Jan Konvalinka

From nonpeptide toward noncarbon protease inhibitors: Metallacarboranes as specific and potent inhibitors of HIV protease

HIV protease (PR) represents a prime target for rational drug design, and protease inhibitors (PI) are powerful antiviral drugs. Most of the current PIs are pseudopeptide compounds with limited bioavailability and stability, and their use is compromised by high costs, side effects, and development of resistant strains. In our search for novel PI structures, we have identified a group of inorganic compounds, icosahedral metallacarboranes, as candidates for a novel class of nonpeptidic PIs. Here, we report the potent, specific, and selective competitive inhibition of HIV PR by substituted metallacarboranes. The most active compound, sodium hydrogen butylimino bis-8,8-[5-(3-oxa-pentoxy)-3-cobalt bis(1,2-dicarbollide)]di-ate, exhibited a Ki value of 2.2 nM and a submicromolar EC50 in antiviral tests, showed no toxicity in tissue culture, weakly inhibited human cathepsin D and pepsin, and was inactive against trypsin, papain, and amylase. The structure of the parent cobalt bis(1,2-dicarbollide) in complex with HIV PR was determined at 2.15 Å resolution by protein crystallography and represents the first carborane-protein complex structure determined. It shows the following mode of PR inhibition: two molecules of the parent compound bind to the hydrophobic pockets in the flap-proximal region of the S3 and S3′ subsites of PR. We suggest, therefore, that these compounds block flap closure in addition to filling the corresponding binding pockets as conventional PIs. This type of binding and inhibition, chemical and biological stability, low toxicity, and the possibility to introduce various modifications make boron clusters attractive pharmacophores for potent and specific enzyme inhibition.

Structure of glutamate carboxypeptidase II, a drug target in neuronal damage and prostate cancer

Membrane‐bound glutamate carboxypeptidase II (GCPII) is a zinc metalloenzyme that catalyzes the hydrolysis of the neurotransmitter N‐acetyl‐L‐aspartyl‐L‐glutamate (NAAG) to N‐acetyl‐L‐aspartate and L‐glutamate (which is itself a neurotransmitter). Potent and selective GCPII inhibitors have been shown to decrease brain glutamate and provide neuroprotection in preclinical models of stroke, amyotrophic lateral sclerosis, and neuropathic pain. Here, we report crystal structures of the extracellular part of GCPII in complex with both potent and weak inhibitors and with glutamate, the product of the enzymes hydrolysis reaction, at 2.0, 2.4, and 2.2 Å resolution, respectively. GCPII folds into three domains: protease‐like, apical, and C‐terminal. All three participate in substrate binding, with two of them directly involved in C‐terminal glutamate recognition. One of the carbohydrate moieties of the enzyme is essential for homodimer formation of GCPII. The three‐dimensional structures presented here reveal an induced‐fit substrate‐binding mode of this key enzyme and provide essential information for the design of GCPII inhibitors useful in the treatment of neuronal diseases and prostate cancer.

A Novel Substrate-Based HIV-1 Protease Inhibitor Drug Resistance Mechanism

Background HIV protease inhibitor (PI) therapy results in the rapid selection of drug resistant viral variants harbouring one or two substitutions in the viral protease. To combat PI resistance development, two approaches have been developed. The first is to increase the level of PI in the plasma of the patient, and the second is to develop novel PI with high potency against the known PI-resistant HIV protease variants. Both approaches share the requirement for a considerable increase in the number of protease mutations to lead to clinical resistance, thereby increasing the genetic barrier. We investigated whether HIV could yet again find a way to become less susceptible to these novel inhibitors. Methods and Findings We have performed in vitro selection experiments using a novel PI with an increased genetic barrier (RO033-4649) and demonstrated selection of three viruses 4- to 8-fold resistant to all PI compared to wild type. These PI-resistant viruses did not have a single substitution in the viral protease. Full genomic sequencing revealed the presence of NC/p1 cleavage site substitutions in the viral Gag polyprotein (K436E and/or I437T/V) in all three resistant viruses. These changes, when introduced in a reference strain, conferred PI resistance. The mechanism leading to PI resistance is enhancement of the processing efficiency of the altered substrate by wild-type protease. Analysis of genotypic and phenotypic resistance profiles of 28,000 clinical isolates demonstrated the presence of these NC/p1 cleavage site mutations in some clinical samples (codon 431 substitutions in 13%, codon 436 substitutions in 8%, and codon 437 substitutions in 10%). Moreover, these cleavage site substitutions were highly significantly associated with reduced susceptibility to PI in clinical isolates lacking primary protease mutations. Furthermore, we used data from a clinical trial (NARVAL, ANRS 088) to demonstrate that these NC/p1 cleavage site changes are associated with virological failure during PI therapy. Conclusions HIV can use an alternative mechanism to become resistant to PI by changing the substrate instead of the protease. Further studies are required to determine to what extent cleavage site mutations may explain virological failure during PI therapy.

Substrate specificity, inhibition and enzymological analysis of recombinant human glutamate carboxypeptidase II

Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21) is a membrane peptidase expressed in a number of tissues such as kidney, prostate and brain. The brain form of GCPII (also known as NAALADase) cleaves N‐acetyl‐aspartyl glutamate to yield free glutamate. Animal model experiments show that inhibition of GCPII prevents neuronal cell death during experimental ischaemia. GCPII thus represents an important target for the treatment of neuronal damage caused by excess glutamate. In this paper we report expression of an extracellular portion of human glutamate carboxypeptidase II (amino acids 44–750) in Drosophila Schneiders cells and its purification to homogeneity. A novel assay for hydrolytic activity of recombinant human GCPII (rhGCPII), based on fluorimetric detection of released alpha‐amino groups was established, and used for its enzymological characterization. rhGCPII does not show dipeptidylpeptidase IV‐like activity assigned to the native form of the enzyme previously. Using a complete set of protected dipeptides, substrate specificity of rhGCPII was elucidated. In addition to the previously described substrates, four novel compounds, Ac‐Glu‐Met, Ac‐Asp‐Met and, surprisingly, Ac‐Ala‐Glu and Ac‐Ala‐Met were identified as substrates for GCPII, and their respective kinetic constants determined. The glycosylation of rhGCPII was found indispensable for the enzymatic activity.

A reliable docking/scoring scheme based on the semiempirical quantum mechanical PM6-DH2 method accurately covering dispersion and H-bonding: HIV-1 protease with 22 ligands.

In this study, we introduce a fast and reliable rescoring scheme for docked complexes based on a semiempirical quantum mechanical PM6-DH2 method. The method utilizes a PM6-based Hamiltonian with corrections for dispersion energy and hydrogen bonds. The total score is constructed as the sum of the PM6-DH2 interaction enthalpy, the empirical force field (AMBER) interaction entropy, and the sum of the deformation (PM6-DH2, SMD) and the desolvation (SMD) energies of the ligand. The main advantage of the procedure is the fact that we do not add any empirical parameter for either an individual component of the total score or an individual protein-ligand complex. This rescoring method is applied to a very challenging system, namely, the HIV-1 protease with a set of ligands. As opposed to the conventional DOCK procedure, the PM6-DH2 rescoring based on all of the terms distinguishes between binders and nonbinders and provides a reliable correlation of the theoretical and experimental binding free energies. Such a dramatic improvement, resulting from the PM6-DH2 rescoring of all the complexes, provides a valuable yet inexpensive tool for rational drug discovery and de novo ligand design.

Expression of glutamate carboxypeptidase II in human brain.

Glutamate carboxypeptidase II (GCPII) is a transmembrane glycoprotein expressed in various tissues. When expressed in the brain it cleaves the neurotransmitter N-acetylaspartylglutamate (NAAG), yielding free glutamate. In jejunum it hydrolyzes folylpoly-gamma-glutamate, thus facilitating folate absorption. The prostate form of GCPII, known as prostate specific membrane antigen (PSMA), is an established cancer marker. The NAAG-hydrolyzing activity of GCPII has been implicated in a number of pathological conditions in which glutamate is neurotoxic (e.g. amyotrophic lateral sclerosis, Huntingtons disease, Alzheimers disease, epilepsy, schizophrenia, and stroke). Inhibition of GCPII was shown to be neuroprotective in tissue culture and in animal models. GCPII is therefore an interesting putative therapeutic target. However, only very limited and controversial data on the expression and localization of GCPII in human brain are available. Therefore, we set out to analyze the activity and expression of GCPII in various compartments of the human brain using a radiolabeled substrate of the enzyme and the novel monoclonal antibody GCP-04, which recognizes an epitope on the extracellular portion of the enzyme and is more sensitive to GCPII than to the homologous GCPIII. We show that this antibody is more sensitive in immunoblots than the widely used antibody 7E11. By Western blot, we show that there are approximately 50-300 ng of GCPII/mg of total protein in human brain, depending on the specific area. Immunohistochemical analysis revealed that astrocytes specifically express GCPII in all parts of the brain. GCPII is enzymatically active and the level of activity follows the expression pattern. Using pure recombinant GCPII and homologous GCPIII, we conclude that GCPII is responsible for the majority of overall NAAG-hydrolyzing activity in the human brain.

Current and Novel Inhibitors of HIV Protease

The design, development and clinical success of HIV protease inhibitors represent one of the most remarkable achievements of molecular medicine. This review describes all nine currently available FDA-approved protease inhibitors, discusses their pharmacokinetic properties, off-target activities, side-effects, and resistance profiles. The compounds in the various stages of clinical development are also introduced, as well as alternative approaches, aiming at other functional domains of HIV PR. The potential of these novel compounds to open new way to the rational drug design of human viruses is critically assessed.

Mouse brain serine racemase catalyzes specific elimination of L‐serine to pyruvate

D‐Serine was previously identified in mammalian brain and was shown to be a co‐agonist at the ‘glycine’ site of the N‐methyl‐D‐aspartate (NMDA)‐type receptors. Racemization of serine is catalyzed by serine racemase, a pyridoxal 5′‐phosphate‐dependent enzyme expressed mainly in brain and liver. NMDA receptor overactivation has been implicated in a number of pathological conditions and inhibitors of serine racemase are thus potentially interesting targets for therapy. We expressed recombinant mouse serine racemase in insect cells and purified it to near homogeneity. The enzyme is a non‐covalent homodimer in solution and requires divalent cations Mg2+, Ca2+ or Mn2+ for activity but not for dimerization. In addition to the racemization it also catalyzes specific elimination of L‐Ser to pyruvate. D‐Serine is eliminated much less efficiently. Both L‐serine racemization and elimination activities of serine racemase are of comparable magnitude, display alkaline pH optimum and are negligible below pH 6.5.

Hydrolysis of synthetic chromogenic substrates by HIV-1 and HIV-2 proteinases.

Kinetic constants (Km,Kcat) are derived for the hydrolysis of a number of chromogenic peptide substrates by the aspartic proteinase from HIV-2. The effect of systematic replacement of the P2 residue on substrate hydrolysis by HIV-1 and HIV-2 proteinases is examined.

Identification of the N-glycosylation sites on glutamate carboxypeptidase II necessary for proteolytic activity.

Glutamate carboxypeptidase II (GCPII) is a membrane peptidase expressed in the prostate, central and peripheral nervous system, kidney, small intestine, and tumor‐associated neovasculature. The GCPII form expressed in the central nervous system, termed NAALADase, is responsible for the cleavage of N‐acetylL‐aspartyl‐L‐glutamate (NAAG) yielding free glutamate in the synaptic cleft, and is implicated in various pathologic conditions associated with glutamate excitotoxicity. The prostate form of GCPII, termed prostate‐specific membrane antigen (PSMA), is up‐regulated in cancer and used as an effective prostate cancer marker. Little is known about the structure of this important pharmaceutical target. As a type II membrane protein, GCPII is heavily glycosylated. In this paper we show that N‐glycosylation is vital for proper folding and subsequent secretion of human GCPII. Analysis of the predicted N‐glycosylation sites also provides evidence that these sites are critical for GCPII carboxypeptidase activity. We confirm that all predicted N‐glycosylation sites are occupied by an oligosaccharide moiety and show that glycosylation at sites distant from the putative catalytic domain is critical for the NAAG‐hydrolyzing activity of GCPII calling the validity of previously described structural models of GCPII into question.