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Which Compound to Select in Lead Optimization? Prospectively Validated Proteochemometric Models Guide Preclinical Development
Gerard J. P. van Westen,
Affiliation Division of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research, Leiden, The Netherlands
Jörg K. Wegner,
Affiliation Tibotec BVBA, Beerse, Belgium
Peggy Geluykens,
Leen Kwanten,
Inge Vereycken,
Anik Peeters,
Adriaan P. IJzerman,
Herman W. T. van Vlijmen,
Affiliations Division of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research, Leiden, The Netherlands, Tibotec BVBA, Beerse, Belgium
Andreas Bender
* E-mail: ab454@cam.ac.uk
Affiliations Division of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research, Leiden, The Netherlands, Unilever Centre for Molecular Science Informatics, University of Cambridge, Cambridge, United Kingdom
Gerard J. P. van Westen Jörg K. Wegner ... Andreas Bender
In quite a few diseases, drug resistance due to target variability poses a serious problem in pharmacotherapy. This is certainly true for HIV, and hence, it is often unknown which drug is best to use or to develop against an individual HIV strain. In this work we applied 'proteochemometric' modeling of HIV Non-Nucleoside Reverse Transcriptase (NNRTI) inhibitors to support preclinical development by predicting compound performance on multiple mutants in the lead selection stage. Proteochemometric models are based on both small molecule and target properties and can thus capture multi-target activity relationships simultaneously, the targets in this case being a set of 14 HIV Reverse Transcriptase (RT) mutants. We validated our model by experimentally confirming model predictions for 317 untested compound – mutant pairs, with a prediction error comparable with assay variability (RMSE 0.62). Furthermore, dependent on the similarity of a new mutant to the training set, we could predict with high accuracy which compound will be most effective on a sequence with a previously unknown genotype. Hence, our models allow the evaluation of compound performance on untested sequences and the selection of the most promising leads for further preclinical research. The modeling concept is likely to be applicable also to other target families with genetic variability like other viruses or bacteria, or with similar orthologs like GPCRs.
Citation: van Westen GJP, Wegner JK, Geluykens P, Kwanten L, Vereycken I, Peeters A, et al. (2011) Which Compound to Select in Lead Optimization? Prospectively Validated Proteochemometric Models Guide Preclinical Development. PLoS ONE 6(11): e27518. https://doi.org/10.1371/journal.pone.0027518
Editor: Mark Wainberg, McGill University AIDS Centre, Canada
Received: July 21, 2011; Accepted: October 18, 2011; Published: November 23, 2011
Copyright: © 2011 van Westen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: GJPvW is a PhD student funded by Tibotec whose role involved contributing to the experimental data, validation of the results, and providing scientific input into the design of the study (such as the improvement of modeling methods). The performing of the data analysis, and the decision to publish was not influenced in any way by the funders. The authors' collaborators from Tibotec helped in the preparation of the manuscript, however, no situation occurred where the academic partners of the team were not able to publish what was desired. AB and API received funding from Dutch Top Institute Pharma (TI Pharma), project number D1-105, who had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have read the journal's policy and have the following conflicts: The Non-Nucleoside Reverse Transcriptase Inhibitiros Etravirine and Rilpivirine are marketed drugs developed by Tibotec, one of the funders of the current study. GJPvW was funded by Tibotec BVBA and JKW, PG, LK, IV, AP and HWTvV are employees of this company. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials, as detailed online in the guide for authors.
Genetic Information is readily available
Over the last decade extensive sequencing efforts have unraveled the human genome and provide an insight into the extent of human genetic variation [1], [2]. On the one hand this provides possible new drug targets that can lead to new drugs [3]–[5]; on the other hand it shows clearly that natural genetic variation needs to be addressed by some form of personalized medicine that works in a particular patient [6]. An exhaustive, individual "pharmacogenomics" approach for a patient, taking the full genetic make-up of a human into account, is unfortunately not feasible in the short term. This is due to the cost of sequencing but even more so to insufficient understanding of biological processes in humans [7]. However, what is already feasible today for every patient is the full sequencing of pathogens such as bacteria and viruses, as these contain a significantly smaller genome with relatively established locations and functions of drug targets. It is now possible through 'Deep Sequencing' technologies, to identify dominant and subdominant viral strains present in an individual patient, paving the way for the development of HIV inhibitors with an optimal potency profile made to target all relevant HIV variants [8], [9].
What is required for the development of an optimal preclinical candidate on the other hand is a knowledge base of the effect mutations have on the binding of current inhibitors. When this information is available it can be used to create a model that allows the user to extrapolate between target sequence variants and predict binding affinities of preclinical compounds on previously untested viral target sequences. While similar models have been trained on this data for clinical drugs, these models have in common that they solely are trained on recognizing patterns of the presence and absence of mutations, thus only considering target information [10]–[15]. They do not take into account structural information of the compound – target interaction; hence they are not able to rationalize why an inhibitor is active on one sequence but not on another. As a result, the application to the discovery of preclinical candidates is rather limited.
How to Choose the Right Drug for a Genotype?
In the current work we present one approach to remedy the situation, by making use of the large amount of structural data available on the binding of HIV Reverse Transcriptase (RT) inhibitors to their targets. We will show using prospective experimental validation on hundreds of data points that we can indeed predict which compound is preferable with regard to activity against particular mutants, compared to other compounds. In particular, our aim was to predict activity of compounds on previously untested genetic variants of the virus. Given our in-depth understanding of the structural differences between viral enzyme sequences we can incorporate this knowledge to arrive at much improved extrapolation abilities, which enables the design of new inhibitors with improved broad activity profiles.
Extrapolating in Target Space
When learning from bioactivity data, and attempting to make predictions for novel chemical structures, statistical and machine learning techniques have a proven ability to 'make sense' of large data sets under certain conditions (such as interpretable variables used in the model) and to relate chemical structure to activity against a protein target. Bioactivity models are generally based on the 'Molecular Similarity Principle' stating that similar compounds (individual compounds or with respect to the distribution of chemistry in a given data set) possess similar properties, such as in this case similar bioactivity [16]–[18]. Yet conventional bioactivity models possess a severe limitation when considering sets of targets, which may be members of a target family such as kinases or G protein-coupled receptors (GPCRs), or as in the case presented here, sequences of viral enzymes. Those models take into account multiple molecules active on a single protein target, yet they completely neglect our extensive knowledge on the similarities of targets to each other. Hence, conventionally a single bioactivity model is generated for every target – neglecting that not only similar compounds show similar bioactivity, but reversely also that similar targets bind similar compounds. In addition, this concept is crucial for the chemogenomics paradigm that has been receiving lots of attention recently [19], [20]. In practice this means that even if a particular ligand-protein target data point is unknown, we can often extrapolate from neighboring activities in both ligand and target space, and not only in ligand space as has previously been done.
In this work we employed a modeling technique called 'proteochemometric modeling' (PCM) to model bioactivity data on a set of 14 enzyme sequences. This technique was introduced by Lapinsh, Wikberg et al. [21], [22] and similar approaches have since appeared [23]. These techniques have been recently reviewed by the authors [24]. However, no large prospective studies have been presented until today. As PCM uses both ligand and target information, the hypothesis of our work was that PCM would be able to extrapolate the activity of compounds encountered in the training set to novel target sequences. The extension of bioactivity modeling and its impact on preclinical drug research, plus the extensive prospective experimental validation performed, is the main contribution of the current work to the bioactivity modeling field. In addition, applications of PCM to novel target families including, but not limited to: Class A, B and C GPCRs; Kinases; Voltage-gated ion channels and others, can also be covered by this concept. The flexibility of the method is of particular interest when taking current multi-target drug paradigms into account [25], [26].
Data set used to build the models
The data set employed comprises 451 compounds and 14 HIV RT sequences and hence 6,314 possible compound – target combinations. The set was generously provided by Tibotec BVBA. For a total of 4,024 of these combinations an activity value in the form of a pEC50 value was available for training the bioactivity model. Compounds with a pEC50 value on a certain sequence closer than 0.3 log units to the toxic concentration for that compound (expressed as pCC50) were discarded (81 compound – sequence pairs). Table 1 shows the point mutations present in the 13 mutated sequences as well as the wild type (HXB2 / IIIB reference strain [27], sequence 1 in the table) and the average pEC50 per sequence. A graphic representation of our data set is shown in Figure 1; a histogram of the pEC50 values of all compounds – sequence pairs is available in Figure S8. A sample dataset, the final full model (in the form of a pipeline pilot component), and a protocol to perform PCM are included in the electronic Supporting information.
Figure 1. Graphical representation of the NNRTI dataset.
(A) Our dataset consisted of 451 compounds (in rows) and 14 sequences (in columns) of HIV Reverse Transcriptase with about 60% of the experimental data pairs known. Black indicates a low pEC50, grey indicates a high pEC50 and white indicates a missing value. (B) The dataset with the missing pEC50 values completed by our model from which 317 experimental validation data points were chosen.
Table 1. Sequence information of the RT sequences in the data set.
Compound and protein descriptors
All descriptors were calculated in the academic version of PipelinePilot 6.1.5 [28]. Ligands were described by Scitegic FCFP_6 circular fingerprints [29], [30], which have previously been shown to capture a large amount of information with respect to compound bioactivity [31], [32]. FCFP_6 descriptors provide individual substructures and treat these as a feature of a compound. These substructures have a maximal diameter of 6 bonds from a central atom. In the final model we can link these substructures to a change in pEC50.
Sequences were encoded based on the binding site sequence in which each amino acid was represented as a single unique feature. The residues used to define the binding site are shown in Figure 2 (PDB Code 2ZD1, HIV RT bound to Rilpivirine [33], created with Molsoft ICM version 3.6-h) and Table 1. The residues used to define the binding site are shown in red and black, where the black residues are the ones that were mutated only in sequence 7. The features describing the binding site were obtained by hashing an array of 58 physicochemical properties obtained from the AAindex database [34]; the used indices can be found in Table S3.
Figure 2. The binding site used in our models.
PDB X-ray structure 2ZD1 of RT with Rilpivirine, an NNRTI like the analog series we modeled and shown in grey. The residues that were used to identify the binding site (and which were used for activity modeling) are shown in black and red. The black residues are only mutated in sequence 7 (heavy mutant) while the red residues are mutated in more than one sequence. Indicated by a green ribbon is the 'A' chain of HIV RT while the 'B' chain is displayed in yellow.
Finally, both ligand and protein fingerprints were converted to a fixed length array of features which were then used in the modeling. The ligands were converted to a fixed length array of 155 features while the sequences were converted to a fixed length array of 24 features using default Pipeline Pilot settings.
Models were constructed in the academic version of Pipeline Pilot 6.1.5 [28] using the R-statistics package. Support vector machines (SVM) as coded in the e1071 package were used for model creation [35]. Parameters gamma and cost were tuned over an exponential range and epsilon was set at 0.2 as this was the given assay error. It has been shown that setting epsilon to the data error is the optimal value for training [36]. The optimal model was determined using 5-fold cross validation before proceeding to experimental prospective validation of the model. The parameters used for validation were RMSE and R02, although the regular R2 was also determined it has been shown that R02 provides better reliability [37], [38]. R02, in contrast to R2, takes into account that the regression line of our model predictions should intersect with the origin. In silico validation on trained models was done via learning curves (see Figure S9).
Prospective Experimental Validation
To assess the prospective capabilities of PCM we used our final model, which was trained on the full data set, to predict the activity of all compounds for which no pEC50 value was available. In total 835 data points were subsequently experimentally validated, 317 of these represented novel predictions and 518 were repeat experiments to establish reproducibility of the assay. The 317 novel predictions included 130 compound – sequence pairs that were predicted to differ 2 standard deviations from either compound average (69 compound – sequence pairs), called compound outliers, or sequence average (61 compound – sequence pairs), called sequence outliers. Therefore we specifically tested the ability of our model to extrapolate outside of the bioactivity space of 'typical' compound – sequence pairs.
The techniques used as a benchmark were QSAR, kNN and pEC50 scaling. Individual QSAR models were trained for all sequences based on the same FCFP_6 compound descriptors as the PCM model. The kNN models were created based on Jensen et al. [39]. In total nine kNN models were created. Three models were based on only compound data using 3, 10 or 20 neighbors, three models on only target data using 3, 10 or 14 neighbors and three models based on both compound and target data using 3, 10 or 20 neighbors. In addition we also performed simple interpolation of pEC50 values ('scaling'), by assuming that the affinity of a compound on a sequence follows the general trend displayed by a series of other compounds. Thus, if a series of compounds on average has a 0.3 log unit lower pEC50 value than on the wild-type sequence, then the new compound is also assigned a 0.3 log unit lower pEC50 value.
Antiviral assays
The antiviral activity of different inhibitors was determined in a cell-based HIV-1 replication assay. Here MT4 cells (150,000 cells/ml), stably transformed with an LTR-EGFP reporter gene, were infected with HIV-1 (IIIB, clinical isolates, or site-directed sequence strains; multiplicity of infection MOI = 0.0025) in the presence or absence of different inhibitor concentrations. After three days of incubation, the amount of HIV replication was quantified by measuring the EGFP fluorescence, and expressed as EC50 values. The toxicity of inhibitors was determined in parallel on mock-infected MT4 (150,000 cells/ml) cells stably transformed with a CMV-EGFP reporter gene and cultured in the presence or absence of test compound concentrations. After three days of incubation, cell proliferation was quantified by measuring the EGFP fluorescence, and expressed as CC50 values (cytotoxicity, 50% inhibitory concentration of cell growth).
Leave-one-sequence-out validation
In order to assess the models' capability to extrapolate between the different sequences we performed 'Leave-one-sequence-out validation' (LOSO). In this approach for each of the 14 sequences all data points of the sequence under consideration are left out of the training set, a model is trained on the remainder and the points left out are used as a test set for that model. Both the RMSE and the R02 values of the validation plot were subsequently determined for each of the 14 sequences in turn.
Model interpretation
To determine the effect of individual residues, for each sequence each residue was mutated back to wild type in silico. Subsequently for all compounds the model prediction on the original mutant sequence was compared with the prediction of the model on the in silico changed mutant sequence. The difference was interpreted as the change in pEC50 induced by that particular residue. From all these changed prediction values the following values were calculated: the average overall, the average per sequence and per individual mutation. This provided the model interpretability. However, changes that led to a 0 value shift in pEC50 were removed in the calculation of the average per position since in all cases this was caused by mutation back to wild type of a residue that was already wild type in that particular sequence. In addition the large amount of 0 value shifts lead to a shift of the average towards 0 when it was calculated thereby masking the actual contribution of mutations.
To interpret the influence of compound substructures a slightly different approach was chosen. Here each FCFP_6 feature, corresponding with a particular substructure, was substituted by the feature representing a single Carbon atom ('0'). Since this carbon atom was already present in all compounds, its effect on binding serves as a calibration. Subsequently the full model was used to predict the pEC50 of the adapted compound fingerprint lacking a certain substructure on all sequences and compared with the model prediction of the original compound fingerprint on all sequences. From all these changed predictions the average overall and per sequence was calculated for that particular feature, or chemical substructure in this case.
Synthesis of the analog series we used in this work has been described in multiple patents. Compound 1 is listed in patent WO 2007/113256 [40], compound 2 is listed in WO 2008/080965 [41] and compounds 3 and 4 are listed in WO 2008/080964 [42]. Compound 6 is listed in patent WO 2008/080965 [41], compounds 5, 7 and 8 are published in WO2007/113254 [43]. In addition the electronic Supporting information to this work contain the structures of 57 of the modeled compounds. For these compounds the full biological activity spectrum on the 14 sequences as we had it available is included along with the pEC50 predicted by our model on each of the 14 sequences (Table S4 and Archive S1).
In the current work PCM was applied to a data set of Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs), which constitutes one of the major classes of anti HIV drugs on the market. Among these are compounds such as Nevirapine [44] and Efavirenz [45], but also novel compounds such as Etravirine [46], which was approved by the FDA as recently as 2008. Since NNRTIs are allosteric binders they have shown considerably fewer side effects than orthosteric drugs [47]. However they are also known for a quick onset of viral resistance due to the accumulation of resistance associated mutations [48], [49]. Hence, the pharmacological profile of NNRTIs is highly desirable, but their effectiveness is hampered by the onset of resistance. This problem is also encountered in other viral infections like Hepatitis B [50], Hepatitis C [51] and Influenza A (H5N1) [52], [53]. Resistance can therefore be considered a universal problem when developing a new anti-viral drug. Thus, when new anti-virals are developed it is important that they retain their effectiveness despite the presence of these mutations. To be able to predict the activity of a preclinical drug candidate on an adapted pathogen would be an important contribution to drug discovery. Here we present an application of PCM that can predict drug performance on unknown sequences or adapted pathogens, when staying within model limitations.
Solving the problem of sparse data sets
Our data set contains compounds that inhibit wild type Reverse Transcriptase (RT), but also some that inhibit a number of RT sequences that are highly resistant against inhibition. NNRTIs are allosteric inhibitors of RT, which is illustrated in Figure 2 [33]. Incomplete bioactivity data sets are common in real-world settings, and this study is no exception. The data set is graphically displayed in Figure 1, with 'blanks' representing unknown data points that we would like to predict using computational methods. A pEC50 value was available for approximately 60% of the data set.
In a preclinical drug discovery setting, the ability to make decisions on a full rather than a sparse matrix increases the likelihood that the best candidate will be selected. This is of vital importance as a SAR table of drug – target interactions does not necessarily show linear relationships, e.g., a substituent in a given compound will not lead to the same increase or decrease in binding on different targets. This is even the case in analog series like our data set [54], [55]. Especially in anti-viral research compounds can display unexpected behavior on the different sequences. It is this behavior we are able to capture and translate into accurate predictions.
Prospective Experimental Model Validation
During model development, learning curves were generated that represent in silico validation, in addition we performed Y-scrambling [56] (Figure S9 and Figure S11). More importantly, we predicted the pEC50 of 317 unknown compound – target pairs. These predictions were subsequently measured experimentally. By predicting first and subsequent experimental validation, we obtain an estimate of model performance when considering novel compound – target pairs. Here we explicitly selected the more 'difficult' compound – target pairs; namely those with predicted pEC50 values that are either atypical for the particular compound tested (compound outliers), or for the particular sequence under consideration (sequence outliers). It is trivial to pick the compounds that are always active or always less active – we precisely removed those cases from our prospective testing and focused on the inhibitors that were predicted to be active against highly resistant sequences, or those which were predicted to be less active against very susceptible sequences.
Figure 3 shows the performance of the model, trained on the full data set, in these prospective validation experiments. During cross-validation the full model achieved a Root-Mean-Square Error (RMSE) of 0.38 log units (with a Q2 of 0.84) while employing 155 ligand features and 23 protein features. When applied to the new untested data, the model achieves an RMSE of 0.62 log units and an R02 of 0.69 in the prospective validation. Our model can predict the pEC50 of untested compound – target pairs with average accuracy of 0.62 log units. This RMSE approaches the reproducibility of the assay which was 0.50 log units (R02 of 0.88).
Figure 3. Model performance in the prospective experimental validation.
Shown are both predictive performance of the model (black dots; R02: 0.69, RMSE: 0.62 log units) and assay reproducibility (grey squares; R02: 0.88, RMSE: 0.50 log units). The continuous lines indicate an error of 1 log unit, while the center dashed line indicates a perfect correlation (see main text for further details).
To benchmark this performance against conventional approaches we applied QSAR modeling, k-Nearest Neighbor (kNN) modeling (using 3, 10 or 20 nearest neighbors and based on compound, target, or compound and target information) and pEC50 scaling to the same data set. The results are shown in Table 2. Here PCM outperforms QSAR and all forms of kNN modeling, while pEC50 scaling seems to perform slightly better. More specifically, PCM had an RMSE of 0.62 log units, while kNN showed 0.90 for the best model and scaling performed slightly better with 0.57. The R02 reached 0.69 for PCM, where kNN showed 0.41 and scaling also reached 0.69. Scaling of pEC50 values performs second best but this method has two major disadvantages. When we consider the sequence and compound outliers, PCM outperformed the simple pEC50 scaling. It is precisely those data points that points are most interesting in research. The sequence outliers represent the inhibitors that inhibit all present HIV mutants, candidates to select in lead selection. The compound outliers can be completely inactive on one particular sequence, candidates to avoid in lead selection. When considering these compounds, the average RMSE was 0.52 for PCM and 0.58 for scaling (R02 was 0.61 for PCM and 0.59 for scaling). A second disadvantage of scaling is that this method cannot be applied to untested viral sequences, whereas PCM can.
Table 2. Performance of different methods in experimental validation.
Hence, we conclude that the prospective experimental validation confirms the validity of our model, and the applicability of PCM to extrapolate in both ligand (chemical) space and target (biological) space. The other benchmarked techniques, kNN and QSAR, do not accurately capture the compound – target interaction space. The ability to model all these situations with a comparable reliability and the possibility to interpret the model from both a sequence and compound perspective, are the major advantages of PCM over kNN, QSAR and scaling methods.
Neighborhood Behavior in Target Space
In the prospective validation we noticed that a number (15) of data points were predicted inaccurately with an error larger than twice the RMSE, all of which involved sequence 7. Trying to elucidate the reason for this behavior the different sequences were clustered based on similarity (Figure S5 for details), where sequence 7 was found to be most dissimilar to all other sequences, containing a rather large number of 13 point mutations (for sequence information see Table 1). In addition, it is the sequence with the smallest number of tested compounds in the training set, thus diminishing the number of compound – target pairings that were used in the model directly (without extrapolation from neighboring sequences), thereby rationalizing the large number of false predictions on this sequence.
In order to systematically investigate the dependence of the prediction error on the sequence similarity we plotted the prediction error as a function of the average Tanimoto similarity of the individual sequences to the rest of the training set (shown in Figure 4). A correlation between model error and average sequence similarity is observed. The prediction error increases when the average similarity decreases between the sequence, for which the compound activity is predicted, and the rest of the training set sequences. This observation is an extension to the 'Molecular Similarity Principle' which states that similar compounds have similar properties, and in this work we are able to show that this paradigm also holds in biological space where similar sequences show similar ligand binding abilities; a concept we are now able to quantify numerically. This extends recent work on 'applicability domains' and 'activity cliffs' by also taking the biological target side into account [54], [55], [57], [58]. An improved distance measure is an ensemble of both the distance between the compounds (Figure S10), as has been previously shown [16], and the distance on the target side. To our knowledge this neighborhood behavior in target space has not been previously shown but is a natural extension of the chemical similarity principle to this new technique.
Figure 4. Extension of the applicability domain to target space.
Prediction error (measured as R02 and RMSE in log units) versus the average similarity of the sequence to the rest of the training set, extending the 'Molecular Similarity Principle' to biological space which is of crucial relevance in PCM. It can be seen that a higher similarity to the training set leads to more accurate predictions. Still, predictions on the most dissimilar sequence have an average error of less than 1 log unit.
The dependence of model accuracy on the average distance provides a useful tool to set the model applicability domain. As this distance is a property dependent on the training set on one hand and the unknown compound – target pair on the other hand it can be measured before any model prediction is made. Furthermore, neighborhood behavior can determine beforehand if the model is capable of predicting pEC50 changes on a previously untested genotype. This approach ensures that only model predictions with certain accuracy are used and that those that do not meet this accuracy are disregarded. For our current model this accuracy is defined as an error in pEC50 prediction (Figure 4) but this can also be an error in pKi value or any other value the model is trained to predict.
How to anticipate bioactivity for novel protein targets?
As we have shown that our model displays neighborhood behavior in target space, here we explore the possibility to use such a model in extrapolation. Conventionally a fraction of the full data set is left out from the training set when testing bioactivity models. The ability of the model to make predictions for the previously untested ('novel') compounds is taken as a predictor of model performance. In our case, we not only extrapolated in compound space, but also in sequence space. Hence, in order to confirm the ability of the model to extrapolate the activity of compounds to related sequences we performed a 'Leave-one-sequence-out' experiment (LOSO). Here, it emulates the prediction of inhibitor activity for a virus with a not previously encountered RT sequence, based solely on bioactivity measurements against other sequences in the data set. While applied to enzyme mutants here, the concept is generally applicable and the authors are currently investigating its performance on other target families such as GPCRs.
Figure 5 shows the R02 and RMSE of the LOSO validation experiment (see Figures S1 and S2 for additional information including error depending on similarity to the training set in sequence space). We observe that for 13 of the 14 sequences an RMSE of less than one log unit was obtained, and five of the 14 sequences even yielded an RMSE of less than 0.5 log units, which is the order of magnitude of assay reproducibility. Most interestingly, the model was able to use the information contained in all the different mutant sequences and predict the affinity of the compounds on the wild type sequence (Sequence 1). This means that this LOSO-1 model was able to deconvolute the individual contributions of the different mutants. Furthermore other LOSO models were also capable of predicting activity on unknown sequences 2–5 and 9–14. All these sequences contain very different mutations (Table 1). Finally, the LOSO-7 model was able to predict the pEC50 of the compounds on the "heavy" mutant (sequence 7), which contains a total of 13 point mutations. These findings underline the ability of PCM to extrapolate in target space, which supports the application of the technique to predict compound activity on different mutants.
Figure 5. Performance of PCM in leave-one-sequence-out experiments.
Performance was measured by R02 and the RMSE in log units. The number below the bar corresponds to the sequence left out of the training set.
Our results show that the model is indeed able to predict the pEC50 values of a known compound on an unknown sequence. However it should be noted that PCM performs lesser in 2 of the 14 mutants considered. The first of the two exceptions is sequence 6, which can be seen as a singleton since one of the mutations it carries (E138G) is only present in this particular sequence. Therefore, bioactivity prediction based on other sequences that do not carry this mutation is not straightforward. It was already known from the full model interpretation (see 'Model Based Interpretation of Mutants') that the impact of this mutant was underestimated. The LOSO-6 model correctly predicted that all compounds have a lower activity on this sequence (giving rise to a small RMSE value); however, the ranking among compounds is not very accurate (explaining the low R02).
The second sequence where our model underperforms is sequence 8. This case is different from the previous one: while the model is able to correctly rank the compounds, leading to an acceptable R02 of about 0.6, it consistently overpredicts the activity of compounds, leading to a high RMSE. In sequence 8 at position 101 a positively charged lysine residue is replaced with a proline, which likely induces conformational changes to the backbone of the protein. K101P is present as a single mutation in sequence 8, as well as in combination with a total of 12 other mutations in sequence 7 (which is then the only sequence that contains K101P in the training set when sequence 8 is left out for LOSO validation). Hence, the model likely underestimates the impact of the K101P mutation due to the large number of other mutations present in sequence 7; it is not able to deconvolute the impact of every single mutation properly. In this case, while the model would overestimate drug activity on this particular target, in a computer-aided compound selection setting the correct candidate would still be identified due to the accurate ranking of the compounds by the model.
Model performance in relation to chemical structure
To get an idea of the ability of the LOSO models' ability to predict individual compounds we have shown representative examples of compounds either predicted accurately or inaccurately in Figure 6. The activities of compounds 1 and 2 on the different sequences were correctly predicted by our leave-one-sequence-out models, while compounds 3 and 4 were predicted inaccurate (see Figures S3 and S4 for the predictions and experimental activities of the compounds discussed here). Please note that we made predictions challenging as we used LOSO models to predict the activity of compounds on the sequences left out of the training of these exact LOSO models. It is therefore a realistic emulation of a preclinical PCM application.
Figure 6. Example structures that where included in the model.
A selection of both compounds containing accurately (1,2) and inaccurately modeled chemistry (3,4). Also shown are compounds containing a substructure positively correlated with pEC50 (5,6) and compounds containing a substructure negatively correlated with pEC50 (7,8). In the upper part, shown are sample compounds that were accurately predicted using the LOSO models, a low RMSE (1, RMSE was 0.22 log units, R02 was 0.96) and a high R02 (2, R02 was 0.89 , RMSE was 0.26). Secondly, sample compounds that were predicted inaccurately using the LOSO models, a high RMSE (3, RMSE was 1.12 log units, R02 was −0.10) and a low R02 (4, R02 was −3.66, RMSE was 0.61). The lower part shows the 17th best substructure (5) and the 30th best substructure (6). Conversely the 3rd worst substructure (7) and the 4th worst substructure (8) are depicted.
From the data we conclude that inaccurately predicted compounds have a large functional group in the 4 position of the pyrimidine ring. Apparently the LOSO models are unable to capture this information correctly. When we further analyzed the individual predictions on the different mutants (shown in Figure S4), we noticed that the compounds are predicted accurately (error <0.5 log units) on the majority of the sequences. The underperformance for compound 3 is caused by overprediction on sequence 8 (2.7 log units, carrying K101P) and the underperformance of compound 4 by underprediction on sequence 2 (carrying V179F).
To explain this behavior, we need to consider the ligand binding mode. A shared binding mode of all compounds is very likely since i), they all share a common chemical scaffold and ii), NNRTIs are known to have a highly homologous binding mode [59]. Hence we can correlate these mispredictions with the protein structure. The substitution position on the pyrimidine ring in the compounds corresponds to the location of residues L100, K101 and V179 in crystal structure 2ZD1. It is known that these residues mutate easily and sequences carrying point mutations in this location are present in our training set [60]. When studying the crystal structure, it can be seen that the side chains of these residues are likely to hamper the binding of compounds with a large functional group in position 4.
Hence, we propose the presence of a large functional group on position 4 on the pyrimidine ring to be a predictor of insufficient model performance in combination with mutations on positions 101 and 179 in the protein. While compounds with a large substituent on the 4 position are accurately predicted on targets not carrying mutations on positions 101 and 179, they tend to be predicted inaccurately when the targets are mutated in these positions. However, if no large substituent is present on the 4 position, compounds are predicted accurately on targets carrying mutations on positions 101 and 179. Using this knowledge we can define an applicability domain when applying this particular model, but this finding should also be taken as a warning when using any PCM model to predict the affinity of known compounds on unknown sequences.
Model based interpretation of mutations
After completion of the full validation, the final full model was subsequently interpreted to explain the differences in activity of the compounds on the individual mutants. This is the model we used to perform the experimental validation and not one of the LOSO models. Firstly we focused on the sequence side of the model (Table 1 and for a multiple sequence alignment see Figure S6). By correlating model predictions of all compounds on each mutant sequence, where all amino acids were replaced in turn by their wild type counterpart on that particular position, an overview of the variation present at all individual residues was created (Figure 7A). The full model explains the lowered activity (pEC50) on mutants mainly by mutations at residues 100, 101, 103, 162, 179, 181, 188, 227, 234 and slightly by residue 138 on the 'b' chain. Interestingly the model interprets mutations at positions 89, 102, 118, 190, 203, 207, 210, 215, 219, 245 to actually slightly increase compound activity. Residues 106, 169 and 214 have little influence in this model and residue 211 does not seem to contribute at all (Figure S7 shows the variation in pEC50 caused by individual mutations).
Figure 7. Interpretation of the full model.
(A) Overview of the contribution of mutations present at all individual residue positions. The full model explains the lowered activity (pEC50 in log units) on mutants mainly by mutations at residues 100, 101, 103, 162, 179, 181, 188, 227, 234 and residue 138 on the 'b' chain. (B) Overview of the contribution of the different chemical substructures. Substructures occurring only in a single compound have been removed and the remaining substructures have been numbered sequentially. Several substructures have been visualized and linked to their position on the overview.
The main contributor to lowered affinity appears to be residue 179 rather than residue 181, while the latter is known to lead to NNRTI cross resistance [60]. Supporting this finding, it is known that clinically used Etravirine, a compound similar to but not included in our data set, is also sensitive to mutations at position 179 (V179D, V179F, and V179T [60]). Secondly, the influence of mutations at position 101 differs widely indicated by the high standard deviation. This is caused mainly by the K101P mutant, which causes a large decrease in pEC50 for some compounds and very little for others, depending on the chemistry (see below). The K101E mutant overall has little influence. This interpretation is in line with the results from the LOSO experiments mentioned above where both residues 101 and 179 were identified as having a high impact on model reliability.
Model based interpretation of ligand substructures
Similar to the mutation interpretation, the full model was interpreted to elucidate the average contribution of individual compound substructures to changes in activity (Figure 7B). Here the substructure that was being investigated was replaced by a single carbon atom in all compounds and the subsequent model predictions were compared to the model predictions for the original compounds. Figure 7B shows the contribution of the substructures after a selection was made to only use substructures that occur in more than one compound in order to lower a bias of a continuously active compound (resulting in 1068 of 2546 substructures). In the figure some examples of substructures that improve activity and substructures that decrease activity are shown. For a full table with the top 15 best and top 15 worst substructures, please see Table S1 and Table S2. Figure 6 (compounds 5,6) shows two examples of compounds that contain a substructure, which has been highlighted, that the model predicts to lead to a good activity and two examples of compounds (7,8) that contain a substructure that the model predicts to have a negative effect on activity. The effect is expressed as an average increase or decrease of all compounds containing that substructure and their activities on all sequences. Compound 5 contains the 17th best substructure, leading to an average increase in pEC50 of 0.14, and compound 6 contains the 30th best substructure, leading to an average increase in pEC50 of 0.12. Therefore these substructures constitute chemistry that is optimally contained in the compound. Compound 7 contains the 3rd worst substructure, leading to an average decrease in pEC50 of 0.23, and compound 8 contains the 4th worst substructure, leading to average decrease in pEC50 of 0.19. These two substructures constitute chemistry that is rather avoided in possible drug candidates.
Application of PCM in preclinical drug research
We have shown that PCM can be applied in a preclinical setting to predict the resistance profile of compounds. In addition we can interpret our final full model and identify favorable and unfavorable substructures, providing insights that can be used in compound design. For this data set we can conclude that the mutations in the protein sequences have a larger impact on pEC50 values than the compound substructures. However, we have also shown that the substructures still possess a significant influence, as PCM was the only technique that was able to combine target and substructures information.
Combining all our results from prospective experimental validation, the LOSO and the model interpretation, we feel confident that our model can be used to estimate the activity of previously untested compound – sequence pairs, with the main limitation (which can be quantified) being the similarity of the target protein. This opens the door for models that are able to predict the changes in activity of different compounds on clinical isolates obtained from patients [13]. PCM can thereby serve as a modeling tool to predict the activity for untested compound – isolate pairs before any assay measurement is performed, providing a quick guidance to medicinal chemists in the development of drugs based on their expected resistance profile. An example of this application is given in Tables 3 and 4, listing which inhibitors show best activity against a particular sequence and for which inhibitors resistance would be expected. (Table S4 lists a total of 57 compounds and their activity on the 14 sequences) Likewise, our method also aids in the development of drugs that have a broad inhibition profile. Noteworthy is that eight of the 28 specific compound - sequence predictions are untested compound – sequence pairs, which underlines the value of PCM to extrapolate to untested drug – sequence pairings, a feature not possible to achieve in conventional (single-target based) bioactivity modeling.
Table 3. Best performing compounds (per sequence and overall).
Table 4. Worst performing compounds (per sequence and overall).
In this work we have shown how to incorporate personalized data (specific viral mutants) as a tool to select optimal candidates in drug development by using proteochemometrics modeling in combination with a large scale experimental validation of inhibitors of HIV Reverse Transcriptase. While applied here to NNRTIs, PCM is a universally applicable method since all it requires is the sequence of a target of interest and structures of ligands. These two prerequisites are something that is available in any preclinical drug research project. We employed a prospective validation of 317 new experimental data points and a new type of 'leave one sequence out' validation (representing the case of a previously untested virus genotype). We were able to predict which compounds are best for a particular HIV RT sequence (with high accuracy in 12 out of the 14 sequences in the data set). We established that distance in biological (target) space is tightly correlated with prediction performance, enabling us to judge where the model likely succeeds, and where it may fail. Hence, in this work, we present a real-world scenario of HIV drug development, make practical steps towards drug design tailored towards specific patients, and we aim to extend this to other target families in the future.
Additional figures (Figures S1, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11), four tables (Tables S1, S2, S3, S4), the final model and 57 of the here modeled compounds with biological activity ( are available online. A protocol to be run in pipeline pilot to apply this model and perform PCM is also available online.
Figure S1.
Correlation parameters plotted against the average similarity with the training set. (A) The R02 of the different leave-one-sequence-out experiments (LOSO) against the average similarity between that specific sequence and the training set. (B) The RMSE of the different leave-one-sequence-out experiments against the average similarity between that specific sequence and the training set.
(TIF)
The distribution of the correlation parameters when validating individual compound predictions using the individual LOSO models. (A) Distribution of the RMSE and the R02 of the individual compound predictions using the LOSO models. The compounds have been ranked by increasing RMSE in (A), the corresponding R02 is shown in (B). Please note that the number on the x-axis is not the name of the compound, it is merely a serial number. Likewise the compounds have been ranked by increasing R02 (C) the corresponding RMSE is shown in (D).
Two compounds that were predicted accurately using the LOSO models. (A) Prediction of the activity (pEC50 value) of compound 1 on the different sequences using the LOSO models (RMSE of 0.22 log units, R02 of 0.96). (B) Prediction of the activity (pEC50 value) of compound 2 on the different sequences using the LOSO models (RMSE of 0.26 log units, R02 of 0.89).
Two compounds that were predicted inaccurately using the LOSO models. (A) Prediction of the activity (pEC50 value) of compound 3 on the different sequences using the LOSO models (RMSE of 1.12 log units, R02 of −0.10). (B) Prediction of the activity (pEC50 value) of compound 4 on the different sequences using the LOSO models (RMSE of 0.61 log units, R02 of −3.66).
Sequences present in the dataset clustered to similarity based on the protein descriptor.
Multiple sequence alignment of the used mutants. Outside the shown alignment the 14 sequences were equal; therefore these residues were omitted from the PCM model.
Average contribution to pEC50 according to the full model of all present mutants. The standard deviation was determined over all calculated pEC50 changes.
Overview of the pEC50 values of all compound – sequence pairs.
PCM and QSAR learning curves. As training is performed on an increasing part of the data set, validation is performed on a decreasing part of the data set. The PCM models are shown by solid lines and the QSAR by dashed lines. The validation parameters were calculated per sequence, using a single PCM model for all sequences, as well as for comparison and dedicated QSAR models for each individual sequence. The error bars indicate the standard deviation over the R02 and RMSE values of the validation on the different sequences. The PCM single models outperform the dedicated QSAR models in each case both measured by the R02 and the RMSE.
Figure S10.
The maximal distance of the compound from the training set plotted to the prediction error. The plot shows that the distance relates to the prediction error as compounds closer than 0.98 are predicted better than compounds further away than 0.98 from the training set.
Y-scrambling plot. In order to rule out chance correlations 100-fold Y-scrambling was performed. After scrambling the pEC50 values, these 100 data sets were in each case divided into a training set consisting of 80% of the total set and a test set consisting of 20%. The models built on permutated data cannot be validated with a Q2, R2 and R02 of approximately 0. After training and validation, Q2, R2 of the training and R02 of the validation were plotted against the similarity of the scrambled dataset with the original training set. This correlation was defined as the % of compounds that had a pEC50 value within 0.3 log units of its true value. A simple linear regression was subsequently performed for Q2, R2 and R02. The regression lines for Q2, R2 of the training and R02 of the validation crossed the y-axis at −0.12, −0.10 and −0.16 respectively. We conclude that it is highly unlikely that our model was created based on chance correlations between the different descriptors. Therefore we are modeling an actual correlation between the pEC50 values on the one hand and the compounds and proteins on the other hand.
The top 15 substructures contributing the most to binding on all sequences. Mean indicates the mean increase in pEC50 by the presence of that particular substructure, StdDev represents the standard deviation of the distribution of all changes in pEC50 correlated with the presence of that substructure and N represents the amount of predicted changes within this distribution. Binvalue is the identifier for that particular substructure (FCFP_6 format) and the properties Variance, Skew and Kurtosis have also been calculated over the distribution to determine the distribution pattern of the changes caused by that substructure.
(DOC)
The top 15 substructures leading on average to a lower pEC50 on all sequences. Mean indicates the mean decrease in pEC50 by the presence of that particular substructure, StdDev represents the standard deviation of the distribution of all changes in pEC50 correlated with the presence of that substructure and N represents the amount of predicted changes within this distribution. Binvalue is the identifier for that particular substructure (FCFP_6 format) and the properties Variance, Skew and Kurtosis have also been calculated over the distribution to determine the distribution pattern of the changes caused by that substructure.
All AAindices used with their reference on the AAindex website. This table lists all indices we used to create a unique hashed value per amino acid.
The complete SAR of 57 of the modeled compounds.
Archive S1.
Model and sample data set.
(RAR)
GvW would like to thank Tibotec BVBA for generously providing the data set.
Conceived and designed the experiments: GJPvW JKW API HWTvV AB. Performed the experiments: GJPvW PG LK IV AP. Analyzed the data: GJPvW JKW HWTvV AB. Contributed reagents/materials/analysis tools: GJPvW JKW PG LK IV AP API HWTvV AB. Wrote the paper: GJPvW JKW API HWTvV AB.
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text:
package org.jf.dexlib2.immutable.reference;

import com.google.common.collect.ImmutableList;
import org.jf.dexlib2.base.reference.BaseMethodReference;
import org.jf.dexlib2.iface.reference.MethodReference;
import org.jf.dexlib2.immutable.util.CharSequenceConverter;
import org.jf.util.ImmutableUtils;

public class ImmutableMethodReference extends BaseMethodReference implements ImmutableReference {
    private static final long serialVersionUID = -1369034637371375747L;
    protected final String definingClass;
     protected final String name;
     protected final ImmutableList<String> parameters;
     protected final String returnType;

    public ImmutableMethodReference( String definingClass,
                                     String name,
                                     Iterable<? extends CharSequence> parameters,
                                     String returnType) {
        this.definingClass = definingClass;
        this.name = name;
        this.parameters = CharSequenceConverter.immutableStringList(parameters);
        this.returnType = returnType;
    }

    public ImmutableMethodReference( String definingClass,
                                     String name,
                                     ImmutableList<String> parameters,
                                     String returnType) {
        this.definingClass = definingClass;
        this.name = name;
        this.parameters = ImmutableUtils.nullToEmptyList(parameters);
        this.returnType = returnType;
    }

    public static ImmutableMethodReference of( MethodReference methodReference) {
        if (methodReference instanceof ImmutableMethodReference) {
            return (ImmutableMethodReference)methodReference;
        }
        return new ImmutableMethodReference(
                methodReference.getDefiningClass(),
                methodReference.getName(),
                methodReference.getParameterTypes(),
                methodReference.getReturnType());
    }

     @Override public String getDefiningClass() { return definingClass; }
     @Override public String getName() { return name; }
     @Override public ImmutableList<String> getParameterTypes() { return parameters; }
     @Override public String getReturnType() { return returnType; }

}

redpajama_set_name: RedPajamaGithub


text:
Denkingen ist eine Gemeinde im Landkreis Tuttlingen in Baden-Württemberg.

Geographie

Geographische Lage
Denkingen liegt auf einer Geländestufe zwischen Großem Heuberg und Baar am Fuße des Klippenecks. Das Gemeindegebiet erstreckt sich von  im Primtal bis zum 1002 Meter hohen Hummelsberg.

Nachbargemeinden
Die Gemeinde grenzt im Norden an Gosheim, im Osten an Böttingen, im Süden an die Stadt Spaichingen und im Westen an Aldingen und Frittlingen.

Gemeindegliederung
Zur Gemeinde Denkingen gehören das Dorf Denkingen und die Häuser Erlenmühle und Klippeneck. Im Gemeindegebiet liegen die abgegangenen Ortschaften Berghof, Hinterhausen, Ipspoche, Niederhofen und Riedheim.

Schutzgebiete

In Denkingen liegt das Naturschutzgebiet Klippeneck und das Landschaftsschutzgebiet Albtrauf zwischen Balgheim und Gosheim mit Dreifaltigkeitsberg, Klippeneck und Lemberg.

Denkingen hat zudem Anteil an den FFH-Gebieten Prim-Albvorland und Großer Heuberg und Donautal sowie am Vogelschutzgebiet Südwestalb und Oberes Donautal. Darüber hinaus gehört Denkingen zum Naturpark Obere Donau.

Geschichte

Altertum
Das relativ ebene Gelände zwischen dem Primtal und dem Steilabfall der Schwäbischen Alb war schon früh bewohnt. Römer und Alemannen siedelten hier, wie Bodenfunde und Grabstätten belegen.

Mittelalter
Erstmals urkundlich erwähnt wurde Denkingen am 10. Oktober 818 in einer Schenkungsurkunde eines gewissen Theotmar von Grundstücken im Dorf Thanchinga zu seiner und seiner Gattin Seelenheil an das Kloster St. Gallen. Verschiedene geistliche und weltliche Herrschaften hatten Besitz am Ort. 1381 kam das Dorf zu Vorderösterreich und blieb dort, bis es 1805 an Württemberg fiel.

Neuzeit
Während des Dreißigjährigen Kriegs brannten Soldaten den Ort fast vollständig ab, bei dem die Weiler Hinterhausen und Rietheim verloren gingen. 1743 entstand auf Grund langer Streitigkeiten zwischen den Bürgern und dem Pfarrer Stockhl ein Aufruhr in der Gemeinde, auf den der Narrentyp Pfarrbachweib zurückgeht.

Das 1806 gegründete Königreich Württemberg ordnete den Ort dem Oberamt Spaichingen zu. 1889 schloss man den Ort an das Wasserleitungsnetz an. Durch eine Brandkatastrophe im Spätsommer 1892 wurde fast die komplette Hintere Gasse zerstört. Von 1928 bis zum 23. September 1968 war Denkingen durch die Heubergbahn ans Eisenbahnnetz geschlossen.

Bei der Gebietsreform während der NS-Zeit in Württemberg gelangte der Ort 1938 zum Landkreis Tuttlingen. Während des Zweiten Weltkriegs blieb Denkingen von Kriegsschäden verschont, wurde aber am 21. April 1945 von französischen Truppen eingenommen. 1945 wurde Denkingen Teil der Französischen Besatzungszone und kam somit zum Nachkriegsland Württemberg-Hohenzollern, welches 1952 im Land Baden-Württemberg aufging. Durch den Zuzug von Heimatvertriebenen und Flüchtlingen wuchs der evangelische Bevölkerungsanteil nach 1948 um 25 Prozent an. 1861 hatte der Ort nur zwei evangelische Einwohner. 1972 bildete Denkingen mit Spaichingen und den umliegenden Gemeinden eine Verwaltungsgemeinschaft. In den Tagen vom 23. und 24. Juni 1975 hatte der Ort mit schweren Hochwasserschäden zu kämpfen.

Politik

Gemeinderat
Der Gemeinderat der Gemeinde Denkingen besteht aus zwölf Mitgliedern. Parteien sind im Denkinger Gemeinderat nicht vertreten. Seit 1983 ist Rudolf Wuhrer Bürgermeister der Gemeinde. Die Gemeinde ist Mitglied der vereinbarten Verwaltungsgemeinschaft der Stadt Spaichingen.

Bürgermeister
Rudolf Wuhrer wurde bei der Bürgermeisterwahl am 28. Juni 2015 mit 52,5 % der Stimmen wiedergewählt, seine Herausforderin Manuela Raichle erreichte 47,4 %. Nach einer Wahlanfechtung ordnete das Landratsamt Tuttlingen eine Wiederholung der Wahl an, da Mitglieder des Wahlausschusses entgegen der gebotenen Unparteilichkeit Wahlkampf für den Amtsinhaber gemacht hatten. Bei der Wiederholungswahl am 6. Dezember 2015 konnte sich Wuhrer mit 53,6 % der Stimmen gegen Raichle (46,2 %) durchsetzen.

Wappen

Banner und Flagge

Partnerschaften
Denkingen unterhält partnerschaftliche Beziehungen zu Kirschau in der Oberlausitz in Sachsen.

Kultur und Sehenswürdigkeiten

Die Kommune ist dem Tourismusverband "Donaubergland" angeschlossen.

 Die Trasse der durch Denkingen verlaufenden, 1966 stillgelegten Heubergbahn ist heute als Rad- und Wanderweg ausgebaut. Sie bietet einen steigungsarmen Aufstieg nach Gosheim mit zahlreichen Aussichtspunkten entlang des Steilhangs des Heubergs.
 Das Segelfluggelände Klippeneck und der Wanderweg entlang der Hangkante zum Dreifaltigkeitsberg bieten bei guten Sichtverhältnissen herrliche Ausblicke über die Baarlandschaft zum Schwarzwald und zu den Alpen.
 Am steilen Hang des Klippenecks gibt es einige geschlossene Bestände von Eiben.

 Die Katholische Pfarrkirche St. Michael wurde 1933 von Hans Lütkemeier und Martin Schilling erbaut, der Turm der Vorgängerkirche blieb dabei stehen.

Wirtschaft

Verkehr
Denkingen lag bis 1966 an der inzwischen abgebauten Heubergbahn. Heute verkehrt die Buslinie 220 (Aldingen – Schömberg (Zollernalbkreis)), in Aldingen besteht Ringzug-Anschluss in Richtung Rottweil und Tuttlingen.
Denkingen ist über die Landesstraße 433 aus Richtung Aldingen / Spaichingen bzw. Wehingen / Albstadt und über die Kreisstraße 5907 aus Richtung Frittlingen / Rottweil zu erreichen.

Ansässige Unternehmen
 Die Firma Paul Kauth GmbH & Co. KG ist mit rund 220 Mitarbeitern einer der größten Arbeitgeber in Denkingen und ist Zulieferer für die Automobilindustrie.
 Die Schwer Fittings GmbH ist ein Hersteller von Verschraubungen, Kugelhähnen, Ventilen, Armaturen, Rohren und Schläuchen aus Edelstahl.

Söhne und Töchter der Gemeinde
 Rudolf Sauter (1925–2013), Erziehungswissenschaftler und Mundartdichter

Literatur

 (Wolfgang Urban): Katholische Pfarrkirche St. Michael in Denkingen. Kunstverlag Fink, Lindenberg 2008, ISBN 978-3-89870-530-1.

Einzelnachweise

Weblinks

 Internetpräsenz von Denkingen

Ort im Landkreis Tuttlingen

Ersterwähnung 818
Ort auf der Schwäbischen Alb

redpajama_set_name: RedPajamaWikipedia


At first when I joined Facebook I didn't like the feature of having other people's posts end up on my wall as game players were swamping my posts there; and then I discovered I could hide all of that and still keep the person's own comments visible. I also have the option of hiding a friend's posts totally from my wall and going to their wall when I am curious about what they are doing. I have yet to chat there, do not do any games, but I do like the photo posting feature. It is where my family photos go now. Facebook works fine for me at least for now.
Virginia, thank you for posting this comprehensive-yet-easy-to-understand introduction to Facebook! As I recall, women over 55 are among the fastest-growing demographics on Facebook, yet I keep running into people who are not familiar with it at all. Your guide can help them get started!
I also use it to send notes to a family member who has a Facebook account but not an email address or a phone. They respond if needed on Facebook. Works for us.
I have a Facebook account, but have never explored how to use it. I only go there when someone friends me or sends a message. I guess I had better give it another look.
Thanks for the information, Jan.
I was "permitted' to sign up for Facebook once my children were out of college; before that they considered me "a stalker". Sharing photos and keeping up with nieces and nephews makes far-flung relatives feel right next door. I play Wordscraper (faux Scrabble) and have developed long-term-on-line-superficial-but-friendly relationships with people all over the globe as we play rematch after rematch. Perhaps the best use of FB was in reconnecting with classmates in preparation for our 40th HS Reunion last fall. Plans were made, hotels reviewed, attendance was encouraged... and it was all free and easy.
I guard my privacy zealously, and keep most of my information restricted. But for those I've let into my circle of love, it's the best thing I've found for staying connected.
As a 71 year old techno-granny, I ignored FB for a long time. Then, I noticed that my Yahoo groups were dwindling in posts and content. I joined FB and found every one was there having a party without me. I have been FBing for a year and love it.
I live on the west coast. My siblings are all on the east coast where I grew up. One of the fringe benefits of FB is keeping in constant contact with them.
Rain, thanks for mentioning the Hide option for keeping the play by play activities of your friends who play games off your page.
I guess I'm lucky that my children and grandchildren have been willing to have me as a friend on Facebook. I especially like having little chats with my granddaughter who lives far away after she gets home from school in the afternoons.
It takes a while to see the benefits of Facebook, and I didn't like it at first. I got used to Facebook and figured out how to manage the privacy settings and so on, and now I love it.
My blog is not a suitable place to talk about my kids' personal lives or to post pix of them, so Facebook is great for picking up on the news and seeing what they are up to, as at my granddaughter's birthday party, etc.
Thanks for the tips, Virginia, which I can use to make my Facebook experience even better.
And the cherry on the cake is that a couple of years ago, when I didn't use FB at all but only had an account that I hardly ever visited, one of my long-lost friends found me. As she lives in the centre of France where I never go, I don't think we would have got together otherwise.
Thank you, Jan...I like FB - it is kind of a grab bag of info. By the way for any Jewish folks on TBG - there is a JewishGen.org that lets you check out your ancestors. Very interesting to check out.
This post discussed a lot of the good with Facebook but it minimizes the privacy issues and problems. First, FB relies on an opt-in marketing strategy, where its users are automatically included in programs. this forces FB members to constantly opt out of things... a time-consuming frustration especially if you are an infrequent FB user.
Second, there are privacy settings in several places within the FB site. I liken this to several gopher holes one must visit to effectively limit what personal data gets shared. And FB can change things anytime which overrides your settings, so you have to revisit those multiple privacy setting places.
Be fully aware of the risk.
George, You make a good point. I would add that Facebook frequently changes things--appearance, privacy settings, and more. If you pay attention to such changes you can keep on top of the privacy settings issues as they arise.
Thanks for the FB info., Virginia. My dtr guided me thru setting up my acct. last Dec. including privacy settings. George's info re FB's ever-changing privacy set up is certainly impt. to be aware of and one that has influenced my limited involvement there.
I've restricted my FB "friends" to only personal friends and family that I don't mix with blogging friends with whom I can have contact through our blogs, other. I don't visit my FB regularly so several months passed before learning someone was looking for me to tell me their non-computer using family member former classmate had others trying to find me. Has been fun to connect these fifty years later and we'll likely try to get together in the future.
I've embraced more tech than I once thought I might including texting, plus I really like Skype and will likely keeping adding whatever evolves in the future -- sooner or later. Am looking forward to possibly adding the next IPad after it shakes down and the price mellows. The IPhone is even attractive to me now though I currently don't have one.
I think Facebook is going to have to change its direction on privacy if it wants to keep its users. They need to get their act together and show some respect for their users.
Here's a Facebook rant by the brilliant danah boyd that you may find interesting.
I think I'll appreciate FaceBook more when my kids are on their own and I want to keep up with them.
Hello Lee, I just love reading this post, keep it up.


text:
Tyler's CC Scores
By quiznos00, June 19, 2016 in High Scores
CCLP2 MS
#85 (Follow Some Stuff): 301 (+3, b-5)
#94 (Checkerboard II): 686 (+119, b-102)
#114 (Joyride II): 78 (+1, b-3)
#146 (Run-a-Muck): 301 (+128, b-18)
Total score: 6,037,770
CC1 MS
#89 (Blockbuster): 361 (+5, b-41)
#132 (Mix Up): 678 (+61, b-5)
#147 (Force Field): [819] (+783, b-151)
#30 (Problem Solver): 232 (+5, b)
#58 (Bowling): 244 (+1, b)
#59 (Air Pocket): 322 (+63, b)
#14 (Window Shopping): 347 (+7, b)
#131 (Mental Blocks): 640 (+243, b-16)
116 (Communism): 520 (+28, b)
Total score: 6,002,460 (100 b)
lol back
#11 (Blue Blocks): 448 (+2, b)
#17 (Lot in Life): 97 (+18, b)
#18 (Replay): 343 (+9, b-2)
#28 (Pac-Man): 392 (+23, b)
#31 (Bouncin' Blocks): 211 (+2, b)
#68 (Incident on a Bridge): 430 (+154, b)
#100 (Packt Like Sardines in a Crushd Tin Box): 230 (+12, b)
#29 (Tool Box): 285 (+78, b)
#86 (To Catch a Thief): 392 (+114, b)
#105 (Caves) 205 (+35, b)
#77 (Oh-Ho!): 191 (+3, b)
#81 (Possible): 113 (+12, b)
#84 (No End in Sight): 346 (+27, b)
#85 (Magi-no Line): 507 (+134, b-6)
#91 (Olly Olly Oxygen): 229 (+29, b)
#148 (Diabolical): 320 (+15, b)
#117 (THE MARSH-IAN): 942 (+30, b)
#125 (DEEZ NUTS): 830 (+85, b)
#130 (CRAZY MOTHERF'ER NAMED ICE CUBE): 933 (+22, b)
#68 (Sadness II): 263 (+32, b-14)
This score report is boring don't read it.
#100 (COLONY): 910 (+15, b)
#8 (Pigeonholed): 111 (+6, b)
#33 (Vague Dream): 40 (+10, b)
#40 (Blazes): 228 (+7, b)
This is also a boring score report, but I'm posting it so I can pass Shane in overall bold count
#12 (My Friend, Sits at the Drum, His Magic Hand, Feels Nothing But Time, Nothing But Time): 125 (+9, b)
#48 (Switch Hit II): 391 (+51, b)
#52 (Dolly Dolly Oxen): 365 (+2, b)
CCLP3 MS:
#60 (Cyan, Magenta and Yellow): 258 (+189, b, thank you pieguy's computer for the bold)
#63 (Pocket Monsters): 230 (+57, b)
#74 (Trevor's Favorite Level): 270 (+6, b)
#75 (Josh Alone: Lost in Acampo): 314 (+54, b)
#89 (Going for Bold II): 398 (+6, b)
#4 (Suction Solution): 245 (+1, b)
#49 (All About Blocks): 306 (+3, b)
#55 (Construct-a-Maze): 237 (+97, b)
#61 (Bump and Run): 318 (+98, b)
#118 (Obstacle Course): 386 (+138, b-8)
Officially in the top 10
I was planning on getting more scores, but I'll probably be busy with CCLP4 stuff now
#71 (How to Get Around in Venice): 406 (+48, b)
#87 (Block and Key): 396 (+44, b-1)
Crappy initial scores
#1 (Molecule): 164
#2 (Pixelated Fire): 246
#3 (Fossilized Snow): 197
#4 (Oasis): 214
#5 (Non-Dimensional Layer): 266
#6 (Proving Grounds): 303
#7 (In the Pool): 93
#8 (The Fourth Dimension ): 321
#9 (Pinball): 235
#10 (Stuck in Emerald): 67
My Poly-Gone and Rivets scores are bad so here are some snipes
#11 (Keyboard Malfunction): 303
#13 (Encased in Carbonite): 184
#15 (Cross Back): 260
#16 (Reservoir Frogs): 274
#17 (The Three Trials): 249
#18 (Inferno Dynamics): 219
#19 (Conservation of Keys): 187
#20 (It's No Skin Off My Teeth): 328
#23 (Western Standards of Living): 349
#66 (Anaconda): 264
CCLP4 MS wowee!
#115 (Overlap): [916]
#135 (Propaganda): 384
#142 (Stratagem): 43
EDIT: Apparently I posted this one minute early according to CCZone but this does not make a difference in confirms/evolution and whatnot
Edited July 15, 2017 by quiznos00
#46 (XOR): 251 (boy, one of the choices is much longer)
#40 (Periodic Lasers): 130
#60 (Flippant): 208
#47 (Anti-Disruptiive Caves): 353
Some alright scores (CCLP4 MS obvs)
#12 (Rivets): 219
#14 (Poly-Gone): 324
#21 (Glacial Palace): 301
#39 (In the Walls of Josh Castle): 434
Some alright scores, the sequel:
#22 (Bodyguards): 238
#24 (It's Easy Being Josh): 371
#25 (Difficulty Switch): 396
#30 (Hoodwinked): 59
#33 (Tool Shed): 211
#34 (Frozen Waffle): 175
#26 (Shrub): 350 (b, tyrsfttspga)
While I'm at it...
#10 (Alone): 6180 118 (b)
#84 (Forsythia): 397 (bc)
#88 (Empty Rooms): 277 (pc, optimization is pain)
Optimization is pain, highness. Anyone who says differently is selling something.
#120 (Build-a-Sokoban Workshop): 639
But wait! There's more!
#120 (same level lol): 644
#51 (Will It Blend? That Is the Question): 392 (bc)
#106 (Grid, Locked): 232 (bc)
The saga continues!
#120 (CaS): 647 (+3)
Might as well get a Lynx confirm
CCLP4 Lynx
#51 (Ice in a Blender): 380 (bc)
#120 (Construct-a-Sokoban): 655 (bc, I'm done with block pushing for a while )
EDIT: CCLP4 MS obvs
#120 (Construct-a-Sokoban): 661 (+6, JB (joint bold) with JB (John Brian))
And now, some initials. CCLP4 MS
#35 (Chasing Chips): 346
#36 (One Who Raids Tombs): 455
#37 (Tropical Hibiscus): 376
#38 (Detonation Station): 138
#41 (Ghetto Piranha): 300
#42 (Nova Prospect): 229
#43 (Coral Reef): 410
#44 (Blobfield): 400
#45 (Seven-Layer Salad): 335
#48 (Key Insight): 287
#49 (Block Parking): [881]
#50 (Secret Underground Society): 317
EDIT: never reported this as well
#27 (Suburban Legend): 376
#28 (Zephyr Heights): 381
#29 (Flipper Departments): 306
#52 (It Suits the Purpose): 90
#53 (Protect Your Fortress): 289
#54 (Split Path): 191
#55 (If I Ran the Zoo): 520
#56 (Fireworks Factory): 539
#57 (Bisection): 446
#58 (Ruinous Plaza): 317
#59 (Blockpick): 315
#61 (Blue Tooth): 523
#62 (Block Unpuzzle): 305
#88 (Empty Rooms): 278 (+1, bc)
#88 (Empty Rooms): 278 (joint bold with J.B. )
#63 (Pneumatic Diversity Vents): 495
#64 (Excuse Me): 291
#65 (Duplex): 535
#67 (Nuclear Energy for Dummies): 296
#68 (Cold Fusion Reactor): [908]
#69 (Ball in an Awkward Place): 391
#70 (Science Museum): 339
#71 (Puuf): 175
#72 (Sewerway): 356
#73 (Sealed Doors in the Spacecraft): 249
#74 (Technopathic): 326
#75 (Unmitigated Hint Factory Disaster): 376
#76 (Flow State): 371
#77 (Brick Block Facility ): 484
#78 (Aquatic Ruins): 473
#79 (Spring ): 372
#80 (Monster Swapper): 282
#81 (Estranged for a Season): 298
#82 (Puzzle Box): [903]
#83 (Frozen Over): 415
EDIT: never submitted these scores from a while ago
#31 (BBA): 382
#32 (BBB): 304
#85 (Nectar Meadow): 429
#86 (Cyprus): 304
#87 (And the Walls Kept Tumbling Down): 508
#89 (Diametric Opposition): 373
#90 (Wretched Hive of Scum and Villainy): 360
#91 (How to Retune Your Harp): 374
#92 (Fire Is My Enemy): 418
#93 (Bombs Are a Beautiful Thing): 370
#94 (Ditchdigger): 487
#95 (Ravaged): 502
#93 (Optimization Is a Beautiful Thing): 384 (+14, bc)
#95 (Sapphire Caverns Part Deux): 514 (+12, bc)
#67 (Nuclear Energy for Smarties): 317 (+21, bc)
#96 (Lean Thinking): [823]
#97 (Lockdown): 259
#98 (Clay Tunnel): 470 (b)
#99 (Ice Cavern): 297
#100 (One Tank's Adventure): 808
#87 (And the Walls Kept Tumbling Down): 524 (+16, bc)
#149 (Mental Marvery Monastery): 510 (b+3)
#149 (Mental Marvel Monastery): 513 (+3, b+1)
#101 (Condo Management): 398
#102 (The Key Issue): 312
#103 (Malachite): 551
#104 (Dual): 189
#105 (Living Things): 407
#107 (Combinations): [867]
#108 (Scatterbrained): 477
#109 (Shemozzle): 17
#110 (Keyrithmetic): [946] (b)
#149 (Mental Marvel Monastery): 514 (+1, JB with JB... Jeffrey Bardon this time!)
#111 (Water Bottle): 174 (b)
#112 (Triple Mint Slurpee): 512
#113 (Half of You, Half of Me): 321
#114 (Repugnant Nonsense): 478
#116 (They're Not Called Blocks for Nothing): 303
#117 (Greenian Motion): 256
#118 (Chip Controls): 412
#119 (Strandquist): 495
#119 (Strandquist): 519 (+24, b-1, pc, optimization is pain)
#121 (Death and Destruction): 381
#122 (Jigsee): 706
#123 (Life Is Not a Puzzle): 619
#124 (Air Bubble): 111
#125 (Beautiful Struggle): 501
#126 (Bind Mender): 132
#127 (Wrong Exit): 218
#128 (Mindless Self-Indulgence): 480
#129 (Undefined Fantastic Object): 202
#130 (Bam Thwok): 496
#133 (Monochrome): 634 (bc)
#131 (Jigsaw): 636
#132 (Monorail): 498
#134 (Pushover): 406
#136 (Seeing Red): 295
#137 (The Longest Track): [857]
#138 (Zipper): 434
#139 (Unravel): [972]
#140 (Repair the Automatic (Caution) Doors): 737
#141 (World of a Thousand Flames): 691
#143 (Color Coordination): 619
#144 (Paradigm Shift): 638
#145 (Hacked Save File): 491
#146 (Japanese Game Show): [818]
#147 (Gimmick Isle): 729
#148 (Gravity Well): 402
6,081,070 (I have some unreported seconds) - 26 b
#141 (World of a Thousand Flames): 712 (+21, bc)
#128 (MSI): 506 (+26, bc)
#56 (Fireworks Factory): 554 (+15, bc)
#143 (CC): 645 (+26, bc)
Edit: Oh yeah here's an easy bold I got
#60 (Flippant): 210 (+2, b)
Optimization is cool again. Here are some quick ones.
#6 (Grooving Pounds): 310 (+7, b)
#21 (Iceland): 303 (+2, b)
#30 (Does Anyone Else Remember This Movie): 61 (+2, b)
#33 (The Sharpest Tool): 215 (+4, b)
#104 (It's Time to Dual): 196 (+7, b)
Optimization coolness has reached maximum capacity, so here are some more easy ones and a medium one.
#2 (Pixelated Fire): 251 (+5, b)
#8 (The Fourth Dimension): 333 (+12, b)
#11 (Keyboard Malfunction): 350 (+47, b)
#13 (Encased in Carbonite): 211 (+27, b)
#37 (Can I Join Da Club): 324 (+2, b)
#127 (Escape from Zartacla): 113 (+1, b, Bacorn is God)
#10 (Graduation): 320 (+2, b)
#127 (In the Pink): 422 (+36, b)
#38 (Up-Down Adventure): 482 (+8, b-1)
#73 (Zzzzzz): 419 (+8, b)
#013 (The Monster Cages): 263 (+2, b)
#090 (Bowling Alleys): 332 (+17, b-11)
#115 (California): 430 (+10, b)
#116 (Communism): 521 (+1, b)
#65 (Squared in a Circle): 428 (+9, b)
#140 (ACD): 821 (+8, b-4)
#141 (Flush): 269 (+3, b)
#87 (And the Walls Kept Crumbling Down): 526 (+2, b, tykl)
#130 (Bam Thwok): 561 (+65, b)
#3 (lol bad score): 216 (+19, b-4)
#14 (Poly-Gone): 335 (+11, b)
#17 (The Three Trials): 254 (+5, b)
#35 (Chasing Chips): 356 (+10, b)
#80 (Monster Swapper): 292 (+10, b)
#119 (Strandquist): 520 (+1, b)
It's boring score report hour
#13 (Road Block): 107 (+10, b)
#44 (Tiny RNG): 217 (+31, b)
#45 (Block Head): 411 (+28, b)
#128 (Caves II): 433 (+68, b)
#54 (huh): 284 (+23, b)
#54 (Good Advice): 286 (+2, b+2, joint bold with James)
Here's one wacky score report
#72 (Reverse Alley): [961] (+7, b)
#100 (Colony): [911] (+1, b)
#103 (Memory): 488 (+1, b)
#133 (Blobdance): [887] (+102, b-62)
#136 (Doublemaze): [855] (+293, b-71)
#138 (Partial Post): 235 (+25, b-5)
#139 (Yorkhouse): [920] (+32, b)
#144 (Fireflies): [832] (+31, b)
#127 (Yeet the Heat): 432 (+72, b-5)
#97 (No!): 263 (+4, b-1)
#106 (Gridlock): 240 (+8, b)
#127 (Wrong Exit): 225 (+7, b)
#29 (Even Flow): 302 (+2, b)
#64 (Yikes): 548 (+1, b)
#12 (Mazed In): 354 (+1, b)
#34 (Cypher II): 404 (+26, b)
#55 (Dangers of Fire and James): 78 (+4, b)
#111 (Bustin' Out): 613 (+144, b-7)
#130 (Four by Four): 311 (+116, b-14)
#145 (Recurring Dream): 735 (+146, b)
#22 (Peer Pressured Into Getting This One): 293 (+3, b)
#97 (I Don't Understand James): 264 (+1, b)
#94 (I see it, I see it, and now it's all within my reach, endless possibility): [906] (+50, b)
#91 (Good Bust tbh): 472 (+98, b)
Boring score report only to get by bold count above 450
#111 (The Sims: Bustin' Out): 623 (+10, b)
#109 (Shemozzle): 22 (+5, b)
#124 (Air Bubble): 112 (+1, b)
#79 (lmao): [958] (+1, b)
CC1 Lynx, most of them are bold
#1 (LESSON 1): 82
#4 (LESSON 4): 116
#9 (NUTS AND BOLTS): 299
#10 (BRUSHFIRE): 51
#13 (SOUTHPOLE): [981]
#16 (CELLBLOCKED): [970] (b-1 LOL)
#17 (NICE DAY): 82
#18 (CASTLE MOAT): 552
#31 (KNOT): 3
#32 (SCAVENGER HUNT): 322 (didn't mean to report this but too late)
#34 (CYPHER): 297
#39 (GLUT): 17
#40 (FLOORGASBORG): 192
#45 (MONSTER LAB): 286
#52 (THE LAST LAUGH): 381
#69 (BOUNCE CITY): 218 (b-2, p)
#75 (STEAM): 479
#102 (ICEHOUSE): 175
#129 (LOBSTER TRAP): 287
CC1 Lynx
#11 (TRINITY): 204
#15 (ELEMENTARY): 87
#16 (CELLBLOCKED): [971] (+1 LOL)
#25 (BLINK): 422
#42 (BEWARE OF BUG): 187
#51 (I SLIDE): 649
#101 (APARTMENT): 240
#105 (SHORT CIRCUIT): 254
More meme scores
#12 (HUNT): 269
#14 (TELEBLOCK): 196
#19 (DIGGER): 171
#20 (TOSSED SALAD): 340
#21 (ICEBERG): 115
#26 (CHCHCHIPS): 254
#27 (GO WITH THE FLOW): 144
#46 (THREE DOORS): 200
#59 (HIDDEN DANGER): 366
#95 (FOUR SQUARE): 333
the years start comin and they don't stop comin
#32 (SCAVENGER HUNT): 379
#38 (SAMPLER): 452
#43 (LOCK BLOCK): 118
#53 (TRAFFIC COP): 448
#62 (SLO MO): 282
#63 (BLOCK FACTORY): 471
#66 (VICTIM): 291
#78 (FORCE SQUARE): 469
#80 (VANISHING ACT): 732
#82 (SOCIALIST ACTION): 969
#84 (WARS): 579
#86 (SUICIDE): 380
#88 (SPIRALS): 317
#91 (JUMPING SWARM): 367
fed to the rules and i hit the ground runnin
#41 (I.C. YOU): 166
#48 (MUGGER SQUARE): 271
#49 (PROBLEMS): 161
#57 (STRANGE MAZE): 228
#65 (AMSTERDAM): 383
#99 (CATACOMBS): 373
#109 (TORTURECHAMBER): 129
#111 (TIME LAPSE): [961]
#112 (FORTUNE FAVOURS THE): [985]
#119 (SLIDE STEP): 178
#121 (PERFECT MATCH): [966]
#127 (SKELZIE): 442
#130 (ICE CUBE): [925]
#137 (GOLDKEY): 381
#140 (ICEDEATH): 247
#142 (PENTAGRAM): [966]
#149 (SPECIAL): 949
I have a lot of unreported CCLP1 Lynx scores it seems. Here are some old bolds plus a few new ones I just got.
#1 (Key Pyramid): 168
#2 (Slip and Slide): 179
#3 (Present Company): 183
#4 (Block Party): 201
#5 (Facades): 232
#7 (Under Pressure): 184
#8 (Switcheroo): 229
#9 (Swept Away): 225
#11 (Basketball): 225
#13 (The Monster Cages): 263
#15 (Twister): 306
#17 (Tiny): [989]
#25 (Mind Lock): 138
#27 (Teleport Depot): 276
#36 (Rhombus): 216
#43 (Gears): 206
#50 (The Grass Is Greener on the Other Side): 166
#63 (New Block in Town): 169
#72 (Breathing Room): 180
#77 (Shuttle Run): 6
#48 (Sundance): 180
#67 (Booster Shots): 386
#79 (Elevators): [958]
#85 (Black Hole): [969]
#96 (Going Underground): 407
#100 (Loose Pocket): 334
#102 (Frozen in Time): [943]
#103 (Portcullis): [978]
#105 (Tunnel Clearance): 235
#123 (The Very Hungry Caterpillar): 58
CCLXP2
#1 (A Fleeting Memory): 348
#4 (Force World): 234
#5 (Suction Ride): 371
#7 (Slightly Mad): 246
#9 (Maze of One Way): 196
#10 (Who Needs a Flipper?): 366
#13 (The Serial Port): 337
#15 (Debug File *): 234
#20 (Ranger Denmark *): 292
#135 (Meme): 495
#36 (LADDER): 241
#50 (DIGDIRT): 319
#55 (POTPOURRI): 68
#56 (DEEPFREEZE): 150
#60 (SCOUNDREL): 232
#70 (NIGHTMARE): 136
#74 (PLAYTIME): 355
#77 (INVINCIBLE CHAMPION): 478
#79 (DRAWN AND QUARTERED): 218
#97 (METASTABLE TO CHAOS): 290
#114 (BAD LEVEL): 174
#125 (Blockade): 207
#131 (Easier Than It Looks): 103
#137 (Thief Street): 178
#6 (Thieves and Teleports): 90
#19 (Super Chip): 168
#25 (Recess): 99
#28 (PING PONG): 236
#30 (MISHMESH): 454
#33 (ON THE ROCKS): [515] (b-116 hehe)
#54 (GRAIL): 319
#58 (LOOP AROUND): 546
#64 (SPOOKS): 547
#71 (CORRIDOR): 351
#83 (UP THE BLOCK): 297
#90 (PLAYHOUSE): 314
#122 (TOTALLY FAIR): 272
#128 (ALL FULL): 289 (b-3)
#131 (TOTALLY UNFAIR): 26
#122 (Jeepers Creepers): 651 (+1, b, thx Sharpeye!!)
#133 (Block Maze): 834 (+2, b, yet another collab with my main man Sharpeye!!)

redpajama_set_name: RedPajamaCommonCrawl


text:
Q: Unity: Set RigidBody velocity in FixedUpdate() or Start()? I'm developing a simple 2D game in Unity (balls hitting bricks), so i have a ball and i want to fire the ball to hit bricks, now to fire the ball i have to set Velocity on the RigidBody component attached to the ball's GameObject.
There are two ways to do that:
Method 1:
Set the velocity inside the Start() method
private void Start()
{
    rb.velocity = direction * speed;
}

Method 2:
Write a FixedUpdate() method and put the statement in there. because:

Use FixedUpdate when using Rigidbody.(Unity Docs)

private void Start()
{
    _setVelocity = true;
}

private void FixedUpdate()
{
    if (!_setVelocity) return;

    rb.velocity = direction * speed;
    rb.angularVelocity = 0;
    rb.gravityScale = 0;
    _setVelocity = false;
}

But the thing is: i don't need the FixedUpdate() method for anything else, because i only fire the ball once in its script's lifespan, so after i fire the ball unity will continue to run the FixedUpdate()method and check for _setVelocity's value and return (exit) every time because it will always be false after i shoot the ball the first time.
And this will happen every fixed frame-rate frame which is expensive i guess for just setting the velocity only once.
SO:

*

*Is it expensive to have a FixedUpdate() method that checks for false value and returns every frame?

*Which of the two methods performs better in this case?

A:
Is it expensive to have a FixedUpdate() method that checks for false value and returns every frame ?

A simple boolean condition such as yours is not that greedy so do not worry about that. However, any line of code that runs decrease your performances. The Update() method itself is more greedy in terms of performance than your "trivial" condition. Disabling your component script or removing the object itself is more optimize than keep it running for nothing. Just keep that in mind :)

Which of the two methods performs better in this case ?

The first one is more efficient because your velocity is fixed, so there is no point of using an update method (Update() or FixedUpdate()). You should initialize your rigidbody's velocity directly in your Start() method as you did in your post, so you can get rid of your boolean and its condition.
Unity's documentation is pretty clear about rigidbody and FixedUpdate() but I have the impression that you misunderstood some aspects.
They tell you that if you want to modify any data of your rigidbody during the run-time, prefer to use FixedUpdate() instead of Update() to avoid weird behavior.
But why should you use FixedUpdate() instead of Update() ?
Because Update() is called at each frame, so if your pc runs at 100 fps, Update() will be called 100 times. And if your pc runs at 200 fps, it'll be called 200 times. It is kind of problematic in some cases, especially when you interact with physics components.
In order to understand, lets admit that we want to apply a force to our rigidbody (rb.AddForce(100)). If you call it in your Update() method and your pc runs at 100 fps, you'll apply 10000 force in one second. However, if your pc runs at 50 fps, then you'll apply 5000 force in one second. It'll cause you some weird behavior that could be avoided thanks to FixedUpdate(), which is called at a fix number of times per second (see unity doc).
However, there is no point of using FixedUpdate() with your rigidbody component if you do not want to modify it. They tell you that if you want to update your rigidbody component, you should do it inside the FixedUpdate method instead of Update().
So for your question, you can simply do the following:
Awake() {
    // getting your components
    rb = GetComponent<Rigidbody2D>();
}

Start() {
    // initiliaze your components
    rb.velocity = direction * speed;
    rb.angularVelocity = 0;
    rb.gravityScale = 0;
}

Hoping that I've helped you, good luck with your project !

redpajama_set_name: RedPajamaStackExchange


That's great Jess and I won't say awesome. I'm glad you're getting involved in different organizations and reaching out to various parts of your community.
Absolutely right. I always forget great grandpa was born in Buffalo. 🙂 Thanks Pink Ma!