic status, namely, increased lipolysis energy, accelerated protein synthesis, accelerated gluconeogenesis, reduced muscle proteolytic rate, and reduced REE expenditure. There is no effect on stress hormone. rhGH exerts a beneficial effect on metabolism in severely burned patients, but hyperglycemia is apt to occur, and water, Na(+), Cl(-) retention are suggested.

Adolescent ; Adult ; Amino Acids ; blood ; Burns ; blood ; drug therapy ; surgery ; Child ; Female ; Growth Hormone ; therapeutic use ; Human Growth Hormone ; genetics ; therapeutic use ; Humans ; Insulin ; blood ; Male ; Middle Aged ; Recombinant Proteins ; therapeutic use ; Sodium Chloride ; blood

OBJECTIVETo observe the effect of parenteral nutrition (PN) with branched-chain amino acid supplementation on protein metabolism after partial hepatectomy in rats with liver cirrhosis.

METHODSEighteen rats with liver cirrhosis were randomly divided into pre-operation group (n=6), post-operation 8.5% Novamin PN group (n=6) and post-operation 10% Hepa PN group (n=6), with 6 normal rats severing as the normal control group. Five days after the operation, serum albumin (ALB), insulin-like growth factor I (IGF-1) and plasma amino acid spectrum were measured, and ALB mRNA level in the liver was assayed using RT-PCR.

RESULTSPostoperative serum ALB was similar between 10% Hepa PN and 8.5% Novamine PN groups, but the rats in the latter group showed significantly increased serum IGF-1 level, Fischer ratio and hepatic ALB mRNA expression (P<0.05).

CONCLUSIONAdministration of PN with branched-chain amino acid supplementation can ameliorate plasma amino acid spectrum and increase protein synthesis in rats with liver cirrhosis after partial hepatectomy.

Albumins ; metabolism ; Amino Acids, Branched-Chain ; administration & dosage ; therapeutic use ; Animals ; Hepatectomy ; Insulin-Like Growth Factor I ; metabolism ; Liver Cirrhosis ; therapy ; Parenteral Nutrition ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo study the effect of 5-fluorouracil-FU in combination with astragalus membranaceus(AM) on amino acid metabolism in mice model of gastric carcinoma induced by 3-methylcholanthrene(MC).

METHODSMice gastric carcinoma models were established by 3-methylcholanthrene induction and randomly divided into different groups, and received 5-FU treatment (group A) 5-FU plus AM (group B), 5-FU plus a high dose of AM(group C), no treatment (group D). Normal mice were used as control (group N). Free amino acid in the tumor specimens were examined.

RESULTSThe levels of free Valine, Methionine, Leucine, Arginine and cystine in the tumor specimens in group D were significantly higher than that in group N(P< 0.05). The levels of free serine in group A, B, C, D were significantly higher than that in group N. The levels of free glutamic acid in group A, B were significantly higher than that in group N(P< 0.05). The levels of free proline in group C, D were significantly higher than that in group P, N(P< 0.05).

CONCLUSIONSThe increasing levels of free serine and proline in tumor specimens in gastric cancer mice model reveals metabolic disturbance of amino acid. 5-FU plus astragalus membranaceus can decrease the level of free glutamic acid in the mice models, and inhibit tumor growth.

Amino Acids ; metabolism ; Animals ; Astragalus membranaceus ; Fluorouracil ; therapeutic use ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Phytotherapy ; Stomach Neoplasms ; drug therapy ; metabolism