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Objectives: The aim of this study was to evaluate whether chronic heart failure (HF) therapy guided by concentrations of amino-terminal pro-B-type natriuretic peptide (NT-proBNP) is superior to standard of care (SOC) management.Background: It is unclear whether standard HF treatment plus a goal of reducing NT-proBNP concentrations improves outcomes compared with standard management alone.Methods: In a prospective single-center trial, 151 subjects with HF due to left ventricular (LV) systolic dysfunction were randomized to receive either standard HF care plus a goal to reduce NT-proBNP concentrations ≤1,000 pg/ml or SOC management. The primary endpoint was total cardiovascular events between groups compared using generalized estimating equations. Secondary endpoints included effects of NT-proBNP-guided care on patient quality of life as well as cardiac structure and function, assessed with echocardiography.Results: Through a mean follow-up period of 10 ± 3 months, a significant reduction in the primary endpoint of total cardiovascular events was seen in the NT-proBNP arm compared with SOC (58 events vs. 100 events, p = 0.009; logistic odds for events 0.44, p = 0.02); Kaplan-Meier curves demonstrated significant differences in time to first event, favoring NT-proBNP-guided care (p = 0.03). No age interaction was found, with elderly patients benefitting similarly from NT-proBNP-guided care as younger subjects. Compared with SOC, NT-proBNP-guided patients had greater improvements in quality of life, demonstrated greater relative improvements in LV ejection fraction, and had more significant improvements in both LV end-systolic and -diastolic volume indexes.Conclusions: In patients with HF due to LV systolic dysfunction, NT-proBNP-guided therapy was superior to SOC, with reduced event rates, improved quality of life, and favorable effects on cardiac remodeling. (Use of NT-proBNP Testing to Guide Heart Failure Therapy in the Outpatient Setting; NCT00351390). [ABSTRACT FROM AUTHOR]

Growth Factors have been evaluated as therapeutic targets for the treatment of a broad spectrum of diseases. Because they are proteins with pleiotropic effects, the quest to harness their beneficial effects has presented challenges. Most Growth Factors operate at the extracellular-receptor level and have natural feedback mechanisms that modulate their effects. As proteins, they are difficult and expensive to manufacture. Frequently proteins must be administered parenterally, may invoke an immune response, and may be neutralized by naturally occurring inhibitors. To circumvent these limitations, we have undertaken an effort to develop mimetics for the Bone Morphogenetic Protein (BMP) signaling pathway effects that incorporate the beneficial effects, eliminate the deleterious effects, and thereby create effective drug-like compounds.To this end, we have designed and tested a family of small peptide BMP mimetics. The design used the three-dimensional structure of BMP-7 to identify likely active surface regions. Lead sequences were then optimized based on in vitro assays that examine the selective binding to BMP receptors, demonstrate the phosphorylation of Smad-1,5,8, detect anti-apoptosis and anti-inflammation, and block the epithelial to mesenchymal transition (EMT) in renal tubular epithelial cells. These sequences were further optimized using in vivo assays of the attenuation of acute kidney injury in a rat-model of unilateral clamp ischemic reperfusion. This process uses a Structure Variance Analysis algorithm (SVA) to identify structure/activity relationships. One member of this family, THR-184, is an agonist of BMP signaling and a potent antagonist of TGFβ signaling. This small peptide mimetic inhibits inflammation, apoptosis, fibrosis and reverses epithelial to mesenchymal transition (EMT) by regulating multiple signaling pathways involved in the cellular injury of multiple organs. Its effects have been shown to control Acute Kidney Injury (AKI). THR-184 has progressed through phase I and II clinical trials for the prevention of Cardio-Vascular Surgery (CVS) associated AKI. This work provides a roadmap for the development of other growth factor mimetics and demonstrates how we might harness their therapeutic potential., Competing Interests: Authors WC, PK, DM and FC were employed by the Therapeutics by Design, Thrasos Therapeutics and Thrasos Innovatioins., (Copyright © 2022 Carlson, Keck, Bosukonda and Carlson.)

Competing Interests: The authors all hold equity in Therapeutics by Design, LLC; and all authors have been listed as inventors on BMP mimetic patents.

Cancer stem cells (CSCs) persist in tumors as a distinct population and may be causative in metastasis and relapse. CSC-rich tumors are associated with higher rates of metastasis and poor patient prognosis. Targeting CSCs therapeutically is challenging, since they seem to be resistant to standard chemotherapy. We have shown that a novel peptide agonist of bone morphogenetic protein (BMP) signaling, P123, is capable of inhibiting the growth of primary tumor cells by interacting with type I receptors selectively [activin receptor-like kinase 2 (ALK2) and ALK3, but not ALK6] and type II BMP receptors, activating SMAD 1/5/8 signaling and controlling the cell cycle pathway. Furthermore, the compound is capable of blocking transforming growth factor-β induced epithelial-to-mesenchymal transition (EMT) in primary tumor cells, a critical step for tumor progression and metastasis. In addition, we have investigated the effects of P123 on self-renewal, growth, differentiation (reversal of EMT) and apoptosis of isolated human breast CSCs. We have shown that P123 and BMP-7 reverse the EMT process in human breast CSCs, and inhibit self-renewal and growth. Moreover, compared with single treatment with paclitaxel, co-treatment with paclitaxel and P123 showed an increase in cell apoptosis. Together, these findings suggest that P123 has the therapeutic potential to suppress both bulk tumor cells and CSCs. We believe that P123 represents a new class of drugs that have the potential to eliminate the primary tumor, prevent reoccurrence and metastasis, and enhance the treatment of breast cancer., (© 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

We report the three-dimensional structure of osteogenic protein 1 (OP-1, also known as bone morphogenetic protein 7) to 2.8-A resolution. OP-1 is a member of the transforming growth factor beta (TGF-beta) superfamily of proteins and is able to induce new bone formation in vivo. Members of this superfamily share sequence similarity in their C-terminal regions and are implicated in embryonic development and adult tissue repair. Our crystal structure makes possible the structural comparison between two members of the TGF-beta superfamily. We find that although there is limited sequence identity between OP-1 and TGF-beta 2, they share a common polypeptide fold. These results establish a basis for proposing the OP-1/TGF-beta 2 fold as the primary structural motif for the TGF-beta superfamily as a whole. Detailed comparison of the OP-1 and TGF-beta 2 structures has revealed striking differences that provide insights into how these growth factors interact with their receptors.

We have obtained trigonal crystals of recombinant human osteogenic protein-1 (hOP-1), a member of the transforming growth factor-beta (TGF-beta) superfamily. hOP-1 (also referred to as BMP-7) is a bone morphogenetic protein and is active as a dimer of M(r) 32 to 36 kDa. The crystals have the symmetry of space group P3(1)21 or the enantiomorph P3(2)21 with unit cell dimensions of a = b = 99.46 A, c = 42.09 A. The crystals diffract to 2.2 A resolution and there is one hOP-1 monomer per asymmetric unit. In this paper we describe the first crystallization of a bone morphogenetic protein and present the results of preliminary X-ray diffraction data from the native protein and two heavy-atom derivatives.

The hemodynamic and humoral effects of the specific human renin inhibitor R-PEP-27 were studied in six normal human subjects on low and high sodium intake diets. An intravenous infusion of R-PEP-27 (0.5 to 16 micrograms/min/kg body wt) reduced blood pressure in a dose-dependent fashion; the mean arterial blood pressure at the end of the infusion fell from 128 +/- 4/83 +/- 4 to 119 +/- 3/71 +/- 3 mm Hg (mean +/- SEM) (P < .01) during the low sodium intake diet. R-PEP-27 had no effect on blood pressure during the high sodium intake diet. R-PEP-27 significantly reduced plasma angiotensin II and aldosterone concentrations. The temporal response to R-PEP-27 suggests that it is a short-lived although highly potent competitive inhibitor of renin; this peptide is a valuable and specific physiologic probe of the renin-angiotensin system.

Quantitative three-dimensional analysis of rock textures is now possible with the use of high-resolution computed x-ray tomography. When applied to metamorphic rocks, this technique provides data on the sizes and positions of minerals that allow mechanisms of porphyroblast crystallization to be identified. Statistical analysis of the sizes and spatial disposition of thousands of garnet crystals in three regionally metamorphosed rocks with diverse mineralogies, in conjunction with simple numerical models for crystallization, reveals in all cases the dominance of crystallization mechanisms whose kinetics are governed by rates of intergranular diffusion of nutrients.

A peptide inhibitor, having the sequence D-His-Pro-Phe-His-Phe psi [CH2-NH]Phe-Val-Tyr, with a reduced bond between the two adjacent phenylalanines, has been diffused into crystals of the aspartic proteinase from Rhizopus chinensis (rhizopuspepsin, EC 3.4.23.6). X-ray diffraction data to 1.8-A resolution have been collected on the complex, which has been subjected to restrained least-squares refinement to an R-factor (R equals the sum of the absolute value of the difference between the observed and calculated structure factor amplitudes divided by the sum of the observed structure factor amplitudes) of 14.7%. The inhibitor lies within the major groove of the enzyme and is clearly defined with the exception of the amino-terminal D-histidine and the carboxyl-terminal tyrosine. The reduced peptide bond is located in the active site with close contacts to the two catalytic aspartyl groups. The active-site water molecule that is held between the two carboxyl groups is displaced by the inhibitor, as are a number of other water molecules seen in the binding groove of the native enzyme. A mechanism of action for this class of enzymes is proposed from these results.

A synthetic nonapeptide corresponding to the predicted amino terminus of human prorenin was synthesized and used as an antigen in the production of mouse hybridomas. Forty-seven clones producing antibody to this peptide were identified and screened for ability to bind to partially purified amniotic fluid inactive renin in a soluble phase assay. None showed activity. One purified antibody, 4D3-3C4, was found to bind by Western blotting to renal and amniotic fluid inactive renins, molecular size 52 kDa, after gel electrophoresis in sodium dodecyl sulphate (SDS) following reduction with mercaptoethanol. In contrast, the anti-renin monoclonal antibody, R3-27-6, recognizes active renin, molecular size 38 kDa, and two inactive renin species, 42 kDa and 52 kDa, in the same renin preparations. Kidney and amniotic fluid apparently contain an inactive renin that possesses the complete prorenin sequence. These data indicate that high molecular weight inactive renin from both kidney and amniotic fluid contain an epitope, accessible to antibody only after denaturation, which represents the amino-terminal octapeptide sequence of prorenin. The inactive renin preparations tested also contain smaller fragments which either possess this epitope or do not, while still reacting with antibodies to active renin. It is likely that all these smaller inactive renin species are the result of proteolysis, either post-synthetic or in vitro.

On the basis of the minimal octapeptide sequence of the renin substrate, a series of peptides was synthesized containing (3S,4S)-4-amino-3-hydroxy-6-methylheptanoic acid (statine) or (3S,4S)-4-amino-3-hydroxy-5-phenylpentanoic acid (AHPPA) at the P1P1' position. Some of these peptides also contained Nin-formyltryptophan at the P5, P3, or P3' position. Renin-inhibitory potency varied over a wide range (from inactive to IC50 = 3 nM). Potency was reduced by at least 10-fold when the peptide was shortened by two residues at either the amino or carboxy terminus. The AHPPA-containing inhibitors were several-fold less potent than the statine-containing inhibitors. Analysis of models for the three-dimensional structure of inhibitors at the active site of human renin suggests that the diminished potency of the AHPPA peptides in comparison with the statine-containing peptides was caused by a shift in the peptide backbone due to a steric conflict between the phenyl ring of the AHPPA residue and the S1 subsite. The importance of the side chain and the 3(S)-hydroxyl group of the statine residue was demonstrated by substituting 5-aminovaleric acid for a dipeptide unit at the P1P1' position, which resulted in a peptide devoid of renin-inhibitory activity. Substitutions of other basic amino acids for histidine at the P2 position caused a great loss in potency, possibly due to disruption of a hydrogen bond as suggested by molecular modeling. Studies on the plasma renins of four nonhuman species suggest that the isoleucine-histidine segment at the P2'P3' position is important to defining the human specificity of the substrate. This work suggests a number of properties important to the design of potent renin inhibitors, and demonstrates the usefulness of three-dimensional models in the interpretation of structure-activity data.

Renin is an aspartyl protease that plays a key role in regulating blood pressure. The aim of this study was to use synthetic peptides to produce antibodies able to recognize the entire human renin molecule and to inhibit its enzymatic activity. Two three-dimensional renin models were used to predict 17 putative epitopes. Then 18 peptides related to 13 potential antigenic determinants were synthesized by solid- or liquid-phase technique, and 11 were shown to be antigenic when tested by their binding to several polyclonal human renin antibodies. The peptides were injected into rabbits, and antisera tested by radio-immunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) showed that nine peptides were immunogenic. The peptide related to the flap region which lies across the renin catalytic cleft showed potentially useful characteristics. Immunoglobulin G, purified from antibodies raised against this epitope, bound labelled renin and inhibited its enzymatic activity in a dose-dependent manner. This approach constitutes the basis for the development of a synthetic antirenin vaccine able to inhibit the renin-angiotensin system specifically.

Antigenic determinants in proteins have been found to coincide with surface regions accessible to a large probe of 1-nm radius, which approximates the size of an antibody binding domain. To predict epitopes of human renin, a computer algorithm was used to calculate the accessibility of a model of the three-dimensional structure of human renin to a 1-nm probe. Of the 17 segments predicted to be epitopes or parts of epitopes, 6 were synthesized and tested for their recognition by 11 polyclonal antibodies prepared against pure human renin. Four peptides, Y-133-144, C-180-188, Y-211-224, and Y-300-310, were bound by some polyclonal anti-renin antibodies when tested in both a solution assay and an enzyme-linked immunosorbent assay (ELISA), and one peptide, C-290-296-G, was bound only in an ELISA. Of the five epitopes identified, peptide Y-211-224 seems dominant, since it was bound by all antisera in the solution assay and by 8 of the 11 in the ELISA. This peptide contains a disulfide loop, and the corresponding linear peptide obtained after reduction of the disulfide was not recognized by any anti-renin antisera in the solution assay. This suggests that the epitope is conformationally dependent and that a disulfide bridge linking cysteines-217 and -221 is present in the native structure of human renin. Purified immunoglobulins raised to peptides Y-133-144 and Y-211-224 (located near, but not at, the catalytic site) were found to inhibit the cleavage of human angiotensinogen by purified human renin but not the cleavage of a tetradecapeptide substrate representing the N-terminal region of angiotensinogen.(ABSTRACT TRUNCATED AT 250 WORDS)

Specific blockade of renin angiotensin system can be obtained both by enzymatic inhibitors and by passive and active immunization against renin. Recent studies have shown that synthetic peptides mimicking a protein segment can be used as immunogens to elicit antibodies which react with the parent molecule. In order to develop synthetic antirenin antigens we have selected peptidic sequences from human active renin and synthesized the corresponding peptides to produce antibodies able to recognize the entire human molecule and to inhibit its enzymatic activity. Three dimensional models of human renin were used to predict 17 putative epitopes. Then 18 peptides related to 13 potential antigenic determinants were synthesized by solid or liquid phase technic and 11 were shown to be antigenic when tested by their binding to several polyclonal and monoclonal human renin antibodies. The peptides were injected into rabbits and antisera tested by radio-immunoassay (RIA) and enzyme linked immunosorbent assay (ELISA) showed that nine peptides were immunogenic. Mainly, antiserum raised against the peptide mimicking the beta-hairpin 81-50 of active human renin which lies across the catalytic cleft, produced a 25 p. 100 inhibition of plasma renin activity at a 1: 50 dilution. Immunoglobulins, purified from antibodies raised against this epitopes, bound labelled renin and inhibited enzymatic activity of pure human renin on its synthetic tetradecapeptide substrate, in a dose dependent manner.

The primary structure of human renin, recently established from the complementary DNA sequence of its messenger RNA, shows a strong homology to other aspartyl proteases. This homology has permitted the construction of a model of the three-dimensional structure of renin based on the crystallographically determined structures of three aspartyl proteases: penicillopepsin, endothiapepsin, and rhizopuspepsin. Using an algorithm in which a spherical probe approximating the size of the antibody-binding domain (1-nm radius) was allowed to contact the surface of the renin model, we predicted 12 to 15 peptides to be immunogenic epitopes. We synthesized peptides corresponding to three different regions of the model: Cys-Gly-Ser-Asp-Pro-Gln-His-Tyr-Glu-Gly-amide (C-180-188), Tyr-Leu-Leu-Cys-Glu-Asp-Gly-Cys-Leu-Ala-Leu-amide (Y-215-224; disulfide bond between cysteines) and Tyr-Gly-Ser-Ser-Thr-Leu-Leu-Cys-Glu-Asp-Gly-Cys-Leu-Ala-Leu-amide (Y-211-224; disulfide bond between cysteines), and Cys-Tyr-Ser-Ser-Lys-Lys-Leu-Cys-Gly (C-290-296-G; disulfide bond between cysteines). All four peptides were tested for their binding to 11 polyclonal and 7 monoclonal antibodies raised against pure human renin, in both a solution assay and an enzyme-linked immunosorbent assay. Peptides Y-215-224 and Y-211-224 bound to all 11 polyclonal antibodies in the solution assay, and peptide Y211-224 bound to eight of them in the enzyme-linked immunosorbent assay. Therefore, region 211-224 can be identified as a major epitope of the human renin molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

The side-chain conformations of topologically equivalent residues in seven pairs of proteins ranging in sequence homology from 16% to 60% are compared. Both identical and mutated residues are included. For proteins with greater than 40% homology, it is found that at least 80% of the side-chain orientations of identical residues and 75% or more of the mutated residues in each pair of proteins have matching gamma atom dihedral angles (+/- 40 degrees); the comparison is not based strictly on chi 1 angles. Further, if a match is obtained at the gamma position, there is a high probability of matching for the delta atom(s) of the side-chain. For proteins with less than 25% homology the percentages are somewhat lower. Trends observed for conservative substitutions are essentially the same as those noted for mutated residues in general. Side-chain accessibility does not affect the probability of matches of identical residues; however, less accessible pairs of mutated residues have 10 to 20% higher matching probabilities than do exposed residues. Mismatches can frequently be related to large B-factors, certain types of amino acid substitutions, or the appearance of multiple minima on the side-chain potential energy surfaces and are most likely to occur for certain small residues (Ser, Thr, Val). Analysis of all the results makes possible the formulation of a set of rules for side-chain positioning in the modeling of homologous proteins.

Three-dimensional models of the octapeptide segment of renin substrate and of several inhibitors characterized by a modified scissile bond were constructed at the active site of human renin. The substrate and inhibitor models were based on the structure of pepstatin solved by x-ray crystallography to a resolution of 1.8 A. The renin structure, previously reported by us, was based on the structures of homologous aspartyl proteases solved by x-ray diffraction techniques to a resolution of 2.1 A or higher. An energy minimization program, CHARMM, was used to refine these structures with respect to the optimal interaction of the enzyme and its inhibitor, and to calculate the energy of each inhibitor-enzyme interaction. The results indicate that the most significant difference between the binding of inhibitors and of the natural substrate is caused by a reduction in electrostatic rather than conformational strain.

Principles for the design of renin inhibitors were examined by utilizing a three-dimensional model of the enzyme with inhibitory peptides in its catalytic site. Optimal conformations were subjected to energy minimization by the computer program CHARMM. The results were then compared with the inhibitory potencies of these peptides as assessed by an in vitro renin assay. The results of the modeling study and enzymatic assay were generally congruent, showing that residues at the P5, P4 and P3 positions contributed importantly to binding, that an acetyl group can replace Pro-His at the P5, P6 position, that the lesser potency of AHPPA-containing as compared with statine-containing peptides may be related to the better fit into the S1 subsite of an isobutyl as opposed to a benzyl side chain, that histidine in the P2 position forms an essential hydrogen bond with residue 230 (which cannot be formed by another basic residue), and that there is a general correlation between inhibitory potency and calculated energy of interaction as deduced from the model. Major determinants of energy of binding are conformational strain and electrostatic interaction.