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Understanding the impacts of changing climate and disturbance regimes on forest ecosystems is greatly aided by the use of process-based models. Such models simulate processes based on first principles of ecology, which requires parameterization. Parameterization is an important step in model development and application, defining the characteristics of trees and their responses to the environment, i.e., their traits. For species-specific models, parameterization is usually done at the level of individual species. Parameterization is indispensable for accurately modeling demographic processes, including growth, mortality, and regeneration of trees, along with their intra- and inter-specific interactions. As it is time-demanding to compile the parameters required to simulate forest ecosystems in complex models, simulations are often restricted to the most common tree species, genera, or plant-functional types. Yet, as tree species composition might change in the future, it is important to account for a broad range of species and their individual responses to drivers of change explicitly in simulations. Thus, species-specific parameterization is a critical task for making accurate projections about future forest trajectories, yet species parameters often remain poorly documented in simulation studies. We compiled and harmonized all existing tree species parameters available for the individual-based forest landscape and disturbance model (iLand). Since its first publication in 2012, iLand has been applied in 50 peer-reviewed publications across three continents throughout the Northern Hemisphere (i.e., Europe, North America, and Asia). The model operates at individual-tree level and simulates ecosystem processes at multiple spatial scales, making it a capable process-based model for studying forest change. However, the extensive number of processes and their interactions as well as the wide range of spatio-temporal scales considered in iLand require intensive parameterization, with tree species characterized by 66 unique parameters in the model. The database presented here includes parameters for 150 temperate and boreal tree species and provenances (i.e., regional variations). Excluding missing values, the database includes a total of 9,249 individual parameter entries. In addition, we provide parameters for the individual susceptibility of tree species to wind disturbance (five parameters) for a subset of 104 tree species and provenances (498 parameter entries). To guide further model parameterization efforts, we provide an estimate of uncertainty for each species based on how thoroughly simulations with the respective parameters were evaluated against independent data. Our dataset aids the future parameterization and application of iLand, and sets a new standard in documenting parameters used in process-based forest simulations. This dataset will support model application in previously unstudied areas and can facilitate the investigation of new tree species being introduced to well-studied systems (e.g., simulating assisted migration in the context of rapid climate change). Given that many process-based models rely on similar underlying processes our harmonized parameter set will be of relevance beyond the iLand community. Our work could catalyze further research into improving the parameterization of process-based forest models, increasing the robustness of projections of climate change impacts and adaptation strategies.

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Understanding the factors influencing species range limits is increasingly crucial in anticipating migrations due to human-caused climate change. In the boreal biome, ongoing climate change and the associated increases in the rate, size, and severity of disturbances may alter the distributions of boreal tree species. Notably, Interior Alaska lacks native pine, a biogeographical anomaly that carries implications for ecosystem structure and function. The current range of lodgepole pine (Pinus contorta var. latifolia) in the adjacent Yukon Territory may expand into Interior Alaska, particularly with human assistance. Evaluating the potential for pine expansion in Alaska requires testing constraints on range limits such as dispersal limitations, environmental tolerance limits, and positive or negative biotic interactions. In this study, we used field experiments with pine seeds and transplanted seedlings, complemented by model simulations, to assess the abiotic and biotic factors influencing lodgepole pine seedling establishment and growth after fire in Interior Alaska. We found that pine could successfully recruit, survive, grow, and reproduce across our broadly distributed network of experimental sites. Our results show that both mammalian herbivory and competition from native tree species are unlikely to constrain pine growth and that environmental conditions commonly found in Interior Alaska fall well within the tolerance limits for pine. If dispersal constraints are released, lodgepole pine could have a geographically expansive range in Alaska, and once established, its growth is sufficient to support pine-dominated stands. Given the impacts of lodgepole pine on ecosystem processes such as increases in timber production, carbon sequestration, landscape flammability, and reduced forage quality, natural or human-assisted migration of this species is likely to substantially alter responses of Alaskan forest ecosystems to climate change.

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Streamflow often increases after fire, but the persistence of this effect and its importance to present and future regional water resources are unclear. This paper addresses these knowledge gaps for the western United States (WUS), where annual forest fire area increased by more than 1,100% during 1984 to 2020. Among 72 forested basins across the WUS that burned between 1984 and 2019, the multibasin mean streamflow was significantly elevated by 0.19 SDs (P < 0.01) for an average of 6 water years postfire, compared to the range of results expected from climate alone. Significance is assessed by comparing prefire and postfire streamflow responses to climate and also to streamflow among 107 control basins that experienced little to no wildfire during the study period. The streamflow response scales with fire extent: among the 29 basins where >20% of forest area burned in a year, streamflow over the first 6 water years postfire increased by a multibasin average of 0.38 SDs, or 30%. Postfire streamflow increases were significant in all four seasons. Historical fire-climate relationships combined with climate model projections suggest that 2021 to 2050 will see repeated years when climate is more fire-conducive than in 2020, the year currently holding the modern record for WUS forest area burned. These findings center on relatively small, minimally managed basins, but our results suggest that burned areas will grow enough over the next 3 decades to enhance streamflow at regional scales. Wildfire is an emerging driver of runoff change that will increasingly alter climate impacts on water supplies and runoff-related risks.

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Fire is an integral component of ecosystems globally and a tool that humans have harnessed for millennia. Altered fire regimes are a fundamental cause and consequence of global change, impacting people and the biophysical systems on which they depend. As part of the newly emerging Anthropocene, marked by human-caused climate change and radical changes to ecosystems, fire danger is increasing, and fires are having increasingly devastating impacts on human health, infrastructure, and ecosystem services. Increasing fire danger is a vexing problem that requires deep transdisciplinary, trans-sector, and inclusive partnerships to address. Here, we outline barriers and opportunities in the next generation of fire science and provide guidance for investment in future research. We synthesize insights needed to better address the long-standing challenges of innovation across disciplines to (i) promote coordinated research efforts; (ii) embrace different ways of knowing and knowledge generation; (iii) promote exploration of fundamental science; (iv) capitalize on the "firehose" of data for societal benefit; and (v) integrate human and natural systems into models across multiple scales. Fire science is thus at a critical transitional moment. We need to shift from observation and modeled representations of varying components of climate, people, vegetation, and fire to more integrative and predictive approaches that support pathways toward mitigating and adapting to our increasingly flammable world, including the utilization of fire for human safety and benefit. Only through overcoming institutional silos and accessing knowledge across diverse communities can we effectively undertake research that improves outcomes in our more fiery future.

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Changing climate and disturbance regimes are increasingly challenging the resilience of forest ecosystems around the globe. A powerful indicator for the loss of resilience is regeneration failure, that is, the inability of the prevailing tree species to regenerate after disturbance. Regeneration failure can result from the interplay among disturbance changes (e.g., larger and more frequent fires), altered climate conditions (e.g., increased drought), and functional traits (e.g., method of seed dispersal). This complexity makes projections of regeneration failure challenging. Here we applied a novel simulation approach assimilating data-driven fire projections with vegetation responses from process modeling by means of deep neural networks. We (i) quantified the future probability of regeneration failure; (ii) identified spatial hotspots of regeneration failure; and (iii) assessed how current forest types differ in their ability to regenerate under future climate and fire. We focused on the Greater Yellowstone Ecosystem (2.9 × 106  ha of forest) in the Rocky Mountains of the USA, which has experienced large wildfires in the past and is expected to undergo drastic changes in climate and fire in the future. We simulated four climate scenarios until 2100 at a fine spatial grain (100 m). Both wildfire activity and unstocked forest area increased substantially throughout the 21st century in all simulated scenarios. By 2100, between 28% and 59% of the forested area failed to regenerate, indicating considerable loss of resilience. Areas disproportionally at risk occurred where fires are not constrained by topography and in valleys aligned with predominant winds. High-elevation forest types not adapted to fire (i.e., Picea engelmannii-Abies lasiocarpa as well as non-serotinous Pinus contorta var. latifolia forests) were especially vulnerable to regeneration failure. We conclude that changing climate and fire could exceed the resilience of forests in a substantial portion of Greater Yellowstone, with profound implications for carbon, biodiversity, and recreation.

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AIM: Simulation models are important tools for quantifying the resilience (i.e., persistence under changed environmental conditions) of forest ecosystems to global change. We synthesized the modelling literature on forest resilience, summarizing common models and applications in resilience research, and scrutinizing the implementation of important resilience mechanisms in these models. Models applied to assess resilience are highly diverse, and our goal was to assess how well they account for important resilience mechanisms identified in experimental and empirical research. LOCATION: Global. TIME PERIOD: 1994 to 2019. MAJOR TAXA STUDIED: Trees. METHODS: We reviewed the forest resilience literature using online databases, selecting 119 simulation modelling studies for further analysis. We identified a set of resilience mechanisms from the general resilience literature and analysed models for their representation of these mechanisms. Analyses were grouped by investigated drivers (resilience to what) and responses (resilience of what), as well as by the type of model being used. RESULTS: Models used to study forest resilience varied widely, from analytical approaches to complex landscape simulators. The most commonly addressed questions were associated with resilience of forest cover to fire. Important resilience mechanisms pertaining to regeneration, soil processes, and disturbance legacies were explicitly simulated in only 34 to 46% of the model applications. MAIN CONCLUSIONS: We found a large gap between processes identified as underpinning forest resilience in the theoretical and empirical literature, and those represented in models used to assess forest resilience. Contemporary forest models developed for other goals may be poorly suited for studying forest resilience during an era of accelerating change. Our results highlight the need for a new wave of model development to enhance understanding of and management for resilient forests.

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In subalpine forests of the western United States that historically experienced infrequent, high-severity fire, whether fire management can shape 21st-century fire regimes and forest dynamics to meet natural resource objectives is not known. Managed wildfire use (i.e., allowing lightning-ignited fires to burn when risk is low instead of suppressing them) is one approach for maintaining natural fire regimes and fostering mosaics of forest structure, stand age, and tree-species composition, while protecting people and property. However, little guidance exists for where and when this strategy may be effective with climate change. We simulated most of the contiguous forest in Grand Teton National Park, Wyoming, USA to ask: (1) how would subalpine fires and forest structure be different if fires had not been suppressed during the last three decades? And (2) what is the relative influence of climate change vs. fire management strategy on future fire and forests? We contrasted fire and forests from 1989 to 2098 under two fire management scenarios (managed wildfire use and fire suppression), two general circulation models (CNRM-CM5 and GFDL-ESM2M), and two representative concentration pathways (8.5 and 4.5). We found little difference between management scenarios in the number, size, or severity of fires during the last three decades. With 21st-century warming, fire activity increased rapidly, particularly after 2050, and followed nearly identical trajectories in both management scenarios. Area burned per year between 2018 and 2099 was 1,700% greater than in the last three decades (1989-2017). Large areas of forest were abruptly lost; only 65% of the original 40,178 ha of forest remained by 2098. However, forests stayed connected and fuels were abundant enough to support profound increases in burning through this century. Our results indicate that strategies emphasizing managed wildfire use, rather than suppression, will not alter climate-induced changes to fire and forests in subalpine landscapes of western North America. This suggests that managers may continue to have flexibility to strategically suppress subalpine fires without concern for long-term consequences, in distinct contrast with dry conifer forests of the Rocky Mountains and mixed conifer forest of California where maintaining low fuel loads is essential for sustaining frequent, low-severity surface fire regimes.

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Subalpine forests in the northern Rocky Mountains have been resilient to stand-replacing fires that historically burned at 100- to 300-year intervals. Fire intervals are projected to decline drastically as climate warms, and forests that reburn before recovering from previous fire may lose their ability to rebound. We studied recent fires in Greater Yellowstone (Wyoming, United States) and asked whether short-interval (<30 years) stand-replacing fires can erode lodgepole pine (Pinus contorta var. latifolia) forest resilience via increased burn severity, reduced early postfire tree regeneration, reduced carbon stocks, and slower carbon recovery. During 2016, fires reburned young lodgepole pine forests that regenerated after wildfires in 1988 and 2000. During 2017, we sampled 0.25-ha plots in stand-replacing reburns (n = 18) and nearby young forests that did not reburn (n = 9). We also simulated stand development with and without reburns to assess carbon recovery trajectories. Nearly all prefire biomass was combusted ("crown fire plus") in some reburns in which prefire trees were dense and small (≤4-cm basal diameter). Postfire tree seedling density was reduced sixfold relative to the previous (long-interval) fire, and high-density stands (>40,000 stems ha-1) were converted to sparse stands (<1,000 stems ha-1). In reburns, coarse wood biomass and aboveground carbon stocks were reduced by 65 and 62%, respectively, relative to areas that did not reburn. Increased carbon loss plus sparse tree regeneration delayed simulated carbon recovery by >150 years. Forests did not transition to nonforest, but extreme burn severity and reduced tree recovery foreshadow an erosion of forest resilience.

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Environmental change is accelerating in the 21st century, but how multiple drivers may interact to alter forest resilience remains uncertain. In forests affected by large high-severity disturbances, tree regeneration is a resilience linchpin that shapes successional trajectories for decades. We modeled stands of two widespread western U.S. conifers, Douglas-fir (Pseudotsuga menziesii var. glauca), and lodgepole pine (Pinus contorta var. latifolia), in Yellowstone National Park (Wyoming, USA) to ask (1) What combinations of distance to seed source, fire return interval, and warming-drying conditions cause postfire tree-regeneration failure? (2) If postfire tree regeneration was successful, how does early tree density differ under future climate relative to historical climate? We conducted a stand-level (1 ha) factorial simulation experiment using the individual-based forest process model iLand to identify combinations of fire return interval (11-100 yr), distance to seed source (50-1,000 m), and climate (historical, mid-21st century, late-21st century) where trees failed to regenerate by 30-yr postfire. If regeneration was successful, we compared stand densities between climate periods. Simulated postfire regeneration were surprisingly resilient to changing climate and fire drivers. Douglas-fir regeneration failed more frequently (55%) than lodgepole pine (28% and 16% for non-serotinous and serotinous stands, respectively). Distance to seed source was an important driver of regeneration failure for Douglas-fir and non-serotinous lodgepole pine; regeneration never failed when stands were 50 m from a seed source and nearly always failed when stands were 1 km away. Regeneration of serotinous lodgepole pine only failed when fire return intervals were ≤20 yr and stands were far (1 km) from a seed source. Warming climate increased regeneration success for Douglas-fir but did not affect lodgepole pine. If regeneration was successful, postfire density varied with climate. Douglas-fir and serotinous lodgepole pine regeneration density both increased under 21st-century climate but in response to different climate variables (growing season length vs. cold limitation). Results suggest that, given a warmer future with larger and more frequent fires, a greater number of stands that fail to regenerate after fires combined with increasing density in stands where regeneration is successful could produce a more coarse-grained forest landscape.

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High-severity, infrequent fires in forests shape landscape mosaics of stand age and structure for decades to centuries, and forest structure can vary substantially even among same-aged stands. This variability among stand structures can affect landscape-scale carbon and nitrogen cycling, wildlife habitat availability, and vulnerability to subsequent disturbances. We used an individual-based forest process model (iLand) to ask: Over 300 years of postfire stand development, how does variation in early regeneration densities versus abiotic conditions influence among-stand structural variability for four conifer species widespread in western North America? We parameterized iLand for lodgepole pine (Pinus contorta var. latifolia), Douglas-fir (Pseudotsuga menziesii var. glauca), Engelmann spruce (Picea engelmannii), and subalpine fir (Abies lasiocarpa) in Greater Yellowstone (USA). Simulations were initialized with field data on regeneration following stand-replacing fires, and stand development was simulated under historical climatic conditions without further disturbance. Stand structure was characterized by stand density and basal area. Stands became more similar in structure as time since fire increased. Basal area converged more rapidly among stands than tree density for Douglas-fir and lodgepole pine, but not for subalpine fir and Engelmann spruce. For all species, regeneration-driven variation in stand density persisted for at least 99 years postfire, and for lodgepole pine, early regeneration densities dictated among-stand variation for 217 years. Over time, stands shifted from competition-driven convergence to environment-driven divergence, in which variability among stands was maintained or increased. The relative importance of drivers of stand structural variability differed between density and basal area and among species due to differential species traits, growth rates, and sensitivity to intraspecific competition versus abiotic conditions. Understanding dynamics of postfire stand development is increasingly important for anticipating future landscape patterns as fire activity increases.

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Macrosystems ecology is an effort to understand ecological processes and interactions at the broadest spatial scales and has potential to help solve globally important social and ecological challenges. It is important to understand the intellectual legacies underpinning macrosystems ecology: How the subdiscipline fits within, builds upon, differs from and extends previous theories. We trace the rise of macrosystems ecology with respect to preceding theories and present a new hypothesis that integrates the multiple components of macrosystems theory. The spatio-temporal anthropogenic rescaling (STAR) hypothesis suggests that human activities are altering the scales of ecological processes, resulting in interactions at novel space-time scale combinations that are diverse and predictable. We articulate four predictions about how human actions are "expanding", "shrinking", "speeding up" and "slowing down" ecological processes and interactions, and thereby generating new scaling relationships for ecological patterns and processes. We provide examples of these rescaling processes and describe ecological consequences across terrestrial, freshwater and marine ecosystems. Rescaling depends in part on characteristics including connectivity, stability and heterogeneity. Our STAR hypothesis challenges traditional assumptions about how the spatial and temporal scales of processes and interactions operate in different types of ecosystems and provides a lens through which to understand macrosystem-scale environmental change.

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CONTEXT: Adefovir dipivoxil is a nucleotide analog that has demonstrated effective antiretroviral activity against human immunodeficiency virus (HIV) with once-daily administration. OBJECTIVE: To determine if adefovir confers antiretroviral or immunologic benefit when added to stable antiretroviral therapy. DESIGN: Multicenter, 24-week, randomized, double-blind, placebo-controlled study. Enrollment was conducted from June 3, 1996, through May 6, 1997. SETTING: Thirty-three US HIV treatment centers. PARTICIPANTS: Of 1171 patients screened, 442 patients infected with HIV receiving stable antiretroviral therapy for at least 8 weeks with plasma HIV RNA greater than 2500 copies/mL and CD4+ cell count above 0.20 x 10(9)/L were randomized. INTERVENTION: Patients were randomized to receive either a single 120-mg/d dose of adefovir dipivoxil (n = 219) or an indistinguishable placebo (n = 223). All patients received L-carnitine, 500 mg/d. Open-label adefovir was offered after 24 weeks and was continued until the end of the study. MAIN OUTCOME MEASURES: Changes in HIV RNA from baseline, based on area under the curve and CD4+ cell levels, adverse events, and effect of baseline genotypic resistance on response to adefovir. RESULTS: Patients assigned to adefovir demonstrated a 0.4-log10 decline from baseline in HIV RNA compared with no change in the placebo group (P<.001), which continued through 48 weeks. CD4+ cell counts did not change. During the initial 24 weeks, elevated hepatic enzyme levels (P<.001), gastrointestinal tract complaints (P<.001), and weight loss (P<.001) were associated with use of adefovir. Between 24 weeks and 48 weeks elevations in serum creatinine occurred in 60% of patients, usually returning to baseline after discontinuation of adefovir. Patients with lamivudine or lamivudine and zidovudine resistance mutations demonstrated anti-HIV effects with adefovir (P< or =.01 vs placebo group). CONCLUSIONS: This study suggests that once-daily adefovir therapy reduces HIV RNA and is active against isolates resistant to lamivudine or lamivudine and zidovudine. Nephrotoxicity occurred when treatment extended beyond 24 weeks but was reversible.

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The efficacy of sequential protease inhibitor therapy was studied in 16 human immunodeficiency virus (HIV) 1-infected persons in whom saquinavir with multiple nucleoside reverse transcriptase (RT) inhibitors (NRTI) had failed. Nelfinavir plus two NRTIs (new or continued) resulted in minimal (0.59 log RNA copies/mL) and transient (8 weeks) suppression of plasma HIV RNA levels. Rapid failure was surprisingly associated with baseline presence of protease gene mutation L90M (P=.04) in the absence of D30N and with RT mutations D67N (P<.01), K70R/S (P=.02), and K219Q/W/R/E (P<.01). Ten patients were subsequently switched to indinavir plus nevirapine and 2 NRTIs, resulting in a median 1.62 log reduction in plasma HIV RNA, with 3 patients maintaining 400 copies/mL for 24 weeks. These results suggest that nelfinavir may have limited utility after saquinavir failure, particularly without potent concomitant therapy. Combining an NRTI with a new protease inhibitor for rescue may improve response.

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BACKGROUND: We compared two combinations of nucleosides with zidovudine alone in patients with advanced human immunodeficiency virus (HIV) infection. METHODS: A total of 1102 patients with the acquired immunodeficiency syndrome or fewer than 200 CD4 cells per cubic millimeter were randomly assigned to receive zidovudine alone or zidovudine combined with either didanosine or zalcitabine. Disease progression, survival, toxic effects, and the CD4 cell response were assessed. RESULTS: After a median follow-up of 35 months, disease progression or death occurred in 62 percent of the 363 patients assigned to zidovudine plus didanosine, 63 percent of the 367 assigned to zidovudine plus zalcitabine, and 66 percent of the 372 assigned to zidovudine only (P=0.24). As compared with zidovudine therapy, treatment with zidovudine plus didanosine was associated with a relative risk of disease progression or death of 0.86 (95 percent confidence interval, 0.71 to 1.03), and treatment with zidovudine plus zalcitabine was associated with a relative risk of 0.92 (95 percent confidence interval, 0.76 to 1.10). Survival was similar in the three groups. In a subgroup analysis, combination therapy delayed disease progression or death in patients who had previously received zidovudine for 12 months or less. Therapy with zidovudine plus didanosine resulted in more gastrointestinal adverse effects, and treatment with zidovudine plus zalcitabine, more neuropathy. The mean increases in CD4 cell counts at two months were higher with combination therapy than with zidovudine alone. CONCLUSIONS: In patients with advanced HIV infection, combination therapy with zidovudine and either didanosine or zalcitabine is not superior to zidovudine therapy alone. However, these combinations may be more effective than zidovudine monotherapy in patients with little or no previous zidovudine treatment.

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OBJECTIVE: To monitor the appearance of HIV-1 variants resistant to inhibition by DMP 266, a benzoxazinone non-nucleoside reverse transcriptase inhibitor using two different protocols for applying drug selective pressure in tissue culture. To compare the phenotype and genotype of viral isolates selected by each method. METHODS: MT-2 cells and fresh donor peripheral blood mononuclear cells (PBMC) were infected with HIV-1 strain RF. The MT-2 cells were infected in the presence of a 50% inhibitory concentration (IC50) of DMP 266 and the concentration was slowly increased during the selection period. The PBMC were infected for 1 week in the absence of inhibitor and then a single concentration was maintained throughout the selection period. Both cultures were passaged for approximately 4 months. Virus and cell pellets were harvested over this in vitro selection period, the RT genes amplified by polymerase chain reaction from the cell pellets, and the proviral DNAs sequenced. Isolated virus was tested for DMP 266 susceptibility in either the AIDS Clinical Trials Group/Department of Defense consensus assay or MT-2 yield reduction assay. RESULTS: Passage in MT-2 cells resulted in accumulation of three substitutions in RT (V179D, L1001, Y181C) after 24 passages associated with 1000-fold reduced susceptibility to DMP 266. In PBMC cultures treated with 0.96 microM DMP 266, virus replication was completely suppressed after 2 weeks; no regrowth occurred in the presence of compound after 10 weeks or in the absence of compound for 3 additional weeks. The 0.096 microM treated cultures had an initial 2.5-log reduction in infectious virus titre followed by rapid regrowth. Virus obtained at week 6 displayed a 28-fold reduction in susceptibility with an L1001 substitution in RT, and by week 11 displayed a 1000-fold reduction in susceptibility with an additional V1081 substitution. CONCLUSIONS: High-level resistance to DMP 266 may develop by at least two pathways and experimental conditions influence the genotype selected. The continued absence of detectable virus in the PBMC cultures grown at 0.96 microM is supportive evidence that maintaining trough plasma levels of DMP 266 which result in sustained antiviral activity in vivo may delay emergence of highly resistant viral variants. Confirmation of this hypothesis will require clinical trials.

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High-resolution X-ray structures of the complexes of HIV-1 protease (HIV-1PR) with peptidomimetic inhibitors reveal the presence of a structural water molecule which is hydrogen bonded to both the mobile flaps of the enzyme and the two carbonyls flanking the transition-state mimic of the inhibitors. Using the structure-activity relationships of C2-symmetric diol inhibitors, computed-aided drug design tools, and first principles, we designed and synthesized a novel class of cyclic ureas that incorporates this structural water and preorganizes the side chain residues into optimum binding conformations. Conformational analysis suggested a preference for pseudodiaxial benzylic and pseudodiequatorial hydroxyl substituents and an enantiomeric preference for the RSSR stereochemistry. The X-ray and solution NMR structure of the complex of HIV-1PR and one such cyclic urea, DMP323, confirmed the displacement of the structural water. Additionally, the bound and "unbound" (small-molecule X-ray) ligands have similar conformations. The high degree of preorganization, the complementarity, and the entropic gain of water displacement are proposed to explain the high affinity of these small molecules for the enzyme. The small size probably contributes to the observed good oral bioavailability in animals. Extensive structure-based optimization of the side chains that fill the S2 and S2' pockets of the enzyme resulted in DMP323, which was studied in phase I clinical trials but found to suffer from variable pharmacokinetics in man. This report details the synthesis, conformational analysis, structure-activity relationships, and molecular recognition of this series of C2-symmetry HIV-1PR inhibitors. An initial series of cyclic ureas containing nonsymmetric P2/P2' is also discussed.

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BACKGROUND: Effective HIV protease inhibitors must combine potency towards wild-type and mutant variants of HIV with oral bioavailability such that drug levels in relevant tissues continuously exceed that required for inhibition of virus replication. Computer-aided design led to the discovery of cyclic urea inhibitors of the HIV protease. We set out to improve the physical properties and oral bioavailability of these compounds. RESULTS: We have synthesized DMP 450 (bis-methanesulfonic acid salt), a water-soluble cyclic urea compound and a potent inhibitor of HIV replication in cell culture that also inhibits variants of HIV with single amino acid substitutions in the protease. DMP 450 is highly selective for HIV protease, consistent with displacement of the retrovirus-specific structural water molecule. Single doses of 10 mg kg-1 DMP 450 result in plasma levels in man in excess of that required to inhibit wild-type and several mutant HIVs. A plasmid-based, in vivo assay model suggests that maintenance of plasma levels of DMP 450 near the antiviral IC90 suppresses HIV protease activity in the animal. We did identify mutants that are resistant to DMP 450, however; multiple mutations within the protease gene caused a significant reduction in the antiviral response. CONCLUSIONS: DMP 450 is a significant advance within the cyclic urea class of HIV protease inhibitors due to its exceptional oral bioavailability. The data presented here suggest that an optimal cyclic urea will provide clinical benefit in treating AIDS if it combines favorable pharmacokinetics with potent activity against not only single mutants of HIV, but also multiply-mutant variants.

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The HIV-1 protease (PR) is essential for the production of mature virions. As such, it has become a target for the development of anti-HIV chemotherapeutics. Multiple passages of virus in cell culture in the presence of PR inhibitors have resulted in the selection of variants with decreased sensitivity to inhibitors of the PR. The most common alteration observed is a single amino acid change at position 82. This particular position has been well characterized by several laboratories as being important for the susceptibility of the virus to inhibitors of PR function. Mutations which result in the substitution of the wild-type valine with alanine, phenylalanine, threonine or isoleucine at position 82 of the PR have been associated with decreased sensitivity to several PR inhibitors. We describe here a clinical strain of HIV-1 that contains an isoleucine at position 82 of the PR instead of the usual valine. This strain is unique in that it was isolated from a patient that was anti-retroviral naive, and in the past, variants at position 82 of the PR have only been found after treatment of patients or cell culture with PR inhibitors. Moreover, this virus remains sensitive to PR inhibitors of the cyclic urea and C-2 symmetrical diol classes.

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Proviral DNAs from 3 laboratory strains and 21 clinical isolates of HIV-1 were extracted from infected cells after proteinase K digestion and the protease gene was PCR amplified and sequenced directly by the Sanger method. In vitro susceptibilities of the virus isolates to protease inhibitors were determined by the ACTG/DoD consensus assay. Four different HIV protease inhibitors were tested including P9941, a C2 symmetrical diol (Du Pont-Merck); A80987, an asymmetric mono-ol (Abbott); XM323, a cyclic urea (Du Pont-Merck); and Ro31-8959, an asymmetric hydroxyethylene isostere (Roche). Maximum sequence variation was 10% at both the nucleic and amino acid levels. Purine-purine substitutions were most common. Five noncontiguous regions were conserved across all isolates and corresponded to amino acids 1-9 (amino terminal), 21-32 (catalytic site), 47-56 ("flap" region), 78-88 (substrate-binding region), and 94-99 (carboxy terminal). All clinical isolates demonstrated in vitro susceptibility to the protease inhibitors. There was no significant difference between the susceptibility of the reference strains and the clinical isolates. These data suggest that the variable regions of protease do not contain sites that are important for interactions with the inhibitors tested.

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