A New Drug Discovery Platform: Application to DNA Polymerase Eta and Apurinic/Apyrimidinic Endonuclease 1.

The ability to quickly discover reliable hits from screening and rapidly convert them into lead compounds, which can be verified in functional assays, is central to drug discovery. The expedited validation of novel targets and the identification of modulators to advance to preclinical studies can significantly increase drug development success. Our SaXPyTM ("SAR by X-ray Poses Quickly") platform, which is applicable to any X-ray crystallography-enabled drug target, couples the established methods of protein X-ray crystallography and fragment-based drug discovery (FBDD) with advanced computational and medicinal chemistry to deliver small molecule modulators or targeted protein degradation ligands in a short timeframe. Our approach, especially for elusive or "undruggable" targets, allows for (i) hit generation; (ii) the mapping of protein-ligand interactions; (iii) the assessment of target ligandability; (iv) the discovery of novel and potential allosteric binding sites; and (v) hit-to-lead execution. These advances inform chemical tractability and downstream biology and generate novel intellectual property. We describe here the application of SaXPy in the discovery and development of DNA damage response inhibitors against DNA polymerase eta (Pol η or POLH) and apurinic/apyrimidinic endonuclease 1 (APE1 or APEX1). Notably, our SaXPy platform allowed us to solve the first crystal structures of these proteins bound to small molecules and to discover novel binding sites for each target.

The novel immunotherapeutic oligodeoxynucleotide IMT504 protects neutropenic animals from fatal Pseudomonas aeruginosa bacteremia and sepsis.

IMT504 is a novel immunomodulatory oligonucleotide that has shown immunotherapeutic properties in early preclinical and clinical studies. IMT504 was tested in a neutropenic rat model of Pseudomonas aeruginosa bacteremia and sepsis. This animal system recapitulates many of the pathological processes found in neutropenic patients with Gram-negative, bacterial infections. The research was conducted in the setting of an academic research laboratory. The test subjects were Sprague-Dawley rats. Animals were rendered neutropenic by administration of cyclophosphamide, colonized with P. aeruginosa by oral feeding, and then randomized to receive IMT504 over a range of doses and treatment regimens representing early and late therapeutic interventions. IMT504 immunotherapy conferred a significant survival advantage over the 12-day study period compared with the results seen with placebo-treated animals when the therapy was administered at the onset of neutropenia and even in the absence of antibiotics and after the onset of fever and systemic infection. Notably, even late salvage IMT504 monotherapy was highly effective (13/14 surviving rats with IMT504 therapy versus 2/14 controls, P=<0.001). Moreover, late salvage IMT504 monotherapy was as effective as antibiotic therapy (13/14 surviving rats versus 21/21 rats, P=0.88). In addition, no antagonism or loss of therapeutic efficacy was noted with combination therapy of IMT504 plus antibiotics. IMT504 immunotherapy provides a remarkable survival advantage in bacteremia and sepsis in neutropenic animals and deserves further study as a new treatment option in patients with, or at risk for, severe Gram-negative bacterial infections and sepsis.

Phylodynamics of HIV-1 from a phase III AIDS vaccine trial in Bangkok, Thailand.

BACKGROUND: In 2003, a phase III placebo-controlled trial (VAX003) was completed in Bangkok, Thailand. Of the 2,546 individuals enrolled in the trial based on high risk for infection through injection drug use (IDU), we obtained clinical samples and HIV-1 sequence data (envelope glycoprotein gene gp120) from 215 individuals who became infected during the trial. Here, we used these data in combination with other publicly available gp120 sequences to perform a molecular surveillance and phylodynamic analysis of HIV-1 in Thailand. METHODOLOGY AND FINDINGS: Phylogenetic and population genetic estimators were used to assess HIV-1 gp120 diversity as a function of vaccination treatment, viral load (VL) and CD4(+) counts, to identify transmission clusters and to investigate the timescale and demographics of HIV-1 in Thailand. Three HIV-1 subtypes were identified: CRF01_AE (85% of the infections), subtype B (13%) and CRF15_AE (2%). The Bangkok IDU cohort showed more gp120 diversity than other Asian IDU cohorts and similar diversity to that observed in sexually infected individuals. Moreover, significant differences (P<0.02) in genetic diversity were observed in CRF01_AE IDU with different VL and CD4(+) counts. No phylogenetic structure was detected regarding any of the epidemiological and clinical factors tested, although high proportions (35% to 50%) of early infections fell into clusters, which suggests that transmission chains associated with acute infection play a key role on HIV-1 spread among IDU. CRF01_AE was estimated to have emerged in Thailand in 1984.5 (1983-1986), 3-6 years before the first recognition of symptomatic patients (1989). The relative genetic diversity of the HIV-1 population has remained high despite decreasing prevalence rates since the mid 1990s. CONCLUSIONS: Our study and recent epidemiological reports indicate that HIV-1 is still a major threat in Thailand and suggest that HIV awareness and prevention needs to be strengthened to avoid AIDS resurgence.

Phylodynamics of HIV-1 from a phase-III AIDS vaccine trial in North America.

In 2003, a phase III placebo-controlled trial (VAX004) of a candidate HIV-1 vaccine (AIDSVAX B/B) was completed in 5,403 volunteers at high risk for HIV-1 infection from North America and the Netherlands. A total of 368 individuals became infected with HIV-1 during the trial. The envelope glycoprotein gene (gp120) from the HIV-1 subtype B viruses infecting 349 patients was sequenced from clinical samples taken as close as possible to the time of diagnosis, rendering a final data set of 1,047 sequences (1,032 from North America and 15 from the Netherlands). Here, we used these data in combination with other sequences available in public databases to assess HIV-1 variation as a function of vaccination treatment, geographic region, race, risk behavior, and viral load. Viral samples did not show any phylogenetic structure for any of these factors, but individuals with different viral loads showed significant differences (P = 0.009) in genetic diversity. The estimated time of emergence of HIV-1 subtype B was 1966-1970. Despite the fact that the number of AIDS cases has decreased in North America since the early 90s, HIV-1 genetic diversity seems to have remained almost constant over time. This study represents one of the largest molecular epidemiologic surveys of viruses responsible for new HIV-1 infections in North America and could help the selection of epidemiologically representative vaccine antigens to include in the next generation of candidate HIV-1 vaccines.

Ethnic differences in the adaptation rate of HIV gp120 from a vaccine trial.

Differences in HIV-1 gp120 sequence variation were examined in North American volunteers who became infected during a phase III vaccine trial using the rgp120 vaccine. Molecular adaptation of the virus in vaccine and placebo recipients from different ethnic subgroups was compared by estimating the dN/dS ratios in viruses sampled from each individual using three different methods. ANOVA analyses detected significant differences in d(N)/d(S) ratios among races (P < 0.02). gp120 sequences from the black individuals showed higher mean d(N)/d(S) ratios for all estimators (1.24-1.45) than in other races (0.66-1.35), and several pairwise comparisons involving blacks remained significant (P < 0.05) after correction for multiple tests. In addition, black-placebo individuals showed significantly (P < 0.02) higher mean d(N)/d(S) ratios (1.3-1.66) than placebo individuals from the other races (0.65-1.56). These results suggest intrinsic differences among races in immune response and highlight the need for including multiple ethnicities in the design of future HIV-1 vaccine studies and trials.

Genome scanning tests for comparing amino acid sequences between groups.

Consider a placebo-controlled preventive HIV vaccine efficacy trial. An HIV amino acid sequence is measured from each volunteer who acquires HIV, and these sequences are aligned together with the reference HIV sequence represented in the vaccine. We develop genome scanning methods to identify positions at which the amino acids in infected vaccine recipient sequences either (A) are more divergent from the reference amino acid than the amino acids in infected placebo recipient sequences or (B) have a different frequency distribution than the placebo sequences, irrespective of a reference amino acid. We consider t-test-type statistics for problem A and Euclidean, Mahalanobis, and Kullback-Leibler-type statistics for problem B. The test statistics incorporate weights to reflect biological information contained in different amino acid positions and mismatches. Position-specific p-values are obtained by approximating the null distribution of the statistics either by a permutation procedure or by a nonparametric estimation. A permutation method is used to estimate a cut-off p-value to control the per comparison error rate at a prespecified level. The methods are examined in simulations and are applied to two HIV examples. The methods for problem B address the general problem of comparing discrete frequency distributions between groups in a high-dimensional data setting.

LC16m8: an attenuated smallpox vaccine.

The frequency of moderate to severe adverse reactions associated with smallpox vaccines currently stockpiled in the US, and the continued threat of bioterrorism have prompted the development of effective vaccines with improved safety profiles. LC16m8, an attenuated, replicating smallpox vaccine derived from the Lister strain of vaccinia, is currently licensed in Japan where it was safely used in over 50,000 children in the 1970s. It has been shown to have markedly less neurotoxicity than unattenuated vaccines in nonclinical studies. LC16m8 is immunogenic after a single dose, and recent studies in two different animal models have demonstrated protective efficacy equivalent to that of the only FDA-licensed smallpox vaccine. This article reviews the history and available scientific literature regarding LC16m8 and provides comparisons to other smallpox vaccines.

Longitudinal population analysis of dual infection with recombination in two strains of HIV type 1 subtype B in an individual from a Phase 3 HIV vaccine efficacy trial.

This study documents a case of coinfection (simultaneous infection of an individual with two or more strains) of two HIV-1 subtype B strains in an individual from a Phase 3 HIV-1 vaccine efficacy trial, conducted in North American and the Netherlands. We examined 86 full-length gp120 (env) gene sequences from this individual collected from nine different time points over a 20-month period. We estimated evolutionary relationships using maximum likelihood and Bayesian methods and inferred recombination breakpoints and recombinant sequences using phylogenetic and substitutional methods. These analyses identified two strongly supported monophyletic clades (clades A and B) of 14 and 69 sequences each and a small paraphyletic recombinant clade of three sequences. We then studied the genetic characteristics of these lineages by comparing estimates of genetic diversity generated by mutation and recombination and adaptive selection within a coalescent and maximum likelihood framework. Our results suggest significant differences on the evolutionary dynamics of these strains. We then discuss the implications of these results for vaccine development.

High incidence of unusual cysteine variants in gp120 envelope proteins from early HIV type 1 infections from a Phase 3 vaccine efficacy trial.

During the course of a large-scale HIV-1 vaccine field trial (VAX004), full-length gp120 sequences were determined for 349 new HIV-1 infections. The data collected represent the largest survey of full-length gp120 sequences from new HIV-1 infections ever assembled. Previous studies have shown that subtype B viruses typically possess 18 cysteine residues that are covalently linked to form 9 conserved disulfide bridges. However, in this study we found that approximately 20% of the trial participants possessed envelope proteins with an unusual number of cysteine residues that could very likely result in unusual protein structures. One class of variants included envelope proteins with two additional cysteine residues in close proximity, potentially yielding additional disulfide-bonded loops. Other classes of variants included envelope proteins where amino acid replacements increased or decreased the number of cysteine residues by one, resulting in molecules with either 19 or 17 cysteines, respectively. Initial functional analysis demonstrated that envelope proteins with 19 cysteine residues bind to CD4 and the CCR5 chemokine coreceptor, and are infectious. These results suggest that the protein structure of gp120 in newly transmitted viruses may be more heterogeneous than previously appreciated and potentially represent a new mechanism of virus variation. The disulfide variation that we report here may have important implications for HIV vaccine and drug development efforts.

Correlation between immunologic responses to a recombinant glycoprotein 120 vaccine and incidence of HIV-1 infection in a phase 3 HIV-1 preventive vaccine trial.

BACKGROUND: An objective of the first efficacy trial of a candidate vaccine containing recombinant human immunodeficiency virus (HIV) type 1 envelope glycoprotein 120 (rgp120) antigens was to assess correlations between antibody responses to rgp120 and the incidence of HIV-1 infection. METHODS: Within the randomized trial (for vaccinees, n=3598; for placebo recipients, n=1805), binding and neutralizing antibody responses to rgp120 were quantitated. A case-cohort design was used to study correlations between antibody levels and HIV-1 incidence. RESULTS: Peak antibody levels were significantly inversely correlated with HIV-1 incidence. The relative risk (RR) of infection was 0.63 (95% confidence interval, 0.45-0.89) per log(10) higher neutralization titer against HIV-1(MN), and the RRs of infection for second-, third-, and fourth-quartile responses of antibody blocking of gp120 binding to soluble CD4 versus first-quartile responses (the lowest responses) were 0.35, 0.28, and 0.22, respectively. CONCLUSIONS: Despite inducing a complex, robust immune response, the vaccine was unable to reduce the incidence of HIV-1. Two interpretations of the correlative results are that the levels of antibodies (i) caused both an increased (low responders) and decreased (high responders) risk of HIV-1 acquisition or (ii) represented a correlate of susceptibility to HIV-1 but had no causal effect on susceptibility. Although the data cannot definitively discriminate between these 2 explanations, (ii) appears to be more likely.

JC virus genotypes in the western Pacific suggest Asian mainland relationships and virus association with early population movements.

Distinct genotypes of human polyomavirus JC (JCV) have remained population associated possibly from the time of dispersal of modern humans from Africa. Seven major genotypes with additional subtypes serve as plausible markers for following early and more recent human migrations in all parts of the world. Phylogenetic trees of JCV sequences from the major continental population groups show a trifurcation at the base indicating early division into European, African, and Asian branches. Here, we have explored JCV relationships in the island populations of the western Pacific. Since these islands were settled from the Asian mainland and islands of Southeast Asia, we expected that their virus genotypes might show an Asian connection. We found that Type 2E (Austronesian) and Type 8 (non-Austronesian) are widely distributed in western Pacific populations. A few south China strains were found (Type 7A). A subtype of Type 8, Type 8A, was confined to Papua New Guinea. In keeping with these assignments we find that phylogenetic analysis by neighbor-joining and maximum parsimony methods places Type 2E in a closer relationship to east Asian mainland strains such as Type 2A and Type 7. Our findings support the Asian origins of the western Pacific JCV strains, and suggest three broad movements: an ancient one characterized by Type 8A, and then Type 8B, followed much later by migrations carrying Type 2E, which may correlate with the arrival of Austronesian-language speakers, the bearers of the "Lapita" cultural complex (approximately 3,500 to 5,000 years ago), and relatively recent movements carrying largely Type 7A (south China) strains directly from the West.

BK virus regulatory region rearrangements in brain and cerebrospinal fluid from a leukemia patient with tubulointerstitial nephritis and meningoencephalitis.

BK virus (BKV) was recovered by polymerase chain reaction (PCR) from brain, kidney, lung, urine, and cerebrospinal fluid (CSF) of a fatal case of BKV tubulointerstitial nephritis with dissemination to lung and brain. Viral regulatory regions in PCR-amplified urine and the lung samples were identical to the archetypal structure, WWT. In the brain and CSF, a rearranged sequence predominated, however. A 94-bp deletion preceded a 71-bp tandem duplication because the same 94-bp segment was deleted from both copies. PCR-amplified regulatory region products were cloned and sequenced to define further the extent of the rearranged structures. Two kidney clones were archetypal, whereas two others were rearranged differently from the brain and from each other. In contrast to the brain clones, the kidney rearrangements seemed to involve deletion after duplication. Three of four brain clones sequenced were identical to the rearrangement found to dominate in the PCR product. A fourth clone showed two short deletions without any duplication. The four CSF clones all showed rearrangements identical to that which was amplified by PCR from CSF and brain. This represents the first molecular analysis of a BKV strain obtained from a central nervous system infection, and it reveals regulatory region rearrangements reminiscent of those described in JC virus from brains with progressive multifocal leukoencephalopathy. We suggest that the presence in the CSF of BKV with a dominant rearranged regulatory region may be useful in the diagnosis of BKV meningoencephalitis secondary to BKV nephritis.

Genotypes of JC virus in East, Central and Southwest Europe.

Distinctive genotypes of JC virus have been described for the major continental landmasses. Studies on European-Americans and small cohorts in Europe showed predominantly Type 1. Types 2 and 7 are found in Asia, and Types 3 and 6 in Africa. These genotypes differ in sequence by about 1--3%. Each genotype may have several subtypes which differ from each other by about 0.5--1%. The genotypes can be defined by a distinctive pattern of nucleotides in a typing region of the VP1 gene. This genotyping approach has been confirmed by phylogenetic reconstruction using the entire genome exclusive of the rearranging regulatory region. In this first large European study, we report on the urinary excretion of JCV DNA of 350 individuals from Poland, Hungary, Germany and Spain. We included Gypsy cohorts in Hungary (Roma), Germany (Sinti), and Spain (Gitano), as well as Basques in Spain. We show that while Type 1 predominates in Europe, the proportions of Type 1A and 1B may differ from East to Southwest Europe. Type 4, closely related to the Type 1 sequence (only approximately 1% difference) was a minor genotype in Germany, Poland and Spain, but represented the majority in Basques. The Gitanos in Spain showed a variant Type 4 sequence termed 'Rom-1'. Interestingly, neither the Gitanos in Spain, nor Sinti or Roma in Germany or Hungary showed the Type 2 or Type 7 genotype that might be expected if their origins were in an Asian population.