Manfred Philipp

Mental retardation linked to mutations in the HSD17B10 gene interfering with neurosteroid and isoleucine metabolism.

Mutations in the HSD17B10 gene were identified in two previously described mentally retarded males. A point mutation c.776G>C was found from a survivor (SV), whereas a potent mutation, c.419C>T, was identified in another deceased case (SF) with undetectable hydroxysteroid (17β) dehydrogenase 10 (HSD10) activity. Protein levels of mutant HSD10(R130C) in patient SF and HSD10(E249Q) in patient SV were about half that of HSD10 in normal controls. The E249Q mutation appears to affect HSD10 subunit interactions, resulting in an allosteric regulatory enzyme. For catalyzing the oxidation of allopregnanolone by NAD+ the Hill coefficient of the mutant enzyme is ≈1.3. HSD10(E249Q) was unable to catalyze the dehydrogenation of 2-methyl-3-hydroxybutyryl-CoA and the oxidation of allopregnanolone, a positive modulator of the γ-aminobutyric acid type A receptor, at low substrate concentrations. Neurosteroid homeostasis is critical for normal cognitive development, and there is increasing evidence that a blockade of isoleucine catabolism alone does not commonly cause developmental disabilities. The results support the theory that an imbalance in neurosteroid metabolism could be a major cause of the neurological handicap associated with hydroxysteroid (17β) dehydrogenase 10 deficiency.

Inhibition of subtilisin by substituted arylboronic acids

Alkyl and arylboronic acids are potent competitive inhibitors of serine proteases [l-S]. These compounds have been known to inhibit hydrolytic enzymes at least since Torssel showed that benzeneboronic acids are potent inhibitors of serum cholinesterase [6]. Crystal structure determinations [7], NMR studies [8], and laser Raman studies [9] have helped to determine the structure of the enzyme-inhibitor complex, while rapid kinetic studies have helped to elucidate the mechanism of association [lo]. Here, we report dissociation constants for a number of different boronic acid inhibitors of the bacterial protease subtilisin Novo (EC 3.4.2 1 .14). One of these, Ndansyl-3aminobenzeneboronic acid (or N-(5 dimethylamino-1 -naphthalenesulfonyl)-3-aminobenzeneboronic acid, abbreviated as Dns-BBA) is a fluorescent boronic acid first prepared in [ 111, for use in cytological experiments as a carbohydrate ligand. We find that the introduction of the dansyl group makes this compound the most potent boronic acid serine protease inhibitor yet found. This inhibitor also exhibits a significant increase in fluorescence intensity upon binding. We also report binding constants for a variety of other newly studied arylboronic acids. Halogenated benzeneboronic acids are good subtilisin inhibitors, with dissociation constants <lo-’ M.

A Novel Mutation in the HSD17B10 Gene of a 10-Year-Old Boy with Refractory Epilepsy, Choreoathetosis and Learning Disability

Hydroxysteroid (17beta) dehydrogenase 10 (HSD10) is a mitochondrial multifunctional enzyme encoded by the HSD17B10 gene. Missense mutations in this gene result in HSD10 deficiency, whereas a silent mutation results in mental retardation, X-linked, syndromic 10 (MRXS10). Here we report a novel missense mutation found in the HSD17B10 gene, namely c.194T>C transition (rs104886492), brought about by the loss of two forked methyl groups of valine 65 in the HSD10 active site. The affected boy, who possesses mutant HSD10 (p.V65A), has a neurological syndrome with metabolic derangements, choreoathetosis, refractory epilepsy and learning disability. He has no history of acute decompensation or metabolic acidosis whereas his urine organic acid profile, showing elevated levels of 2-methyl-3-hydroxybutyrate and tiglylglycine, is characteristic of HSD10 deficiency. His HSD10 activity was much lower than the normal control level, with normal β-ketothiolase activity. The c.194T>C mutation in HSD17B10 can be identified by the restriction fragment polymorphism analysis, thereby facilitating the screening of this novel mutation in individuals with intellectual disability of unknown etiology and their family members much easier. The patients mother is an asymptomatic carrier, and has a mixed ancestry (Hawaiian, Japanese and Chinese). This demonstrates that HSD10 deficiency patients are not confined to a particular ethnicity although previously reported cases were either Spanish or German descendants.

Rational design and characterization of d-phe-pro-d-arg-derived direct thrombin inhibitors.

The tremendous social and economic impact of thrombotic disorders, together with the considerable risks associated to the currently available therapies, prompt for the development of more efficient and safer anticoagulants. Novel peptide-based thrombin inhibitors were identified using in silico structure-based design and further validated in vitro. The best candidate compounds contained both l- and d-amino acids, with the general sequence d-Phe(P3)-Pro(P2)-d-Arg(P1)-P1′-CONH2. The P1′ position was scanned with l- and d-isomers of natural or unnatural amino acids, covering the major chemical classes. The most potent non-covalent and proteolysis-resistant inhibitors contain small hydrophobic or polar amino acids (Gly, Ala, Ser, Cys, Thr) at the P1′ position. The lead tetrapeptide, d-Phe-Pro-d-Arg-d-Thr-CONH2, competitively inhibits α-thrombins cleavage of the S2238 chromogenic substrate with a Ki of 0.92 µM. In order to understand the molecular details of their inhibitory action, the three-dimensional structure of three peptides (with P1′ l-isoleucine (fPrI), l-cysteine (fPrC) or d-threonine (fPrt)) in complex with human α-thrombin were determined by X-ray crystallography. All the inhibitors bind in a substrate-like orientation to the active site of the enzyme. The contacts established between the d-Arg residue in position P1 and thrombin are similar to those observed for the l-isomer in other substrates and inhibitors. However, fPrC and fPrt disrupt the active site His57-Ser195 hydrogen bond, while the combination of a P1 d-Arg and a bulkier P1′ residue in fPrI induce an unfavorable geometry for the nucleophilic attack of the scissile bond by the catalytic serine. The experimental models explain the observed relative potency of the inhibitors, as well as their stability to proteolysis. Moreover, the newly identified direct thrombin inhibitors provide a novel pharmacophore platform for developing antithrombotic agents by exploring the conformational constrains imposed by the d-stereochemistry of the residues at positions P1 and P1′.

A 5-methylcytosine hotspot responsible for the prevalent HSD17B10 mutation

Approximately half of the cases of hydroxysteroid (17β) dehydrogenase X (HSD10) deficiency are due to a missense C>T mutation in exon 4 of the HSD17B10 gene. The resulting HSD10 (p.R130C) loses most or all catalytic functions, and the males with this mutation have a much more severe clinical phenotype than those carrying p.V65A, p.L122V, or p.E249Q mutations. We found that the mutated cytosine which is +2259 nucleotide from the ATG of the gene, is >90% methylated in both the active and inactive X chromosomes in two normal females as well as in the X chromosome of a normal male. Since 5-methylcytosine is prone to conversion to thymine by deamination, the methylation of this cytosine in normal X chromosomes provides an explanation for the prevalence of the p.R130C mutation among patients with HSD10 deficiency. The substitution of arginine for cysteine eliminates several hydrogen bonds and reduces the van der Waals interaction between HSD10 subunits. The resulting disruption of protein structure impairs some if not all of the catalytic and non-enzymatic functions of HSD10. A meta-analysis of residual HSD10 activity in eight patients with the p.R130C mutation showed an average 2-methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD) activity of only 6 (±5) % of the normal control level. This is significantly lower than in cells of patients with other, clinically milder mutations and suggests that the loss of HSD10/MHBD activity is a marker for the disorder.

Isothermal titration calorimetry and inhibition of platelets aggregation by [D-Phe/(transcinnamoyl)-Pro-D-Arg-P1'-CONH2] peptides inhibitors of thrombin.

Introduction Thrombin is the major product of the plasma coagulation “cascade” of sequential “zymogen-to-protease” steps. As the result of a series of proteolytic cleavages, thrombin converts fibrinogen into fibrin, which deposits at the site of bleeding or thrombosis as the fibrinous portion of a haemostatic plug or thrombotic mass(1). Thrombin also stimulates the platelets through its protein G-coupled receptors PARs (protease activated receptors) (PAR1,4 in humans) being their most potent activator. Thrombin induced-platelets activation plays a critical role in the pathophysiology of thrombosis (1). Activated platelets bind to fibrinogen, causing platelets to aggregate at the site of a cardiovascular injury to form a thrombus that is further stabilized by thrombin-generated fibrin network (1). The discovery of new antithrombotic drugs was primarily focused on finding competitive inhibitors of thrombin, however new approaches target thrombin receptor PAR-1 (such as the PAR-1 structural antagonists) offering additional cardiovascular utility in the treatment of atherosclerosis and restenosis (1). We previously reported the discovery of new tetrapeptides with the sequence space D-Phe-Pro-D-Arg-P1’-CONH2 as reversible competitive inhibitors of thrombin (2) (were P1’ requires small, polar natural or unnatural aminoacids). To further evaluate their potential as antithrombotic drugs and as potential PAR-1 antagonists we synthesized new peptides based on the same sequence space but with D-configuration in P1’. The actual paper presents the structure-activity relationship (SAR) for some L/D tetrapeptides isomers and their evaluation as thrombin inhibitors using biophysical/biochemical approaches (isothermal titration calorimetry and kinetics of thrombin inhibition) and cell-based assays (thrombin mediated platelets aggregation). The synthesis of the D-isomers was performed only for the lead L-peptides (Ki (inhibitory constant) less then 20 μM) (2). Additional tetrapeptides were synthesized with the transcinnamoyl group at P3 position (instead of D-Phe) based on their prediction as high affinity competitive binders of thrombin (2).

In silico-mediated virtual screening and molecular docking platforms for discovery of non β-lactam inhibitors of y-49 β-lactamase from Mycobacterium tuberculosis

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is a worldwide health concern. The world health organization in its 2017 tuberculosis report states, “TB is the ninth leading cause of death worldwide and the leading cause from a single infectious agent, ranking above HIV/AIDS” [1-3]. The failure to control TB is due to the emergence of Mtb strains that are resistant to first line beta lactam antibiotics, because of overuse. One of the most effective resistance mechanisms to β-lactam antibiotics involves the production of β-lactamases which cleave the amide bond in the target β-lactam ring hydrolyzing the drug before it reaches its target. The beta-lactamases are classified into four classes: A, B, C and D. These classes are based on conserved and distinguishing amino acid motifs [1-3]. Classes A, C, and D include enzymes that hydrolyze their substrates by forming an acyl enzyme through an active site serine. Class B β-lactamases are metalloenzymes that utilize at least one active-site zinc ion to facilitate β-lactam hydrolysis. One of the most efficient and prevalent mechanisms of resistance to β-lactam antibiotics is the production of β-lactamases in both Gram-negative and Gram-positive bacteria that hydrolyze the drugs before they can reach their target and exert the desired effect. These resistance mechanisms are important, and each bacterium can create a combination of defenses depending on the selective pressures placed on it [1-4]. The intrinsic resistance to β-lactam antibiotics was demonstrated to be mainly due to the presence of a chromosomally-encoded gene (blaC) in M. tuberculosis for a Class A, Ambler β-lactamase (BlaC). The BlaC enzyme has already been validated as one of the leading targets of tuberculosis therapy. This enzyme is extremely active against Volume 7 Issue 1 2018

Structure-Based Design, Synthesis and Evaluation of Novel Peptidic Inhibitors of Thrombin-Induced Activation of Platelets Aggregation

Janet Gonzalez, Anna Babinska, Ebenezer L.V. Ewul, Edem Timpo, Alhassan Jallow, Zhiyong Qiu, Radoslaw Bednarek, Maria Swiatkowska, Moro O. Salifu, Manfred Philipp, and Cristina C. Clement Department of Natural Sciences, LaGuardia Community College, New York, NY, 11104, USA; Division of Nephrology, Department of Medicine, State University of New York, Downstate Medical Center, Brooklyn, NY, 11203, USA; Department of Chemistry, Lehman College of the City University of New York, Bronx, NY, 10468, USA; Medical University of Lodz Department of Cytobiology and Proteomics, Lodz, Poland

pH-Dependent Binding Constants for the Inhibition of Thrombin by Transition State Analogs

Transition state analogs provide a unique view into the course of enzyme catalysis by providing a model of the enzyme’s state during the reaction. Studying the pH dependencies of binding by such inhibitors promises a better understanding of the course of catalysis, especially when these dependencies are compared to those of substrates and other inhibitors.

NONCODING INSERTS IN HEMOGOLOBIN AND SIMIAN VIRUS 40 GENES ARE BOUNDED BY SELF-COMPLEMENTARY REGIONS

Publisher Summary This chapter examines noncoding inserts in hemoglobin and simian virus 40 genes bounded by self-complementary regions. Studies have shown that many eukaryotic genes contain noncoding internal space regions called introns. Several programs are prepared that analyze DNA and RNA sequences. These programs help determine whether these primary sequences contain self-complementarities that might aid in ligation of the coding, exon regions that remain in the final mRNAs. The presence of complementary regions in exons that are eventually ligated to form mRNAs may facilitate such ligation by bringing areas to be ligated into close physical proximity and by providing recognition sufficient to ensure that appropriate exons are ligated. Extensive complementary regions were found in both rabbit β-globin mRNA and SV-40 DNA. The potentially most stable self-complementarity found in rabbit β-globin mRNA extends from nucleotide number 231 to nucleotide number 488. The known rabbit intron is excised at the central, nonbase-paired loop, which may be formed by this self-complementarity.