The creation of enzymes capable of catalyzing any desired chemical reaction is a grand challenge for computational protein design. Using new algorithms that rely on hashing techniques to construct ...active sites for multistep reactions, we designed retro-aldolases that use four different catalytic motifs to catalyze the breaking of a carbon-carbon bond in a nonnatural substrate. Of the 72 designs that were experimentally characterized, 32, spanning a range of protein folds, had detectable retroaldolase activity. Designs that used an explicit water molecule to mediate proton shuffling were significantly more successful, with rate accelerations of up to four orders of magnitude and multiple turnovers, than those involving charged side-chain networks. The atomic accuracy of the design process was confirmed by the x-ray crystal structure of active designs embedded in two protein scaffolds, both of which were nearly superimposable on the design model.
Enzyme catalysts of a retroaldol reaction have been generated by computational design using a motif that combines a lysine in a nonpolar environment with water‐mediated stabilization of the ...carbinolamine hydroxyl and β‐hydroxyl groups. Here, we show that the design process is robust and repeatable, with 33 new active designs constructed on 13 different protein scaffold backbones. The initial activities are not high but are increased through site‐directed mutagenesis and laboratory evolution. Mutational data highlight areas for improvement in design. Different designed catalysts give different borohydride‐reduced reaction intermediates, suggesting a distribution of properties of the designed enzymes that may be further explored and exploited.
The design of new enzymes for reactions not catalysed by naturally occurring biocatalysts is a challenge for protein engineering and is a critical test of our understanding of enzyme catalysis. Here ...we describe the computational design of eight enzymes that use two different catalytic motifs to catalyse the Kemp elimination-a model reaction for proton transfer from carbon-with measured rate enhancements of up to 10(5) and multiple turnovers. Mutational analysis confirms that catalysis depends on the computationally designed active sites, and a high-resolution crystal structure suggests that the designs have close to atomic accuracy. Application of in vitro evolution to enhance the computational designs produced a >200-fold increase in k(cat)/K(m) (k(cat)/K(m) of 2,600 M(-1)s(-1) and k(cat)/k(uncat) of >10(6)). These results demonstrate the power of combining computational protein design with directed evolution for creating new enzymes, and we anticipate the creation of a wide range of useful new catalysts in the future.
Cancer is increasingly common among persons with HIV.
To examine calendar trends in cumulative cancer incidence and hazard rate by HIV status.
Cohort study.
North American AIDS Cohort Collaboration ...on Research and Design during 1996 to 2009.
86 620 persons with HIV and 196 987 uninfected adults.
Cancer type-specific cumulative incidence by age 75 years and calendar trends in cumulative incidence and hazard rates, each by HIV status.
Cumulative incidences of cancer by age 75 years for persons with and without HIV, respectively, were as follows: Kaposi sarcoma, 4.4% and 0.01%; non-Hodgkin lymphoma, 4.5% and 0.7%; lung cancer, 3.4% and 2.8%; anal cancer, 1.5% and 0.05%; colorectal cancer, 1.0% and 1.5%; liver cancer, 1.1% and 0.4%; Hodgkin lymphoma, 0.9% and 0.09%; melanoma, 0.5% and 0.6%; and oral cavity/pharyngeal cancer, 0.8% and 0.8%. Among persons with HIV, calendar trends in cumulative incidence and hazard rate decreased for Kaposi sarcoma and non-Hodgkin lymphoma. For anal, colorectal, and liver cancer, increasing cumulative incidence, but not hazard rate trends, were due to the decreasing mortality rate trend (-9% per year), allowing greater opportunity to be diagnosed. Despite decreasing hazard rate trends for lung cancer, Hodgkin lymphoma, and melanoma, cumulative incidence trends were not seen because of the compensating effect of the declining mortality rate.
Secular trends in screening, smoking, and viral co-infections were not evaluated.
Cumulative cancer incidence by age 75 years, approximating lifetime risk in persons with HIV, may have clinical utility in this population. The high cumulative incidences by age 75 years for Kaposi sarcoma, non-Hodgkin lymphoma, and lung cancer support early and sustained antiretroviral therapy and smoking cessation.
The creation of novel enzymes capable of catalyzing any desired chemical reaction is a grand challenge for computational protein design. Here we describe two new algorithms for enzyme design that ...employ hashing techniques to allow searching through large numbers of protein scaffolds for optimal catalytic site placement. We also describe an in silico benchmark, based on the recapitulation of the active sites of native enzymes, that allows rapid evaluation and testing of enzyme design methodologies. In the benchmark test, which consists of designing sites for each of 10 different chemical reactions in backbone scaffolds derived from 10 enzymes catalyzing the reactions, the new methods succeed in identifying the native site in the native scaffold and ranking it within the top five designs for six of the 10 reactions. The new methods can be directly applied to the design of new enzymes, and the benchmark provides a powerful in silico test for guiding improvements in computational enzyme design.
Background. It is unclear whether immunosuppression leads to younger ages at cancer diagnosis among people living with human immunodeficiency virus (PLWH). A previous study found that most cancers ...are not diagnosed at a younger age in people with AIDS, with the exception of anal and lung cancers. This study extends prior work to include all PLWH and examines associations between AIDS, CD4 count, and age at cancer diagnosis. Methods. We compared the median age at cancer diagnosis between PLWH in the North American AIDS Cohort Collaboration on Research and Design and the general population using data from the Surveillance, Epidemiology and End Results Program. We used statistical weights to adjust for population differences. We also compared median age at cancer diagnosis by AIDS status and CD4 count. Results. After adjusting for population differences, younger ages at diagnosis (P < .05) were observed for PLWH compared with the general population for lung (difference in medians = 4 years), anal (difference = 4), oral cavity/pharynx (difference = 2), and kidney cancers (difference = 2) and myeloma (difference = 4). Among PLWH, having an AIDS-defining event was associated with a younger age at myeloma diagnosis (difference = 4; P = .01), and CD4 count <200 cells/μL (vs ≥500) was associated with a younger age at lung cancer diagnosis (difference = 4; P = .006). Conclusions. Among PLWH, most cancers are not diagnosed at younger ages. However, this study strengthens evidence that lung cancer, anal cancer, and myeloma are diagnosed at modestly younger ages, and also shows younger ages at diagnosis of oral cavity/pharynx and kidney cancers, possibly reflecting accelerated cancer progression, etiologic heterogeneity, or risk factor exposure in PLWH.
Abstract
Background
Persons living with human immunodeficiency virus (HIV; PLWH) experience a high burden of cancer. It remains unknown which cancer types are reduced in PLWH with earlier initiation ...of antiretroviral therapy (ART).
Methods
We evaluated AIDS-free, ART-naive PLWH during 1996–2014 from 22 cohorts participating in the North American AIDS Cohort Collaboration on Research and Design. PLWH were followed from first observed CD4 of 350–500 cells/µL (baseline) until incident cancer, death, lost-to-follow-up, or December 2014. Outcomes included 6 cancer groups and 5 individual cancers that were confirmed by chart review or cancer registry linkage. We evaluated the effect of earlier (in the first 6 months after baseline) versus deferred ART initiation on cancer risk. Marginal structural models were used with inverse probability weighting to account for time-dependent confounding and informative right-censoring, with weights informed by subject’s age, sex, cohort, baseline year, race/ethnicity, HIV transmission risk, smoking, viral hepatitis, CD4, and AIDS diagnoses.
Results
Protective results for earlier ART were found for any cancer (adjusted hazard ratio HR 0.57; 95% confidence interval CI, .37–.86), AIDS-defining cancers (HR 0.23; 95% CI, .11–.49), any virus-related cancer (HR 0.30; 95% CI, .16–.54), Kaposi sarcoma (HR 0.25; 95% CI, .10–.61), and non-Hodgkin lymphoma (HR 0.22; 95% CI, .06–.73). By 15 years, there was also an observed reduced risk with earlier ART for virus-related NADCs (0.6% vs 2.3%; adjusted risk difference −1.6; 95% CI, −2.8, −.5).
Conclusions
Earlier ART initiation has potential to reduce the burden of virus-related cancers in PLWH but not non-AIDS-defining cancers (NADCs) without known or suspected viral etiology.
Earlier antiretroviral therapy initiation has potential to reduce the burden of virus-related cancers in people living with Human Immunodeficiency Virus, but not cancers without known or suspected viral etiology.
With combination-antiretroviral therapy, HIV-infected individuals live longer with an elevated burden of cancer. Given the high prevalence of smoking among HIV-infected populations, we examined the ...risk of incident cancers attributable to ever smoking cigarettes.
Observational cohort of HIV-infected participants with 270 136 person-years of follow-up in the North American AIDS Cohort Collaboration on Research and Design consortium. Among 52 441 participants, 2306 were diagnosed with cancer during 2000-2015.
Estimated hazard ratios and population-attributable fractions (PAF) associated with ever cigarette smoking for all cancers combined, smoking-related cancers, and cancers that were not attributed to smoking.
People with cancer were more frequently ever smokers (79%) compared with people without cancer (73%). Adjusting for demographic and clinical factors, cigarette smoking was associated with increased risk of cancer overall hazard ratios = 1.33 (95% confidence interval: 1.18-1.49); smoking-related cancers hazard ratios = 2.31 (1.80-2.98); lung cancer hazard ratios = 17.80 (5.60-56.63); but not nonsmoking-related cancers hazard ratios = 1.12 (0.98-1.28). Adjusted PAFs associated with ever cigarette smoking were as follows: all cancers combined, PAF = 19% (95% confidence interval: 13-25%); smoking-related cancers, PAF = 50% (39-59%); lung cancer, PAF = 94% (82-98%); and nonsmoking-related cancers, PAF = 9% (1-16%).
Among HIV-infected persons, approximately one-fifth of all incident cancer, including half of smoking-related cancer, and 94% of lung cancer diagnoses could potentially be prevented by eliminating cigarette smoking. Cigarette smoking could contribute to some cancers that were classified as nonsmoking-related cancers in this report. Enhanced smoking cessation efforts targeted to HIV-infected individuals are needed.
We report the cocrystal structures of a computationally designed and experimentally optimized retro-aldol enzyme with covalently bound substrate analogs. The structure with a covalently bound ...mechanism-based inhibitor is similar to, but not identical with, the design model, with an RMSD of 1.4 Å over active-site residues and equivalent substrate atoms. As in the design model, the binding pocket orients the substrate through hydrophobic interactions with the naphthyl moiety such that the oxygen atoms analogous to the carbinolamine and β-hydroxyl oxygens are positioned near a network of bound waters. However, there are differences between the design model and the structure: the orientation of the naphthyl group and the conformation of the catalytic lysine are slightly different; the bound water network appears to be more extensive; and the bound substrate analog exhibits more conformational heterogeneity than typical native enzyme–inhibitor complexes. Alanine scanning of the active-site residues shows that both the catalytic lysine and the residues around the binding pocket for the substrate naphthyl group make critical contributions to catalysis. Mutating the set of water-coordinating residues also significantly reduces catalytic activity. The crystal structure of the enzyme with a smaller substrate analog that lacks naphthyl ring shows the catalytic lysine to be more flexible than in the naphthyl–substrate complex; increased preorganization of the active site would likely improve catalysis. The covalently bound complex structures and mutagenesis data highlight the strengths and weaknesses of the de novo enzyme design strategy.
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► We report x-ray structure of a computationally designed enzyme with substrate analog. ► We determined the contributions of the active site residues to catalysis. ► The study gave insights into strengths and limitations of computational enzyme design.
Kaposi sarcoma (KS) remains common among HIV-infected persons. To better understand KS etiology and to help target prevention efforts, we comprehensively examined a variety of CD4 T-cell count and ...HIV-1 RNA viral load (VL) measures, as well as antiretroviral therapy (ART) use, to determine independent predictors of KS risk.
North American AIDS Cohort Collaboration on Research and Design.
We followed HIV-infected persons during 1996-2009 from 18 cohorts. We used time-updated Cox regression to model relationships between KS risk and recent, lagged, trajectory, and cumulative CD4 count or VL measures, as well as ART use. We used Akaike's information criterion and global P values to derive a final model.
In separate models, the relationship between each measure and KS risk was highly significant (P < 0.0001). Our final mutually adjusted model included recent CD4 count hazard ratio (HR) for <50 vs. ≥500 cells/μL = 12.4; 95% confidence interval (CI): 6.5 to 23.8, recent VL (HR for ≥100,000 vs. ≤500 copies/mL = 3.8; 95% CI: 2.0 to 7.3), and cumulative (time-weighted mean) VL (HR for ≥100,000 vs. ≤500 copies/mL = 2.5; 95% CI: 1.0 to 5.9). Each P-trend was <0.0001. After adjusting for these measures, we did not detect an independent association between ART use and KS risk.
Our results suggested a multifactorial etiology for KS, with early and late phases of development. The cumulative VL effect suggested that controlling HIV replication promptly after HIV diagnosis is important for KS prevention. We observed no evidence for direct anti-KS activity of ART, independent of CD4 count and VL.
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