[Clinical characteristics and prognosis of 49 newly diagnosed primary central nervous system diffuse large B-cell lymphoma].

Objective: The clinical characteristics of patients with primary central nervous system lymphoma-diffuse large B-cell lymphoma (PCNSL-DLBCL) and the effects of different treatment schemes on their survival and prognosis were analyzed retrospectively. Methods: A total of 49 patients with PCNSL-DLBCL who presented at the Tianjin Medical University General Hospital from July 2014 to December 2020 were included, and their clinical data were retrospectively analyzed. They were divided into four groups: the MTX group, the R-CDOP group, the BTKi-R-MTX group, and the RLZT group. The median overall survival (OS) and progression-free survival (PFS) were calculated, and the survival prognosis was compared by univariate and multivariate prognostic analysis. Results: The median OS time of the MTX group, the R-CDOP group, the BTKi-R-MTX group, and the RLZT group was 16.5 months, 4.5 months, 42 months, and not reached, respectively (P<0.001) . The median PFS time of the MTX group, the R-CDOP group, the BTKi-R-MTX group, and the RLZT group was 7 months, 1.5 months, 20 months, and 5 months, respectively (P=0.005) . Multivariate prognostic analysis showed that double expressor lymphoma, IESLG risk grade, and different treatment methods were the prognostic factors of PCNSL-DLBCL. Conclusion: The survival and prognosis of PCNSL-DLBCL are affected by different treatment schemes. The role of CD20 monoclonal antibody in the treatment of PCNSL-DLBCL is still controversial. The treatment scheme containing BTKi has great potential for PCNSL-DLBCL. RLZT scheme has a good prospect for elderly patients who cannot tolerate high-dose chemotherapy and radiotherapy.

[Clipping for large paraclinoid internal carotid artery aneurysms by non-fluoroscopic occlusion via a micro-bone window frontolateral approach].

Objective: To evaluate the clinical value and outcomes of technical improvement of hybrid operatical clipping for large paraclinoid internal carotid artery aneurysms. Methods: A review was conducted on 18 cases of large paraclinoid internal carotid artery aneurysm which were clipped by balloon non-fluoroscopic occlusion of the parent artery via a micro-bone window frontolateral approach in hybrid operating room at Neurosurgery Department of Tianjin Medical University General Hospital from June 2014 to December 2017. There were 8 males and 10 females with age of (63±4) years. There were 6 cases of unruptured aneurysm and 12 cases of ruptured aneurysm of subarachnoid hemorrhage (6 cases of grade Ⅱ, 4 cases of grade Ⅲ and 2 cases of grade Ⅳ in Hunt-Hess classification). Frontolateral approach incision (average length of about 5 cm) and bone window about 3 cm×3 cm were performed. No incision of the neck was needed to expose the internal carotid artery for temporary occlusion. In the operation, the balloon was slowly pushed to the preset position of the internal carotid artery under non-fluoroscopy. The balloon was expanded to block the blood flow of internal carotid artery. Then aneurysm was clipped. The balloon was loosened and retraced to the guiding catheter after clipping. The clipping condition was examined by cerebral angiography. If there was residual aneurysm neck or stenosis of the parent artery, the balloon was pushed under non-fluoroscopy again to temporary occlusion and the clip was adjusted until the aneurysm neck was clamped satisfactorily. Results: Eighteen aneurysms were successfully clipped in hybrid operating room. Fourteen aneurysms showed complete occlusion of the aneurysm neck and no stenosis of the parent artery. Four cases showed residual aneurysm neck after clipping by intraoperative angiography, then aneurysms were clipped satisfy by adjusting the aneurysm clip. The patients were followed up for 3 months to 1 year. Ten patients recovered well (modifed Rankin score (mRS): 0), and 3 patients had no obvious disability (mRS: 1). Two patients with Hunt-Hess grade Ⅲ were slightly disabled (mRS: 2). 1 patients with Hunt-Hess grade Ⅲ were moderately disabled (mRS: 3). 1 patients with Hunt-Hess grade Ⅳ were severely disabled (mRS: 4). One elderly patients with Hunt-Hess grade Ⅳ were seriously disabled (mRS: 5). Conclusions: Application of balloon non-fluoroscopic occlusion clipping for large paraclinoid internal carotid artery aneurysm via a micro-bone window frontolateral approach is safe, effective and minimally invasive.

[Clinical analysis of 10 patients of chronic subdural hematoma associated with arachnoid cyst].

Objective: To investigate the clinical characteristics, pathogenesis and surgical strategy for the chronic subdural hematoma associated with arachnoid cyst (AC). Method: Ten patients of chronic subdural hematoma associated with AC were retrospectively enrolled from the Neurosurgery Department of Tianjin Medical University General Hospital from January 2012 to September 2015, with a mean age of 27.5±5.6 years (range, 18-37 years). All patients simply performed a burr hole drainage of hematoma and left the AC intact, then followed up for 12 to 18 months after discharge respectively. Results: In this study, the AC in 8 of 10 cases occurs in the middle cranial fossa, and the other 2 cases root in the cerebral hemisphere.The AC of 10 patients all locate near the hematoma cavity.Nine patients had a full recovery, and only one patient had a recurrent subdural hematoma with a secondary operation, then recovery in 3 months postoperation.All patients lived completely free of neurological symptom and showed no recurrence in the follow-up period with a Barthel index more than 90. Conclusion: Simply burr hole drainage of hematoma and leave intact AC achieves satisfied outcome and provides a reliable therapy strategy for chronic subdural hematoma associated with arachnoid cyst.

[Stereotactic brain biopsy guided by iMRI co-registration combined with PET/CT].

OBJECTIVE: To explore the clinical value of intraoperative magnetic resonance imaging (iMRI) coregistration combined with position emission tomography/computed tomography (PET/CT) in stereotactic brain biopsy. METHODS: Forty nine patients with intracranial lesions were operated by stereotactic biopsy from June 2010 to June 2015 in Tianjin Medical University General Hospital. Seventeen patient's operation was guided by iMRI only (group A), thirty two patients' operation was guided by iMRI and PET/CT (group B). The diagnosis success rate and operation related complications were compared between the two groups. RESULTS: PET/CT and iMRI were integrated successfully in all cases of group B. Fourteen patients (82.4%) of group A and all 32 patients (100%) of group B had final diagnosis confirmed by histopathological and immunohistochemical observation. The diagnosis success rate of group B was higher than group A (P<0.05). There were 5 patients in total who had postoperative complication, 2 (11.8%) in group A and 3 (9.3%) in group B, but the difference was not statistically significant. CONCLUSIONS: PET/CT based metabolic imaging can be automatically integrated with standard MRI guided stereotactic biopsy. Compared with iMRI only, the combined treatment improves diagnosis success rate without increasing complications; it's safe, and has high clinical efficacy.

Intracranial aneurysm risk factor genes: relationship with intracranial aneurysm risk in a Chinese Han population.

Few studies have examined the genes related to risk fac-tors that may contribute to intracranial aneurysms (IAs). This study in Chinese patients aimed to explore the relationship between IA and 28 gene loci, proven to be associated with risk factors for IA. We recruited 119 patients with aneurysms and 257 controls. Single factor and logistic regression models were used to analyze the association of IA and IA rup-ture with risk factors. Twenty-eight single nucleotide polymorphisms (SNPs) in 22 genes were genotyped for the patient and control groups. SNP genotypes and allele frequencies were analyzed by the chi-square test. Logistic regression analysis identified hypertension as a factor that increased IA risk (P = 1.0 x 10(-4); OR, 2.500; 95%CI, 1.573-3.972); IA was associated with two SNPs in the TSLC2A9 gene: rs7660895 (P = 0.007; OR, 1.541; 95%CI, 1.126-2.110); and in the TOX gene: rs11777927 (P = 0.013; OR, 1.511; 95%CI, 1.088-2.098). Subsequent removal of the influence of family relationship identified between 12 of 119 patients enhanced the significant association of these SNPs with IA (P = 0.001; OR, 1.691; 95%CI, 1.226-2.332; and P = 0.006; OR, 1.587; 95%CI, 1.137-2.213 for rs7660895 and rs11777927, respectively). Fur-thermore, the minor allele of rs7660895 (A) was also associated with IA rupture (P = 0.007; OR, 2.196; 95%CI, 1.230-3.921). Therefore, hypertension is an independent risk factor for IA. Importantly, the TSL-C2A9 (rs7660895) and TOX (rs11777927) gene polymorphisms may be associated with formation of IAs, and rs7660895 may be associated with IA rupture.

Evaluation of the effect of energy conservation techniques in the performance of activity of daily living tasks.

OBJECTIVE: To determine whether energy conservation techniques during common activity of daily living tasks actually result in lower energy expenditure, and to document subjective comments regarding any differences in the perceived level of effort. DESIGN: Descriptive study comparing energy expenditure in three tasks with and without energy conservation techniques, taking into account the effect of age. SETTING: Occupational therapy department of a rehabilitation hospital in Hong Kong. SUBJECTS: One hundred and eight subjects (30 < 60 years; 78 > or = 60 years) were recruited from staff and members of an elderly social centre in the community. MEASUREMENTS: Energy expenditure was measured using a portable indirect calorimetry system for three tasks (shopping, washing clothes and hanging laundry) with and without energy conservation techniques. Dyspnoea, fatigue and perceived exertion were measured using visual analogue scales. RESULTS: Reduction in energy expenditure using energy conservation techniques for shopping and hanging laundry was documented in younger subjects only (O2 consumption fell from 13.8 +/- 3.7 to 12.2 +/- 3.8 mL/min per kg for shopping, P < 0.001 and 5.9 +/- 1.2 to 5.0 -/+ 1.2 mL/min per kg, P < 0.001 for hanging laundry), although the older subjects experienced less perceived exertion with the energy conservation techniques. For washing clothes, no reduction in energy expenditure was observed in either age groups. CONCLUSION: Measurable benefits were observed with use of labour-saving equipment and avoidance of overhead reaching in younger subjects only.

Locally linear embedding for dimensionality reduction in QSAR.

Current practice in Quantitative Structure Activity Relationship (QSAR) methods usually involves generating a great number of chemical descriptors and then cutting them back with variable selection techniques. Variable selection is an effective method to reduce the dimensionality but may discard some valuable information. This paper introduces Locally Linear Embedding (LLE), a local non-linear dimensionality reduction technique, that can statistically discover a low-dimensional representation of the chemical data. LLE is shown to create more stable representations than other non-linear dimensionality reduction algorithms, and to be capable of capturing non-linearity in chemical data.

New diarylmethylpiperazines as potent and selective nonpeptidic delta opioid receptor agonists with increased In vitro metabolic stability.

Nonpeptide delta opioid agonists are analgesics with a potentially improved side-effect and abuse liability profile, compared to classical opioids. Andrews analysis of the NIH nonpeptide lead SNC-80 suggested the removal of substituents not predicted to contribute to binding. This approach led to a simplified lead, N, N-diethyl-4-[phenyl(1-piperazinyl)methyl]benzamide (1), which retained potent binding affinity and selectivity to the human delta receptor (IC(50) = 11 nM, mu/delta = 740, kappa/delta > 900) and potency as a full agonist (EC(50) = 36 nM) but had a markedly reduced molecular weight, only one chiral center, and increased in vitro metabolic stability. From this lead, the key pharmacophore groups for delta receptor affinity and activation were more clearly defined by SAR and mutagenesis studies. Further structural modifications on the basis of 1 confirmed the importance of the N, N-diethylbenzamide group and the piperazine lower basic nitrogen for delta binding, in agreement with mutagenesis data. A number of piperazine N-alkyl substituents were tolerated. In contrast, modifications of the phenyl group led to the discovery of a series of diarylmethylpiperazines exemplified by N, N-diethyl-4-[1-piperazinyl(8-quinolinyl)methyl]benzamide (56) which had an improved in vitro binding profile (IC(50) = 0.5 nM, mu/delta = 1239, EC(50) = 3.6 nM) and increased in vitro metabolic stability compared to SNC-80.

N,N-Diethyl-4-(phenylpiperidin-4-ylidenemethyl)benzamide: a novel, exceptionally selective, potent delta opioid receptor agonist with oral bioavailability and its analogues.

The design, synthesis, and pharmacological evaluation of a novel class of delta opioid receptor agonists, N, N-diethyl-4-(phenylpiperidin-4-ylidenemethyl)benzamide (6a) and its analogues, are described. These compounds, formally derived from SNC-80 (2) by replacing the piperazine ring with a piperidine ring containing an exocyclic carbon carbon double bond, were found to bind with high affinity and exhibit excellent selectivity for the delta opioid receptor as full agonists. 6a, the simplest structure in the class, exhibited an IC(50) = 0.87 nM for the delta opioid receptors and extremely high selectivity over the mu receptors (mu/delta = 4370) and the kappa receptors (kappa/delta = 8590). Rat liver microsome studies on a selected number of compounds show these olefinic piperidine compounds (6) to be considerably more stable than SNC-80. This novel series of compounds appear to interact with delta opioid receptors in a similar way to SNC-80 since they demonstrate similar SAR. Two general approaches have been established for the synthesis of these compounds, based on dehydration of benzhydryl alcohols (7) and Suzuki coupling reactions of vinyl bromide (8), and are herewith reported.

The glycine residue in cyclic lactam analogues of galanin(1-16)-NH2 is important for stabilizing an N-terminal helix.

The neuropeptide galanin is a 29- or 30-residue peptide whose physiological functions are mediated by G-protein-coupled receptors. Galanin's agonist activity has been shown to be associated with the N-terminal sequence, galanin(1-16). Conformational investigations previously carried out on full-length galanin have, furthermore, indicated the presence of a helical conformation in the neuropeptide's N-terminal domain. Several cyclic lactam analogues of galanin(1-16)-NH2 were prepared in an attempt to stabilize an N-terminal helix in the peptide. Here we describe and compare the solution conformational properties of these analogues in the presence of SDS micelles as determined by NMR, CD, and fluorescence spectroscopy. Differences in CD spectral profiles were observed among the compounds that were studied. Both c[D4, K8]Gal(1-16)-NH2 and c[D4,K8]Gal(1-12)-NH2 adopted stable helical conformations in the micelle solution. On the basis of the analyses of their respective alpha H chemical shifts and NOE patterns, this helix was localized to the first 10 residues. The distance between the aromatic rings of Trp2 and Tyr9 in c[D4, K8]Gal(1-16)-NH2 was determined to be 10.8 +/- 3 A from fluorescence resonance energy transfer measurements. This interchromophore spacing was found to be more consistent with a helical structure than an extended one. Removal of the Gly1 residue in compounds c[D4,K8]Gal(1-16)-NH2 and c[D4, K8]Gal(1-12)-NH2 resulted in a loss of helical conformation and a concomitant reduction in binding potency at the GalR1 receptor but not at the GalR2 receptor. The nuclear Overhauser enhancements obtained for the Gly1 deficient analogues did, however, reveal the presence of nascent helical structures within the N-terminal sequence. Decreasing the ring structure size in c[D4, K8]Gal(1-16)-NH2 by replacing Lys8 with an ornithine residue or by changing the position of the single lysine residue from eight to seven was accompanied by a complete loss of helical structure and dramatically reduced receptor affinity. It is concluded from the data obtained for the series of cyclic galanin(1-16)-NH2 analogues that both the ring structure size and the presence of an N-terminal glycine residue are important for stabilizing an N-terminal helix in these compounds. However, although an N-terminal helix constitutes a predominant portion of the conformational ensemble for compounds c[D4,K8]Gal(1-16)-NH2 and c[D4, K8]Gal(1-12)-NH2, these peptides nevertheless are able to adopt other conformations in solution. Consequently, the correlation between the ability of the cyclic galanin analogues to adopt an N-terminal helix and bind to the GalR1 receptor may be considered as a working hypothesis.

Novel "restoration of function" mutagenesis strategy to identify amino acids of the delta-opioid receptor involved in ligand binding.

A novel "restoration of function" mutagenesis strategy was developed to identify amino acid sequence combinations necessary to restore the ability to bind delta-selective ligands to an inactive delta/mu receptor chimera in which 10 amino acids of the third extracellular loop of the delta receptor were replaced by the corresponding amino acids from the mu receptor (delta/mu291-300). This chimera binds a nonselective opioid ligand but is devoid of affinity for delta-selective ligands. A library of mutants was generated in which some of the 10 amino acids of the mu sequence of delta/mu291-300 were randomly reverted to the corresponding delta amino acid. Using a ligand binding assay, we screened this library to select mutants with high affinity for delta-selective ligands. Sequence analysis of these revertants revealed that a leucine at position 300, a hydrophobic region (amino acids 295-300), and an arginine at position 291 of the human delta-opioid receptor were present in all revertants. Single and double point mutations were then introduced in delta/mu291-300 to evaluate the contribution of the leucine 300 and arginine 291 residues for the binding of delta-selective ligands. An increased affinity for delta-selective ligands was observed when the tryptophan 300 (mu residue) of delta/mu291-300 was reverted to a leucine (delta residue). Further site-directed mutagenesis experiments suggested that the presence of a tryptophan at position 300 may block the access of delta-selective ligands to their docking site.

Involvement of Trp-284, Val-296, and Val-297 of the human delta-opioid receptor in binding of delta-selective ligands.

Given the high homology in amino acid sequence between the delta-opioid receptor and the two other types (mu and kappa), distinct residues in this receptor may confer its selectivity to some ligands. In order to identify molecular determinants in the human delta receptor responsible for the selectivity of delta-selective ligands, two different delta/mu chimeras were constructed. In the first one, the delta sequence from the top of transmembrane 5 to the C terminus was replaced by the equivalent mu sequence, and in the second one, 13 consecutive residues in the third extracellular loop region of the delta receptor were replaced by the mu counterpart. These two chimeras retained the ability to bind the nonselective bremazocine but completely lost the ability to bind different delta-selective ligands. These results suggested that the region of the third extracellular loop of the delta receptor is crucial for the type selectivity. Furthermore, an alanine scan was performed by site-directed mutagenesis of 20 amino acids located in or proximal to the third extracellular loop. Among all the point mutations, only mutations of Trp-284, Val-296, or Val-297 significantly decreased the binding of delta-selective ligands tested. Moreover, combined mutation of Trp-284, Val-296, and Val-297 considerably decreased the affinities of the receptor for delta-selective ligands compared with the single point mutations. These findings suggest that Trp-284, Val-296, and Val-297 are crucial residues involved in the delta receptor type selectivity.

Calculation of relative binding free energies and configurational entropies: a structural and thermodynamic analysis of the nature of non-polar binding of thrombin inhibitors based on hirudin55-65.

Free energy calculations were carried out on a series of exosite-binding inhibitors of thrombin. These inhibitors are based on the C-terminal fragment of hirudin and have the sequence Phe-Glu-Glu-IleH59-Pro-Glu-Glu-Tyr- Leu, where the superscript over Ile indicates its relative position in the natural sequence of hirudin. In this study, the effect of replacing IleH59 with ten other non-polar amino acids was examined. Three preferred interaction sites for methyl/methylene groups for the various XaaH59 side-chains in the complex were identified from conformational search calculations. The corresponding thermodynamic changes were determined using a combination of systematic search and energy minimization in a manner that locates the local minima in the system and in the process simultaneously builds up the partition function. The free energy, internal energy and entropic contributions are readily calculated from the partition function. Very good agreement in the resulting relative binding free energies was obtained between theory and experiment. The calculations allowed us to dissect out the enthalpic, entropic and solvation contributions to delta delta G. The contribution from desolvation was found to be relatively weak. The binding of these non-polar side-chains to thrombin is found to be driven mainly by favorable protein-ligand interactions rather than by the desire for non-polar groups to be desolvated. We also find that the configurational entropy contributes about 0.48 kcal/mol (0.81 kappa T) in average for each torsional angle "frozen" in binding.

Interactions of hirudin-based inhibitor with thrombin: critical role of the IleH59 side chain of the inhibitor.

Hirudin is the most potent and specific thrombin inhibitor from medicinal leech with a Ki value of 2.2 x 10(-14) M. It consists of an active site inhibitor segment, hirudin1-48, a fibrinogen-recognition exosite inhibitor segment, hirudin55-65, and a linker, hirudin49-54, connecting these inhibitor segments. The role of the side chain of the hirudin 59th residue, Ile, is studied by using a series of synthetic bivalent thrombin inhibitors, which mimic the binding mode of hirudin. The synthetic inhibitors based on the hirudin sequence have a general sequence of Ac-(D-Phe)-Pro-Arg-Pro-(4-aminobutyric acid)-(7-amino-heptanoic acid)-Asp-Phe-Glu-Glu-Xaa-Pro-Glu-Glu-Tyr-Leu-Gln-OH, in which the 59th residue, Xaa, is substituted by various natural and unnatural L-amino acids. For example, substitution of IleH59 by Val, which is equivalent to removing the delta-methyl group of IleH59, reduces the affinity of the inhibitor 5.7-fold (delta delta G0 = 1.0 kcal/mol) to a Ki value of 4.7 nM compared to that (Ki = 0.82 nM) of the corresponding inhibitor with IleH59. Removal of the entire side chain of IleH59, i.e., a substitution of IleH59 by Gly, reduces the affinity of the inhibitor 6300-fold, revealing the critical role of the IleH59 side chain in the inhibitor binding. Theoretical free energy calculation successfully reproduces the binding free energy of most of the analogs. It suggests that intra- and intermolecular van der Waals interactions of delta-CH3, gamma-CH3, and gamma-CH2 of IleH59 play the major role in the binding affinity.(ABSTRACT TRUNCATED AT 250 WORDS)

Design of potent bivalent thrombin inhibitors based on hirudin sequence: incorporation of nonsubstrate-type active site inhibitors.

Hirudin from medicinal leech is the most potent and specific thrombin inhibitor from medicinal leech with a K(i) value of 2.2 x 10(-14) M. It consists of an active site blocking moiety, hirudin1-48, a fibrinogen-recognition exo-site binding moiety, hirudin55-65, and a linker, hirudin49-54, connecting these inhibitor moieties. Synthetic inhibitors were designed based on the C-terminal portion of hirudin. The bulky active site blocking moiety, hirudin1-48, was replaced by small nonsubstrate-type active site inhibitors of thrombin, e.g., dansyl-Arg-(D-pipecolic acid). The linker moiety was replaced by omega-amino acids of (12-aminododecanoic acid)-(4-aminobutyric acid), and hirudin55-65 was used as a fibrinogen-recognition exo-site binding moiety in most of the inhibitors. The crystal structure of the inhibitor in complex with human alpha-thrombin showed that dansyl, Arg, and D-pipecolic acid of the active site blocking moiety occupy S3, S1, and S2 subsites of thrombin, respectively, and were therefore designated as P3, P1, and P2 residues. The use of dansyl-Arg-(D-pipecolic acid) improved the affinity (K(i)) of the inhibitor 10-100-fold (down to 1.70 x 10(-11) M) compared to that of the similar compounds having D-Phe-Pro-Arg as their substrate-type inhibitor moiety (K(i) = 10(-9)-10(-10) M). The linker connected to P2 residue eliminated the scissile peptide bond. The inhibitor was also stable against human plasma proteases. Further inhibitor design revealed that the toxic dansyl group could be replaced by 4-tert-butylbenzenesulfonyl group and 1- or 2-naphthalenesulfonyl group for in vivo studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular dynamics simulations of thrombin inhibitors at the S' subsites of thrombin.

Molecular dynamics were carried out to simulate binding interactions between S' subsites of thrombin and hirudin-based thrombin inhibitors. These inhibitors include three segments: an active-site segment, N alpha-acetyl-(D-Phe)-Pro-Arg-Pro-; a fibrinogen-recognition exo-site segment, hirudin 55-65; and a 13-atom-long linker. These linkers have been reported (Szewczuk et al. (1993) Biochemistry 32, 3396) to influence the binding potency while keeping the same active and exo-site segments. The study found that, by combining different omega-amino acids, the potency could be increased 8-fold or decreased 4-fold compared to the native hirudin linker, -Gln-Ser-His-Asn-Asp-Gly-. Five typical linkers were simulated and compared. Analyzing the trajectory files led to the classification of three different dynamic behaviours for the linkers. The flexible linkers had no influence on the antithrombin activity. Other linkers formed hydrogen bonds with the thrombin S' subsite residues Glu39, leu40, and Gln 151. Formations of some hydrogen bonds enhanced the potency of the inhibitor. In other cases, the hydrogen bonds caused the distortion of the inhibitor conformation while affected the binding potency. Based on these observations, a general binding mode in the S' subsites of thrombin is proposed and potential applications are discussed.

Design of a linker for trivalent thrombin inhibitors: interaction of the main chain of the linker with thrombin.

N alpha-Acetyl[D-Phe45,Arg47]hirudin45-65 (P53) is a bivalent thrombin inhibitor (Ki = 5.6 nM) that consists of an active site inhibitor segment, [N alpha-acetyl-(dF)PRP]; a fibrinogen recognition exo site inhibitor segment, hirudin55-65 (DFEEIPEEYLQ-OH); and a linker, hirudin49-54 (QSHNDG), connecting these inhibitor segments (DiMaio et al., 1990). The structure-function relationships of the linker were studied using a combination of various omega-amino acids, which modified the length of the linker as well as the number and the locations of peptide bonds. Linkers with 14-18 atoms (counting only the atoms contributing to the length of the linker) showed a competitive inhibition with Ki = 1.7-3.4 nM. The potency of the inhibitors with 12-13-atom linkers was sensitive to the chemical structure of the linker. The high-potency inhibitors showed a competitive inhibition, while the low-potency inhibitors showed a hyperbolic inhibition. Among them, an inhibitor with a 13-atom linker showed the highest potency (Ki = 0.51 nM, an 11-fold improvement from that of P53 above), indicating that this is an optimal linker length. Since linkers with 6-10 atoms failed to bridge the active site and exo site inhibitor segments, a minimum of 11 atoms was required to bridge them, even though the potency of the inhibitor with an 11-atom linker was weak (Ki = 26 nM). Molecular dynamics simulation of the inhibitors with 13-atom linkers suggested that some linkers serve as a functional domain with the amide bond of the linker interacting with thrombin through hydrogen bonds.(ABSTRACT TRUNCATED AT 250 WORDS)

Conformationally restricted thrombin inhibitors resistant to proteolytic digestion.

A new type of thrombin exo-site inhibitor has been designed with enhanced inhibitory potency and increased metabolic stability. With the aid of the model of the structure of the thrombin-hirudin fragment complex [Yue, S.-Y., DiMaio, J., Szewczuk, Z., Purisima, E. O., Ni, F., & Konishi, Y. (1992) Protein Eng. 5, 77-85], cyclic analogs of the hirudin fragment (hirudin55-65) were designed and synthesized. In these analogs, the side chains of appropriately substituted residues, 58 and 61, were joined in order to restrict the conformation of the inhibitor. An analog with an 18-membered lactam ring showed higher antithrombin activity (IC50 = 0.57 microM) than the corresponding analogs with 17- or 16-membered rings and was 2-fold more potent than its linear counterpart. Even 4-fold greater enhancement was obtained when a shorter fragment, hirudin 55-62, was cyclized. This cyclization not only improved the potency but, more importantly, dramatically increased the resistance to proteolytic digestion. Remarkable enhancement of stability to proteolysis was observed for peptide bonds located in the exocyclic linear peptide segments. These results are discussed using molecular modeling.

Synthesis of a homologous series of ketomethylene arginyl pseudodipeptides and application to low molecular weight hirudin-like thrombin inhibitors.

The design of low molecular weight thrombin inhibitors IIa-d (hirutonins) that bind concurrently with the enzyme's catalytic site and auxiliary "anion-binding exosite" for fibrinogen recognition is reported. A practical synthesis of the required homologous ketomethylene arginyl dipeptide inserts [Arg psi CO(CH2)nCO] (n = 1-4) corresponding to the P1-P1' scissile position of hirutonins is described. The substitution of the scissile amide function by a ketomethylene group is compatible with the enzyme active site and conferred complete plasma proteolytic stability. This modification also enhanced enzyme affinity up to 20-fold with hirutonin-4 (IIb, n = 4) displaying highest affinity (Ki = 140 +/- 20 pM). Hirutonins 1-4 exhibited potent inhibition of plasma prothrombin time (PT) and activated partial thromboplastin time (aPTT). The inhibition was biphasic and showed good correlation with the corresponding Ki. Hirutonin-2 inhibited thrombin-mediated platelet aggregation and exhibited a strong antithrombotic effect comparable to r-hirudin in an in vivo rat arteriovenous shunt model (ED15 = 1.20 mg/kg for hirutonin-2 and 1.14 mg/kg for r-hirudin). Lower molecular weight inhibitors were obtained by substituting the six native amino acid residues (Q-S-H-N-D-G), connecting the active site and the auxiliary exosite binding elements with a variable number of interening omega-aminopentenoyl units. In addition, the exosite component was reduced to seven amino acid residues (D-F-E-P-I-P-L). Incorporation of these modifications into the bifunctional format resulted in nanomolar thrombin inhibitory peptides (IIIa-c). The resulting inhibitors were studied by molecular modeling with alpha-thrombin, and the bimolecular interactions served to explain the retention of high enzyme affinity.

Characterization of the interactions of a bifunctional inhibitor with alpha-thrombin by molecular modelling and peptide synthesis.

A potent thrombin inhibitor, [D-Phe45, Arg47] hirudin 45-65, that contains an active site-directed sequence D-Phe-Pro-Arg-Pro, an exosite specific fragment hirudin 55-65 (H55-65) and a linker portion hirudin 49-54, was designed based on the hirudin sequence [DiMaio et al. (1990) J. Biol. Chem., 265, 21698-21798]. A three-dimensional model of the complex between the B-chain of human thrombin and the inhibitor [D-Phe45, Arg47] hirudin 45-65 was constructed using molecular modelling starting from the X-ray C alpha coordinates of the thrombin-hirudin complex and the NMR-derived structure of the thrombin-bound hirudin 55-65. The contribution of the H49-54 fragment to the thrombin-inhibitor interaction was deduced by examining a series of analogs containing single glycine substitution and analogs with reduced number of residues within the linker. The results were consistent with the molecular modelling observations i.e. the H49-54 fragment serves the role of a spacer in the binding interaction and could be replaced by four glycine residues. The studies on the interaction of the exosite-directed portion of the inhibitor with thrombin using a series of synthetic H55-65 analogs demonstrated that residues AspH55 to ProH60 play a major role in binding to human thrombin where the side chains of PheH56, IleH59 and GluH57 showed critical contributions. Molecular modelling suggested that these side chains may contribute to inter- and intramolecular hydrophobic and electrostatic interactions, respectively.