Tetramic acid based alkaloids from Aspergillus amoenus Roberg strain UP197 - antibiotic properties and new pyranterreones.

The fungus Aspergillus amoenus Roberg strain UP197 was shown to produce antibacterial tetramic acid based alkaloids. Two new compounds, pyranterreone I and J (1 and 2), were isolated and characterized, in addition to the known compounds cordylactam, 7-hydroxycordylactam, pyranterreone C, D, F and G. Neither the pyranterreones nor the cordylacctams had previously been tested for antimicrobial activity. Thus, all isolated compounds were tested against a panel of clinically important bacteria and fungi. Pyranterreone C was active against Gram-positive and Gram-negative bacteria, with minimal inhibitory concentrations (MIC) between 1 and 8 µg/mL, whereas the MICs for all other compounds were >32 µg/mL. Pyranoterreone C was cytotoxic towards HepG2 cells, and since pyranterreone C reacted rapidly with the nucleophile cysteine, it is likely that the observed antibacterial activity is due to the chemical reactivity rather than enzymatic affinity, making it unsuitable for development as an antibacterial drug.

Selective Isolation of Multidrug-Resistant Pedobacter spp., Producers of Novel Antibacterial Peptides.

Twenty-eight multidrug-resistant bacterial strains closely related or identical to Pedobacter cryoconitis, Pedobacter lusitanus and Pedobacter steynii were isolated from soil samples by selection for multidrug-resistance. Approximately 3-30% of the selected isolates were identified as Pedobacter, whereas isolation without antibiotics did not yield any isolates of this genus. Next generation sequencing data showed Pedobacter to be on 69th place among the bacterial genera (0.32% of bacterial sequences). The Pedobacter isolates produced a wide array of novel compounds when screened by UHPLC-MS/MSMS, and hierarchical cluster analysis resulted in several distinct clusters of compounds produced by specific isolates of Pedobacter, and most of these compounds were found to be peptides. The Pedobacter strain UP508 produced isopedopeptins, whereas another set of strains produced pedopeptins, which both are known cyclic lipodepsipeptides produced by Pedobacter sp. Other Pedobacter strains produced analogous peptides with a sequence variation. Further strains of Pedobacter produced additional novel antibacterial cyclic lipopeptides (ca 800 or 1400 Da in size) and/or linear lipopeptides (ca 700-960 Da in size). A 16S rRNA phylogenetic tree for the Pedobacter isolates revealed several distinct clades and subclades of isolates. One of the subclades comprised isolates producing isopedopeptin analogs, but the isopedopeptin producing isolate UP508 was clearly placed on a separate branch. We suggest that the non-ribosomal peptide synthases producing pedopeptins, isopedopeptins, and the analogous peptides, may derive from a common ancestral non-ribosomal peptide synthase gene cluster, which may have been subjected to a mutation leading to changed specificity in one of the modules and then to a modular rearrangement leading to the changed sequence found in the isopedopeptins produced by isolate UP508.

In-peptide amino acid racemization via inter-residue oxazoline intermediates during acidic hydrolysis.

Isopedopeptins are antibiotic cyclic lipodepsipeptides containing the subsequence L-Thr-L-2,3-diaminopropanoic acid-D-Phe-L-Val/L-3-hydroxyvaline. Acidic hydrolysis of isopedopeptins in D2O showed the D-Phe residues to racemize extensively in peptides with L-3-hydroxyvaline but not in peptides with L-Val. Similarly, one Leu residue in pedopeptins, which are related peptides containing the subsequence Leu-2,3-diaminopropanoic acid-Leu-L-Val/L-3-hydroxyvaline, was found to racemize in peptides with L-3-hydroxyvaline. Model tetrapeptides, L-Ala-L-Phe-L-Val/3-hydroxyvaline-L-Ala, gave the corresponding results, i.e. racemization of L-Phe only when linked to a L-3-hydroxyvaline. We propose the racemization to proceed via an oxazoline intermediate involving Phe/Leu and the L-3-hydroxyvaline residues. The 3-hydroxyvaline residue may form a stable tertiary carbocation by loss of the sidechain hydroxyl group as water after protonation. Elimination of the Phe/Leu H-2 and ring-closure from the carbonyl oxygen onto the carbocation results in the suggested oxazoline intermediate. The reversed reaction leads to either retained or inversed configuration of Phe/Leu. Such racemization during acidic hydrolysis may occur whenever a 3-hydroxyvaline residue or any amino acid that can form a stable carbocation on the C-3, is present in a peptide. The proposed mechanism for racemization was supported by incorporation of 18O in the 3-hydroxyvaline sidechain when the acidic hydrolysis was performed in H2O/H218O (1:1). The 2,3-diaminopropanoic residues of isopedopeptins and pedopeptins were also found to racemize during acidic hydrolysis, as previously described. Based on the results, the configuration of the Leu and 2,3-diaminopropanoic acid residues of the pedopeptins were reassigned to be L-Leu and D-Leu, and 2 × L-2,3-diaminopropanoic acid.

Isopedopeptins A-H: Cationic Cyclic Lipodepsipeptides from Pedobacter cryoconitis UP508 Targeting WHO Top-Priority Carbapenem-Resistant Bacteria.

Pedobacter cryoconitis strain UP508 was isolated from a soil sample using a mixture of ampicillin, kanamycin, and nalidixic acid for selection. UP508 was found to produce >30 unknown antibacterial peptides, of which eight, isopedopeptins A-H (1-8), were isolated by bioassay-guided fractionation and characterized with respect to structures and biological properties. Compounds 1-8 were all composed of nine amino acid residues and one 3-hydroxy fatty acid residue, and the structures were ring-closed via an ester bond from the C-terminal aspartic acid to the 3-hydroxy fatty acid. The differences between the peptides were the size and branching of the 3-hydroxy fatty acid and the presence of a valine or a 3-hydroxyvaline residue. The isopedopeptins mainly had activity against Gram-negative bacteria, and isopedopeptin B (2), which had the best combination of antibacterial activity, in vitro cytotoxicity, and hemolytic properties, was selected for further studies against a larger panel of Gram-negative bacteria. Isopedopeptin B was found to have good activity against strains of WHO top-priority Gram-negative bacteria, i.e., carbapenem-resistant Acinetobacter baumannii, Escherichia coli, and Pseudomonas aeruginosa, with minimal inhibitory concentrations (MIC) down to 1, 2, and 4 µg/mL, respectively. Furthermore, compound 2 had activity against colistin-resistant strains of A. baumannii, E. coli, and Klebsiella pneumoniae, with a MIC down to 8, 2, and 4 µg/mL, respectively. Compound 6 was tested in an E. coli liposome system where it induced significant leakage, indicating membrane disruption as one mechanism involved in isopedopeptin antibacterial activity. Isopedopeptin B stands out as a promising candidate for further studies with the goal to develop a new antibiotic drug.

Antibacterial pyrrolidinyl and piperidinyl substituted 2,4-diacetylphloroglucinols from Pseudomonas protegens UP46.

In the search for new antibiotic compounds, fractionation of Pseudomonas protegens UP46 culture extracts afforded several known Pseudomonas compounds, including 2,4-diacetylphloroglucinol (DAPG), as well as two new antibacterial alkaloids, 6-(pyrrolidin-2-yl)DAPG (1) and 6-(piperidin-2-yl)DAPG (2). The structures of 1 and 2 were determined by nuclear magnetic resonance spectroscopy and mass spectrometry. Compounds 1 and 2 were found to have antibacterial activity against the Gram-positive bacteria Staphylococcus aureus and Bacillus cereus, with minimal inhibitory concentration (MIC) 2 and 4 µg ml-1, respectively, for 1, and 2 µg ml-1 for both pathogens for 2. The MICs for 1 and 2, against all tested Gram-negative bacteria, were >32 µg ml-1. The half maximal inhibitory concentrations against HepG2 cells for compounds 1 and 2 were 11 and 18 µg ml-1, respectively, which suggested 1 and 2 be too toxic for further evaluation as possible new antibacterial drugs. Stable isotope labelling experiments showed the pyrrolidinyl group of 1 to originate from ornithine and the piperidinyl group of 2 to originate from lysine. The P. protegens acetyl transferase (PpATase) is involved in the biosynthesis of monoacetylphloroglucinol and DAPG. No optical rotation was detected for 1 or 2, and a possible reason for this was investigated by studying if the PpATase may catalyse a stereo-non-specific introduction of the pyrrolidinyl/piperidinyl group in 1 and 2, but unless the PpATase can be subjected to major conformational changes, the enzyme cannot be involved in this reaction. The PpATase is, however, likely to catalyse the formation of 2,4,6-triacetylphloroglucinol from DAPG.

Antibacterial Isoquinoline Alkaloids from the Fungus Penicillium Spathulatum Em19.

In the search for new microbial antibacterial secondary metabolites, two new compounds (1 and 2) were isolated from culture broths of Penicillium spathulatum Em19. Structure determination by nuclear magnetic resonance and mass spectrometry identified the compounds as 6,7-dihydroxy-5,10-dihydropyrrolo[1,2-b]isoquinoline-3-carboxylic acid (1, spathullin A) and 5,10-dihydropyrrolo[1,2-b]isoquinoline-6,7-diol (2, spathullin B). The two compounds displayed activity against both Gram-negative and -positive bacteria, including Escherichia coli, Acinetobacter baumannii, Enterobacter cloacae, Klebsiella pneumonia, Pseudomonas aeruginosa, and Staphylococcus aureus. Compound 2 was more potent than 1 against all tested pathogens, with minimal inhibitory concentrations down to 1 µg/mL (5 µM) against S. aureus, but 2 was also more cytotoxic than 1 (50% inhibitory concentrations 112 and 11 µM for compounds 1 and 2, respectively, towards Huh7 cells). Based on stable isotope labelling experiments and a literature comparison, the biosynthesis of 1 was suggested to proceed from cysteine, tyrosine and methionine via a non-ribosomal peptides synthase like enzyme complex, whereas compound 2 was formed spontaneously from 1 by decarboxylation. Compound 1 was also easily oxidized to the 1,2-benzoquinone 3. Due to the instability of compound 1 and the toxicity of 2, the compounds are of low interest as possible future antibacterial drugs.

Antibacterial 3,6-Disubstituted 4-Hydroxy-5,6-dihydro-2H-pyran-2-ones from Serratia plymuthica MF371-2.

Bioassay-guided fractionation of culture extracts of Serratia plymuthica strain MF371-2 resulted in the isolation of two new antibacterial compounds with potent activity against Gram-positive bacteria, including Staphylococcus aureus LMG 15975 (MRSA). A spectroscopic investigation, in combination with synthesis, enabled the characterization of the compounds as 3-butyryl-4-hydroxy-6-heptyl-5,6-dihydro-2H-pyran-2-one (plymuthipyranone A, 1) and 3-butyryl-4-hydroxy-6-nonyl-5,6-dihydro-2H-pyran-2-one (plymuthipyranone B, 2). The MIC values for 1 and 2 against S. aureus LMG 15975 were determined to be 1-2 µg mL-1 and 0.8 µg mL-1, respectively. Compound 2 was found to have potent activity against many strains of S. aureus, including several mupirocin-resistant strains, other species of Staphylococcus, and vancomycin-resistant enterococci. Compound 2 was slightly cytotoxic for human cells, with CC50 values between 4.7 and 40 µg mL-1, but the CC50/MIC ratio was ≥10 for many tested combinations of human cells and bacteria, suggesting its possible use as an antibacterial agent. Several analogues were synthesized with different alkyl groups in the 3- and 6-positions (6-13), and their biological properties were evaluated. It was concluded that the activity of the compounds increased with the lengths of the alkyl and acyl substituents.

Inhibition of HIV-1 by non-nucleoside reverse transcriptase inhibitors via an induced fit mechanism-Importance of slow dissociation and relaxation rates for antiviral efficacy.

The importance of slow dissociation of non-nucleoside reverse transcriptase inhibitors (NNRTIs) for antiviral effect has been investigated. The kinetic characteristics of a series of NNRTIs interacting with wild type and drug resistant variants of HIV-1 RT (EC 2.7.7.49) were analyzed by SPR biosensor technology. The antiviral effect was determined in MT-4 and peripheral blood mononuclear cells. Due to extremely slow dissociation rates and a complex interaction mechanism, rate constants could not be quantified. Instead, interaction characteristics were qualitatively analyzed using simulated sensorgrams. The simplest model describing these interactions adequately was an induced fit mechanism, i.e. a mechanism involving the formation of an initial enzyme-inhibitor complex subsequently transformed into a more stable complex. Differences in rates of dissociation from the initial complex and rates of relaxation from the induced complex explained (1) the differences in the amounts of formed complex, (2) the stability of the complex and (3) the antiviral efficacies of the compounds. The effect of NNRTI binding site mutations also correlated with these kinetic characteristics. MIV-170 was the most effective inhibitor of wild type and mutant HIV-1 in cell culture, a property that was associated with the formation of the largest amount of complex and the slowest relaxation and dissociation rates. This study supports the hypothesis that the efficacy of anti-HIV drugs is dependent on slow dissociation from the target, thereby maximizing the duration of the inhibitory effect. It also illustrates the strength of simulating interaction data for qualitative analysis of tight-binding drugs and the importance of resolving the kinetic mechanism of drug-target interactions.

Prevention of ulcerative lesions by episodic treatment of recurrent herpes labialis: A literature review.

There are substantial difficulties involved in carrying out clinical studies of recurrent herpes labialis, since the disease has a rapid onset, short-lasting viral shedding period and is rapidly self-healing. The aim of this paper was to critically assess published reports of episodic treatment of herpes labialis and to review biological and methodological problems involved in such studies. Limited, but statistically significant, results have been shown with topical antivirals, such as acyclovir and penciclovir, improving healing times by approximately 10%. Orally administrated antivirals, such as valaciclovir and famciclovir, have subsequently found clinical use. However, these two oral medications have different profiles in phase 3 studies. Famciclovir showed additional improvement of efficacy in terms of lesion healing time, but no effect on prevention of ulcerative lesions, while valaciclovir appeared to have similar efficacy to that of acyclovir cream on lesion healing, but some additional efficacy with respect to prevention of ulcerative lesions. A formulation of acyclovir/hydrocortisone showed further improvement in prevention of ulcerative lesions, while retaining efficacy with respect to lesion healing.

Preclinical assessment of the treatment of second-stage African trypanosomiasis with cordycepin and deoxycoformycin.

BACKGROUND: There is an urgent need to substitute the highly toxic compounds still in use for treatment of the encephalitic stage of human African trypanosomiasis (HAT). We here assessed the treatment with the doublet cordycepin and the deaminase inhibitor deoxycoformycin for this stage of infection with Trypanosoma brucei (T.b.). METHODOLOGY/PRINCIPAL FINDINGS: Cordycepin was selected as the most efficient drug from a direct parasite viability screening of a compound library of nucleoside analogues. The minimal number of doses and concentrations of the drugs effective for treatment of T.b. brucei infections in mice were determined. Oral, intraperitoneal or subcutaneous administrations of the compounds were successful for treatment. The doublet was effective for treatment of late stage experimental infections with human pathogenic T.b. rhodesiense and T.b. gambiense isolates. Late stage infection treatment diminished the levels of inflammatory cytokines in brains of infected mice. Incubation with cordycepin resulted in programmed cell death followed by secondary necrosis of the parasites. T.b. brucei strains developed resistance to cordycepin after culture with increasing concentrations of the compound. However, cordycepin-resistant parasites showed diminished virulence and were not cross-resistant to other drugs used for treatment of HAT, i.e. pentamidine, suramin and melarsoprol. Although resistant parasites were mutated in the gene coding for P2 nucleoside adenosine transporter, P2 knockout trypanosomes showed no altered resistance to cordycepin, indicating that absence of the P2 transporter is not sufficient to render the trypanosomes resistant to the drug. CONCLUSIONS/SIGNIFICANCE: Altogether, our data strongly support testing of treatment with a combination of cordycepin and deoxycoformycin as an alternative for treatment of second-stage and/or melarsoprol-resistant HAT.

Profound antiviral effect of oral administration of MIV-210 on chronic hepadnaviral infection in a woodchuck model of hepatitis B.

MIV-210 is a prodrug of 3'-fluoro-2',3'-dideoxyguanosine with high oral bioavailability in humans and potent activity against hepatitis B virus (HBV). Woodchucks infected with woodchuck hepatitis virus (WHV) represent an accurate model of HBV infection that is utilized for evaluation of the efficacy and safety of novel anti-HBV agents. Oral administration of MIV-210 at 20 or 60 mg/kg of body weight/day induced a rapid virological response in chronically infected woodchucks, reducing serum WHV DNA levels by 4.75 log10 and 5.72 log10, respectively, in 2 weeks. A progressive decline in WHV viremia occurred throughout the 10-week therapy, giving final reductions of 7.23 log10 and 7.68 log10 in the 20- and 60-mg/kg/day groups, respectively. Further, a daily dose of 10 mg/kg decreased the serum WHV load 400-fold after 4 weeks of treatment, and a dose of 5 mg/kg/day was sufficient to maintain this antiviral effect during the following 6-week period. MIV-210 at 20 or 60 mg/kg/day reduced the liver WHV DNA load 200- to 2,500-fold from pretreatment levels and, importantly, led to a 2.0 log10 drop in the hepatic content of WHV covalently closed circular DNA. The treatment with 60 mg/kg/day was well tolerated. Liver biopsy specimens obtained after the 10-week treatment with 20 or 60 mg/kg/day and after the 10-week follow-up showed hepatocyte and mitochondrial ultrastructures comparable to those in the placebo-treated group. It was concluded that MIV-210 is highly effective against chronic WHV infection. These findings, together with the previously demonstrated inhibitory activity of MIV-210 against lamivudine-, adefovir-, and entecavir-resistant HBV variants, make MIV-210 a highly valuable candidate for further testing as an agent against chronic hepatitis B.

Screening for NNRTIs with slow dissociation and high affinity for a panel of HIV-1 RT variants.

A lead optimization library consisting of 800 HIV-1 nonnucleoside reverse transcriptase inhibitors (NNRTIs) was screened in parallel against 4 clinically relevant variants of HIV-1 RT (Wt, L100I, Y181C, and K103N) using a surface plasmon resonance-based biosensor. The aim was to identify inhibitors suitable in specific topical microbicides efficient for preventing the transmission of a range of clinically significant strains of HIV-1. The authors hypothesized that such compounds should have high affinity and slow dissociation rates for multiple variants of the target. To efficiently analyze the large amount of real-time data (sensorgrams) that were generated in the screening, they initially used signals from 3 selected time points to identify compounds with high affinity and slow dissociation for the complete panel of enzyme variants. Hits were confirmed by visually inspecting the complete sensorgrams. Two structurally unrelated compounds fulfilled the hit criteria, but only 1 compound was found to (a) compete with a known NNRTI for binding to the NNRTI site, (b) inhibit HIV-1 RT activity, and (c) inhibit HIV-1 replication in cell culture, for all 4 enzyme variants. This novel screening methodology offers high-resolution real-time kinetic data for multiple targets in parallel. It is expected to have broad applicability for the discovery of compounds with defined kinetic profiles, crucial for optimal therapeutic effects.

Rational design of polymerase inhibitors as antiviral drugs.

Almost all viruses have polymerases which are suitable targets for antiviral drugs. The development of selective polymerase inhibitors started with screening of compounds in virus-infected cell cultures and the mechanism of action was investigated once an inhibitor had been found. Especially nucleoside analogs were screened as their triphosphates were potential substrates for polymerases. However, the stepwise phosphorylation by cellular, and sometimes viral, kinases to the active triphosphate prevented a truly rational design of polymerase inhibitors. Nucleotide analogs offers a type of compounds which could be designed in a more rational way than nucleoside analogs since the first, most selective, phosphorylation step is eliminated in the path to the active inhibitor. The development of pyrophosphate analogs made rational design possible since these compounds act directly on the viral enzyme, but the room for structural variation was limited. The non-nucleoside HIV reverse transcriptase inhibitors are direct inhibitors and can thus be designed in a truly rational way by use of structure information on the enzyme-inhibitor complex by use of X-ray and NMR. This rational design of allosteric inhibitors is also being used in the development of inhibitors to other viral polymerases.

Achievements and challenges in antiviral drug discovery.

The last 40 years have seen the development of several antiviral drugs with therapeutic value in treating life-threatening or debilitating diseases such as those caused by HIV, hepatitis B virus, herpesviruses (such as herpes simplex virus and varicella zoster virus) and influenza virus. These relatively recent advances have been due to technical breakthroughs in the cultivation of viruses in the laboratory, identification of viral enzymes and, more recently, their molecular biology. We describe here the antecedence of several of the existing antivirals and their strengths and weaknesses. We indicate where the major challenges lie for future improvements of current therapies and possible new indications, such as hepatitis C virus and papillomavirus. We also describe how current antiviral therapies are restricted to a rather limited number of viral diseases of sufficient interest to the pharmaceutical industry. Finally we describe the potential threat of emerging viruses and bio-weapons and the challenges that they present to therapy.

MIV-310 reduces HIV viral load in patients failing multiple antiretroviral therapy: results from a 4-week phase II study.

BACKGROUND: Drug resistance is an increasing problem in the treatment of HIV infection. MIV-310 (alovudine), a nucleoside reverse transcriptase inhibitor, potently inhibits the replication of highly mutated strains of HIV in vitro. We examined the efficacy of MIV-310 in highly pretreated patients. METHODS: In a phase II pilot study, 15 patients failing a current antiretroviral therapy with at least two thymidine-associated mutations (TAM) were given MIV-310 7.5 mg once daily for 4 weeks, in addition to their ongoing treatment. The primary endpoint was the plasma viral load reduction at week 4. RESULTS: At baseline, the median viral load was 3.93 log10 copies/ml and the median CD4 cell count was 360 cells/mm3. After 4 weeks of MIV-310 administration, the median decrease in viral load was -1.13 log10. Interestingly, the median reduction was only -0.57 log10 in the four patients on stavudine, contrasting with a median reduction of -1.88 log10 in the 11 patients not receiving concomitant stavudine. The viral load fell by a median of -1.60 log10 in patients with two to three TAM (n = 7), and by -1.88 log10 in patients with four or five TAM (n = 8). The viral load rebounded in all patients after MIV-310 cessation. No mutations were found in the reverse transcriptase coding region during MIV-310 treatment. MIV-310 was well tolerated, with no serious adverse events and no treatment withdrawals. CONCLUSION: MIV-310 7.5 mg/day efficiently reduced the HIV viral load in patients failing a multiple-drug regimen. Further studies with different dosages and longer administration times are urgently needed.

The phenylmethylthiazolylthiourea nonnucleoside reverse transcriptase (RT) inhibitor MSK-076 selects for a resistance mutation in the active site of human immunodeficiency virus type 2 RT.

The phenylmethylthiazolylthiourea (PETT) derivative MSK-076 shows, besides high potency against human immunodeficiency virus type 1 (HIV-1), marked activity against HIV-2 (50% effective concentration, 0.63 microM) in cell culture. Time-of-addition experiments pointed to HIV-2 reverse transcriptase (RT) as the target of action of MSK-076. Recombinant HIV-2 RT was inhibited by MSK-076 at 23 microM. As was also found for HIV-1 RT, MSK-076 inhibited HIV-2 RT in a noncompetitive manner with respect to dGTP and poly(rC).oligo(dG) as the substrate and template-primer, respectively. MSK-076 selected for A101P and G112E mutations in HIV-2 RT and for K101E, Y181C, and G190R mutations in HIV-1 RT. The selected mutated strains of HIV-2 were fully resistant to MSK-076, and the mutant HIV-2 RT enzymes into which the A101P and/or G112E mutation was introduced by site-directed mutagenesis showed more than 50-fold resistance to MSK-076. Mapping of the resistance mutations to the HIV-2 RT structure ascertained that A101P is located at a position equivalent to the nonnucleoside RT inhibitor (NNRTI)-binding site of HIV-1 RT. G112E, however, is distal to the putative NNRTI-binding site in HIV-2 RT but close to the active site, implying a novel molecular mode of action and mechanism of resistance. Our findings have important implications for the development of new NNRTIs with pronounced activity against a wider range of lentiviruses.

ME-609: a treatment for recurrent herpes simplex virus infections.

Studies in conventional murine models of HSV infection use immunologically naive animals. These models thus mimic primary infections rather than recurrent infections in humans. We have, therefore, used a newly developed mouse model that more closely mimics recurrent HSV infection in humans. In this model, the mice are infected, and zosteriform HSV-1 infection develops in the presence of a primed immune response using adoptive transfer of immunity (ATI) as we have described previously. Using the ATI mouse model, it has been shown that a more beneficial therapy for recurrent mucocutaneous HSV infection could be achieved by controlling both the viral replication and the inflammatory response to the virus. Topical treatment was initiated in this model at the time of first occurrence of symptoms and was given three times daily for 4 days. Topical treatment with ME-609 (which contains 5% acyclovir and 1% hydrocortisone) in the ATI mouse model was substantially more efficacious than 5% Zovirax cream, 1% hydrocortisone or no treatment, respectively. The beneficial properties of ME-609 were also found to be superior to those of Zovirax cream when tested in the standard guinea pig model, representing a primary HSV infection. ME-609 represents a novel treatment principle of recurrent HSV infections and the present paper summarizes the preclinical and early clinical experience of ME-609.

Optimization of P1-P3 groups in symmetric and asymmetric HIV-1 protease inhibitors.

HIV-1 protease is an important target for treatment of AIDS, and efficient drugs have been developed. However, the resistance and negative side effects of the current drugs has necessitated the development of new compounds with different binding patterns. In this study, nine C-terminally duplicated HIV-1 protease inhibitors were cocrystallised with the enzyme, the crystal structures analysed at 1.8-2.3 A resolution, and the inhibitory activity of the compounds characterized in order to evaluate the effects of the individual modifications. These compounds comprise two central hydroxy groups that mimic the geminal hydroxy groups of a cleavage-reaction intermediate. One of the hydroxy groups is located between the delta-oxygen atoms of the two catalytic aspartic acid residues, and the other in the gauche position relative to the first. The asymmetric binding of the two central inhibitory hydroxyls induced a small deviation from exact C2 symmetry in the whole enzyme-inhibitor complex. The study shows that the protease molecule could accommodate its structure to different sizes of the P2/P2' groups. The structural alterations were, however, relatively conservative and limited. The binding capacity of the S3/S3' sites was exploited by elongation of the compounds with groups in the P3/P3' positions or by extension of the P1/P1' groups. Furthermore, water molecules were shown to be important binding links between the protease and the inhibitors. This study produced a number of inhibitors with Ki values in the 100 picomolar range.