**7. References**

176 Advanced Topics in Measurements

Step-height: 10 µm

○: center of fingertip ●: top of fingertip

Fig. 10. Relationship among the fingertip region, the upper threshold for a 10 µm step-

according to the PEST algorithm and presented the step pairs to the subjects.

In this study, to measure the finger's fine step-height discrimination capability the computer-controlled measurement system that presents fine step-heights of 0 to 1000 µm to subjects' fingers was developed. Using the measurement system the paper examined the effects of the touching manner of human finger, finger's motion direction, and fingertip region on the tactile recognition of fine step-heights. In the psychophysical experiments, to determine the difference thresholds and subjective equalities for fine step-heights the measurement system calculated the amounts of step-height of the step pairs by computer

Presentation angle of step deg

First, the upper thresholds for the step-heights of 10 to 100 µm were determined in the active- and passive-touch experiments. The resulting thresholds became larger as the magnitude of step-height increased. Also the threshold of active-touch manner for each of the step-heights larger than 50 µm was smaller than that of passive-touch manner and the thresholds of the touching manners for each of the step-heights smaller than about 40 µm were almost equal regardless of the touching manners. Therefore it was found that the fingertip's discrimination ability of the fine step-heights depends on the amounts of stepheight and if a step-height is larger than 50 µm, the finger's tactile sense can increase the

height, and the presentation angle.

0

1

2

Upper threshold for step-height,

*ΔU* µm

3

4

5

6

sensitivity in active-touch manner.

**6. Conclusion**

**Overview of Novel Post-Processing**

**Techniques to Reduce Uncertainty**

Manuel Sierra-Castañer, Alfonso Muñoz-Acevedo,

*Technical University of Madrid (Universidad Politécnica de Madrid - UPM),*

The error analysis has been investigated since 1975 since recent publications, as shown in references as (Newell et al., 1975) where the classical 18 terms for planar near field systems where established. It is also worth mentioning the more recent studies developed inside the actions under the "Antenna Measurement" activity of the "Antenna Centre of Excellence (ACE)" within the sixth framework research program of the European Union. In particular, that work pretended to establish common error calculation criteria in spherical near-field and far-field antenna measurement systems. The results of that research were summarized in an exhaustive deliverable (Alexandridis et al., 2007), which detailed the observations stated by several research institutions. In that deliverable it was agreed that the causes of uncertainties and errors in a spherical near-field antenna measurement are divided in six

1. **Mechanical uncertainties and errors**: this group includes the axes intersection, the axes orthogonality, the horizontal pointing, the probe vertical position, the probe horizontal

2. **Electrical uncertainties and errors**: this class contains the amplitude and phase drift, the amplitude and phase noise, the leakage and crosstalk, the amplitude non-linearity

3. **Probe-related uncertainties and errors**: this kind of uncertainties takes into account the channel balance amplitude and phase, the polarization amplitude and phase and the

4. **Stray signals**: this type of uncertainties consists of the multiple reflections, the room

5. **Acquisitions errors**: this group involves the scan area truncation and the sampling

6. **Processing uncertainties and errors**: in this group the spherical mode truncation and

The second part of the chapter will deal with the description of four different classes of

and vertical pointing and the measurement distances.

and the amplitude and phase shift in rotary joints.

scattering and the AUT support scattering.

the total radiated power are considered.

correction techniques for antenna measurements.:

**1. Introduction**

categories:

pattern knowledge.

point offset.

**in Antenna Measurements**

Francisco Cano-Fácila and Sara Burgos

*Spain*

Vallbo, Å.B. & Johansson, R.S. (1984). Properties of Cutaneous Mechanoreceptors in the Human Hand Related to Touch Sensation, *Human Neurobiology*, Vol. 3, pp. 3-14. **9**


**Preface to "Orthodoxy and Evangelicalism: Contemporary Issues in Global Perspective"**

Since the 1990s, the Eastern Orthodox and Protestant Evangelical communities have had more direct contact with each other than at any other time. This book focuses on current developments and issues in specific regions of the world.

With the fall of communism in Eastern Europe, Western Evangelical missionaries began flooding the former Soviet Union, Romania, and other Eastern Bloc countries, often without consulting the local Evangelical communities. Partly in response to this wave of missions, a new paradigm of ecumenical relations emerged among professional theologians in North America when the *Society for the Study of Eastern Orthodoxy and Evangelicalism* (SSEOE) was formed in 1990. It focused on comparative theology, spirituality, and missions. In 1993, the World Council of Churches (WCC) opened international dialogues between the Orthodox and Evangelical communities, fueled by concerns regarding Evangelical proselytism in Russia and Eastern Europe but also shared concerns regarding perceived theological trends within the WCC itself. Publications included *Proclaiming Christ Today: Orthodox–Evangelical Consultation* (1995) and *Turn to God, Rejoice in Hope* (1998). From 2000 to 2006, a series of seminars at the Ecumenical Institute, Bossey, resulted in the publication of *Building Bridges: Between the Orthodox and Evangelical Traditions* (2012). In the UK, the Evangelical Alliance initiated a dialogue process that resulted in *Evangelicalism and the Orthodox Church* (2001), which aimed to lay a foundation for mutual understanding by comparing and contrasting Orthodox and Evangelical beliefs and practices. Since then, the main ongoing exchange between Orthodox and Evangelical communities has been that initiated in 2010 by leaders within the Lausanne Movement and the Oriental and Eastern Orthodox churches, which resulted in the formation of the Lausanne–Orthodox Initiative. Unlike other dialogues, this focuses on exploring how the two traditions can cooperate in the mission of God. A number of papers from its gatherings appear in *The Mission of God: Studies in Orthodox and Evangelical Mission* (2015) and *Living the Gospel of Jesus Christ* (2021).

However, in spite of all the work that has been conducted, there remains scope for further scholarly investigation; for example, few regional studies have examined areas outside the Anglophone world, or the political and legal aspects of relationships between these traditions. Current environmental and moral concerns present new challenges but also new opportunities to learn from one another. Hence, the purpose of this volume.

This volume presents a collection of scholarly essays on current issues and developments in Orthodox–Evangelical relations, at both global and national levels, which will inform the ongoing dialogue. The essays evaluate regional meetings, discuss the history of relationships, address current missiological challenges, political and legal issues, comparative theology, and related topics. They range through Russia, Eastern and Western Europe, the Middle East, Africa, North America, Australia, and Greece. A particular strength of the volume is the number of contributions from Eastern European writers. Nuances of difference are recognized here between Eastern and Western Evangelicals, and between Eastern Orthodox and Oriental Orthodox communities.

We wish to thank the editors of the Religions journal for their invitation to edit this collection of essays. Their support and editorial efficiency have made our work much easier. Finally, we were delighted at the level of interest in contributing to this volume, and we thank the authors for their patience with the editorial process. Such a degree of interest bodes well for the future development of relationships between these two Christian traditions.

> **Bradley Nassif, Tim Grass** *Editors*


**Coupled Enzyme Activity and Thermal Shift Screening of the Maybridge Rule of 3 Fragment Library Against Trypanosoma brucei Choline Kinase; A Genetically Validated Drug Target**

Louise L. Major, Helen Denton and Terry K. Smith

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52668

# **1. Introduction**

Infectious diseases caused by parasitic protozoa affect approximately 15% of the global pop‐ ulation, and more than 65% of the population in the Third and developing world, yet cur‐ rent drug therapies for protozoal infections are woefully inadequate. As protozoal infections take their toll predominantly in the developing world, market forces are insufficient to pro‐ mote the development of novel anti-protozoal drugs. In 2000, only ca. 0.1% of global invest‐ ment in health research was spent on drug discovery for tropical diseases [1].

One such neglected parasitic disease is Human African Trypanosomiasis (HAT) or African sleeping sickness, which is caused by the protozoan parasite *Trypanosoma brucei* and is trans‐ mitted by the bite of the Tsetse fly. The WHO estimates that HAT constitutes a serious health risk to 60 million people in sub-Saharan Africa, 300,000-500,000 of whom become in‐ fected each year, with an estimated 10,000 fatalities. The related disease in cattle, Cattle Try‐ panosomiasis or Nagana, also represents a major health concern due to its devastating economic, social and nutritional impact on African families, estimated by the WHO as an annual economic loss of ~US\$ 4 billion. As such, the total burden of Trypanosomiasis trans‐ lates into 1,598,000 Disability-Adjusted Life Years, this is on a par with big killers such as *Mycobacterium tuberculosis* and Malaria [2, 3].

Treatment of HAT is dependent upon four drugs: suramin, melarsoprol, pentamidine and eflornithine.These therapies are often toxic, difficult to administer and increasingly have an acquired drug resistance [4, 5].Developed before the 1950s suramin and melarsoprol are

© 2013 Major et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

used for chemotherapy of early stages of the disease, as is pentamidine. The arsenical melar‐ soprol is extremely toxic, with death for ~1 in 20 of cases and treatment failures as high as 30% in certain areas [4, 6]. Treatment of the second stage of the disease, where the parasites cross the blood-brain barrier and invade the central nervous system, is limited to melarso‐ prol and eflornithine [7].The WHO as a desperate measure recently introduced nifurtimoxeflornithine combination therapy for the treatment of HAT. This is despite nifurtimox, a drug often used to treat Chagas' disease (caused by the related protozoan the South Ameri‐ can *Trypanosoma cruzi*), having low efficacy against HAT [8].

Hence there is an urgent need for new, more effective, less toxic, cheap and easy to adminis‐ ter therapeutic agents to treat African sleeping sickness and other closely related parasitic diseases, e.g. Chagas' disease and Leishmaniasis, whose current treatments suffer from simi‐ lar limitations.

*T. brucei* is able to survive and multiply in the harsh environment of a mammalian hosts' bloodstream. This is due to the parasite's dense cell-surface coat of the glycosylphosphatidy‐ linositol anchored variant surface glycoprotein (5 X 106 dimers/cell) [9-11], which protects the parasite in two ways. Firstly by acting as a diffusion barrier, such that complement is unable to reach and attack the plasma membrane of *T. brucei*. Secondly *T. brucei* is able to undergo antigenic variation, where by it is able to express a new variant surface glycopro‐ tein from a repertoire of more than 1000 different genes, before the hosts' innate immune system is able to catch up [12, 13]. This antigenic variation is why a vaccine against this par‐ asite is not a viable option as a therapy.

Phospholipids account for ~80% of total lipids in *T. brucei* with a significant proportion of these containing a choline-phosphate headgroup; phosphatidylcholine (PC) (~48%) and sphingomyelin (~15%) [14,15]. Sphingomyelin is made from PC via the sphingomyelin syn‐ thases transferring the choline-phosphate headgroup from PC to a ceramide lipid moiety [16]. These lipids contribute to the structural integrity of the membrane and in addition de‐ termine membrane fluidity and cell surface charge. Unsurprisingly, the biosynthesis and utilisation of these choline-containing molecules are implicated in a variety of cellular proc‐ esses, including signaling, intracellular cellular protein sorting and transport [reviewed in 16]. Phosphocholine has been reported to be a required mitogen for DNA synthesis induced by growth factors [17]. Recently we have shown that the essential *T. brucei* neutral sphingo‐ myelinase is actively involved in post Golgi sorting of the glycosylphosphatidylinositol anchored variant surface glycoprotein mentioned earlier [18].

Most eukaryotes have three alternative pathways by which PC can be synthesised [19 and reviewed in 20]. The first two pathways both involve three consecutive methylations of PE by S-adenosyl-L-methionemethyltransferases [20]. The PE can be derived from two alterna‐ tive pathways, either from the concerted actions of the CDP-DAG dependantphosphatidyl‐ serine synthase and phosphatidylserine decarboxylase, or via the CDP-ethanolamine branch of the Kennedy pathway. This involves phosphorylation of ethanolamine by an ethanola‐ mine kinase, its activation to CDP-ethanolamine by an ethanolamine-phosphate cytidyl‐ transferase and its transfer to diacylglycerol by an ethanolamine phosphotransferase. The presence of this branch of the Kennedy pathway was demonstrated in *T. brucei* [22], howev‐ er only recently have the constituent enzymes been characterized [23].

The trypanosomal genomes have revealed that *T. brucei* does not contain homologues for any methyltransferase(s) required to convert PE to PC [24] (neither does *T. cruzi*, but *Leish‐ mania* do). *Plasmodium falciparum* have an alternative single plant-like S-adenosyl-L-methio‐ nemethyltransferase [25-27], responsible for phosphoethanolamine conversion to phosphocholine, however there are no trypanosomatid homologues. This rather surprising absence of PE to PC methylation has been confirmed by *in vivo* labellings by ourselves and others [15, 22, 28].

The third alternative pathway for *de novo* synthesis of PC, and the only pathway by which *T. brucei* can *de novo* synthesise PC, utilises the CDP-choline branch of the Kennedy pathway [19, 29-33]. This involves the phosphorylation of choline by a choline kinase, its activation to CDP-choline by a choline-phosphate cytidyltransferase and its transfer to diacylglycerol by a choline phosphotransferase. Biochemical characterisation of the two choline/ethanolamine kinases involved in the initial steps of the Kennedy pathway show that unusually amongst eukaryotes only one of the kinases is able to phosphorylate choline [23].

Collectively this evidence of an absence of redundancy of *de novo* PC synthesis in *T. brucei*, compared with other organisms (including humans), suggests *T. brucei* has a vulnerability to inhibition of their only way to synthesise PC, i.e. the Kennedy pathway. Recently we have exploited this fact by genetically validating the only *T. brucei* choline kinase (*Tb*CK) as a drug target both in culture and in an animal model [34]. Chemical intervention of the *Tb*CK enzyme activity is likely to interfere with the parasite's biology in multiple ways and *Tb*CK is therefore of interest as a target for novel chemotherapeutics.

In this study we interrogate ~630 compounds of the Maybridge Rule of 3 Fragment Library for compounds that interact with, and inhibit *Tb*CK.The Maybridge Rule of 3 Fragment Li‐ brary is a small collection of quantifiable diverse [35, 36], pharmacophoric rich, chemical en‐ tities that comply with the following criteria; MW ≤ 300, cLogP ≤ 3, H-Bond Acceptors ≤ 3, H-Bond Donors ≤ 3, Rotatable bonds (Flexibility Index) ≤ 3, Polar Surface Area ≤ 60 Å2 and aqueous solubility ≥ 1 mM using LogS and high purity (≥ 95%). Comparisons between two different screening methods, a coupled enzyme activity assay and differential scanning fluo‐ rimetry,has allowed identification of compounds that interact and inhibit the *T. brucei* chol‐ ine kinase, several of which possess selective trypanocidal activity.

## **2. Experimental**

#### **2.1. Materials**

All materials unless stated were purchased either from Sigma/Aldrich or Invitrogen. An in house Maybridge Rule of 3 Fragment Library kept in master plates at 200 mM in DMSO (100%), was transferred into working plates with compounds occupying the central 80 wells of a 96-well plate, at 10 mM in 5% DMSO, allowing the two outside columns for positive and negative controls.

#### **2.2. Recombinant expression and purification of** *Tb***CK**

Large-scale recombinant expression and purification of *Tb*CK was conducted using the con‐ struct pET-15bTEV-*Tb*CK in BL21 Rosetta (DE3) cells as described previously [23], except the cells were grown in tryptone phosphate broth [37], harvested by centrifugation at 3500 g for 20 min at 4°C and affinity purified with either a HisTrap™ FF crude column (enzyme activity assay) or a HisTALON Cartridge (thermal shift analysis).

Briefly, pelleted cells were suspended in buffer A (50 mMTris/HCl, pH 8.0, 300 mMNaCl and 10 mM imidazole) and lysed in the presence of DNase I by sonication. The lysate was cleared by centrifugation at 35000 g for 30 min at 4°C and applied to a 1 ml HisTrap™ FF crude column column (GE Healthcare) pre-loaded with Ni2+. Unbound proteins were re‐ moved by washing the column with 15 column volumes of buffer A containing 32.5 mM imidazole and *Tb*CK was eluted with 250 mM imidazole in the same buffer. Using a PD10 column, *Tb*CK was buffer exchanged into 50 mMTris/HCl, pH 8.0, 300 mMNaCl, glycerol (15% w/v) and stored at -80°C.

Alternatively, pelleted cells were suspended in 50 mMTris/HCl, pH 8.0, 300 mMNaCl and 5 mM imidazole and lysed by sonication. The lysate was cleared by centrifugation at 35000 g for 30 min at 4°C and applied to a 1 ml HisTALON Cartridge (Clontech).Unbound proteins were removed by washing the column with 10 column volumes of loading buffer, *Tb*CK was eluted with 15 mM imidazole and a final clearing wash of 250 mM imidazole in the same buffer. Using a PD10 column, *Tb*CK was buffer exchanged into 50 mM HEPES pH 8.0, 300 mMNaCl and 15% glycerol prior to storage at -80°C.

Typical yields were > 10 mg per litre of bacterial culture,*Tb*CK was stable and freeze thaw‐ ing did not lead to any significant loss of activity.

#### **2.3.** *T. brucei* **choline kinase activity assay**

High throughput screening of the Maybridge Rule of 3 Fragment Library was carried out at a final test concentration of 0.5 mM in 96-well plates (final assay volume 200 μl) using a spectrophotometric assay that has been described previously [23]. The screened library working plates consisted of compounds arrayed in 96 well plates at 10 mM in 5% DMSO; columns 1 and 12 contained 5% DMSO only. For high throughput screening, 10 μl from each well of the working plates was added to 110 μl of buffer containing 50 mM MOPS (pH 7.8), 150 mMKCl and 6 mM MgCl2 . 3 μg of purified *Tb*CK was added to each well in 30 μl of the same buffer and the plates were mixed and incubated for 5 min at room temperature. A fur‐ ther 30 μl of buffer containing PEP (1 mM final), ATP (0.5 mM final), NADH (0.5 mM final) and pyruvate kinase and lactate dehydrogenase(PK/LDH) (5 units/ml final) was added, and the reaction was started by addition of 30 μl choline (0.5 mM final) to rows 1-11, 30 μl buffer alone was added to row 12 (negative control) and this was used as an intra-plate control (background rate) in conjunction with row 1 (maximal rate). Following mixing the change in absorbance at 341 nm was monitored for 10 min at room temperature. For testing inhibition of the coupling enzymes (PK/LDH), standard buffer conditions were used but the assay con‐ tained 1 mM PEP, 0.1 mM ADP and 0.5 mM NADH. The PK/LDH was titrated to give a change in absorbance of approximately 0.05 absorbance units/min in the absence of inhibi‐ tor.

#### **2.4. Differential scanning fluorimetry with** *Tb***CK**

Differential scanning fluorimetry was set up in 96 well PCR plates using a reaction volume of 100 μL. Samples contained 2.1 μM *Tb*CK, 6 mM MgCl2 , 50 mM HEPES pH 8.0, 80 mMNaCl, 5.25% glycerol (v/v) and 1.4 x Sypro Orange (Invitrogen), Maybridge Ro3 com‐ pounds were screened at 1 mM concentration with a final DMSO concentration of 0.5% (v/ v).Two controls with eight repetitions per plate were used for the thermal shift experiments: 0.5% DMSO; 0.5 mM ATP, 0.5% DMSO.

Differential fluorimetric scans were performed in a realtime PCR machine (Stratagene Mx3005P with software MxPro v 4.01) using a temperature scan from 25°C to 95°C at 0.5°C min-1.Data were then exported to Excel for analysis using "DSF analysis" modified from the template provided by Niesen et al. [38].Tm values were calculated by non-linear regression, fitting the Boltzmann equation to the denaturation curves using GraFit. *Tb*CK Tm, in the presence of 6 mM MgCl2 and 0.5% DMSO, 41.21 ± 0.03°C (n > 60), Tm for *Tb*CK and 0.5 mM ATP = 44.46 ± 0.05°C (n > 60).

## **3. Results and discussion**

Screening for inhibitors of the genetically validated drug target *Tb*CK is problematic due to the difficulty in following the reaction either continuously or directly. A direct choline kin‐ ase activity assay assessing the production of phosphocholine, utilising a modified method of Kim *et. al.* [39], using *Tb*CK and radiolabelled choline has been performed previously [23]. However this is not suitable for screening purposes, so choline kinase activity was measured by a spectrophotometric coupled assay (Figure 1). This coupled enzyme assay utilises regen‐ eration of ATP from the ADP by-product of the choline kinase by pyruvate kinase, and sub‐ sequent oxidation of NADH as the resulting pyruvate is converted to lactate, by lactate dehydrogenase. This assay using coupled enzymes is also problematic, as a compound could potentially inhibit the coupled enzymes giving rise to a false positive.

An alternative approach for screening is differential scanning fluorimetry (Figure 2), allow‐ ing identification of compounds that interact with the *Tb*CK protein, either to stabilise or de‐ stabilise it, therefore influencing the protein's Tm (melting point) [38-40].

Initially *Tb*CK was subjected to differential scanning fluorimetry to ascertain if this approach was possible. Known components required for enzyme activity were tested to see if thermal shifts were observed.In the presence of 6 mM MgCl2, a Tm of 41.2°C was obtained (Figure 1C, solid dark line). The addition of 0.5 mM ATP resulted in a > 3°C Tm shift for *Tb*CK(Figure 1C, dashed-line). These encouraging results showed *Tb*CK was amenable to differential scanning fluorimetry and allowed validation of this screening method.It is worth noting the surprising low Tm of *Tb*CK, considering that these parasites live within the bloodstream of a mammalian host, i.e. 37°C, or higher with a fever. However, the presence of physiological

**Figure 1.** Schematic of the TbCK reaction and coupled assay.

*T. brucei* choline kinase (*Tb*CK) catalyses the ATP dependent phosphorylation of choline, the ADP is converted back to ATP by pyruvate kinase (PK), which converts phosphoenolpyru‐ vate (PEP) to pyruvate in the process. The resulting pyruvate is reduced to lactate by the NADH dependent lactate dehydrogenase (LDH). The resulting conversion of NADH to NAD+ is monitored, by measuring the reduction in absorbance at 341 nM.

**Figure 2.** Thermal shift assay; typical differential fluorimetry scans of TbCK.

Differential fluorimetric scans were performed and analysed as described in Experimental. *Tb*CK + DMSO (control) solid dark line, *Tb*CK + 0.5 μM ATP (positive control) dashed line, *Tb*CK + 1 mM compound 242, solid light line, *Tb*CK + 1 mM compound 269, dotted line.Tm of *Tb*CK in the presence of 0.5%DMSO is 41.21 ± 0.03°C (control); Tm of *Tb*CK and 0.5 mM ATP is 44.46 ± 0.05°C (positive control). Insert: schematic representation of the thermal shift assay.A protein will unfold exposing hydrophobic domains as it is denatured due to the in‐ creasing temperature. Dyes such as sypro orange (star) are able to bind to these exposed hy‐ drophobic areas giving rise to fluorescence. A plot of this increased fluorescence versus temperature allows determination of Tm (melting point) of the protein. If a compound (squares) is able to interact with the protein it may alter the protein's Tm and thus a library of compounds can be screened to see if they stabilise (increase in Tm) or destabilise (decrease in Tm) the target protein.

An alternative approach for screening is differential scanning fluorimetry (Figure 2), allow‐ ing identification of compounds that interact with the *Tb*CK protein, either to stabilise or de‐ stabilise it, therefore influencing the protein's Tm (melting point) [38-40].

Initially *Tb*CK was subjected to differential scanning fluorimetry to ascertain if this ap‐ proach was possible. Known components required for enzyme activity were tested to see if thermal shifts were observed. In the presence of 6 mM MgCl2, a Tm of 41.2°C was obtained (Figure 1C, solid dark line). The addition of 0.5 mM ATP resulted in a > 3°C Tm shift for *Tb*CK(Figure 1C, dashed-line). These encouraging results showed *Tb*CK was amenable to differential scanning fluorimetry and allowed validation of this screening method.It is worth noting the surprising low Tm of *Tb*CK, considering that these parasites live within the blood‐ stream of a mammalian host, i.e. 37°C, or higher with a fever. However, the presence of physiological relevant levels of ATP does stabilize *Tb*CK by > 3 °C, which may prolong the half-life of the protein in the parasite.

The respective controls in both assay types allowed Z-factors to be determined for all of the plates screened (Figure 3). Both the coupled enzyme activity assay and the thermal shift analysis showed Z-factors to be above 0.5 for all plates, except for plate 5 for the thermal shift assay (but still above 0.45), this is indicative of good reliable assays, with meaningful results [41].

The MayBridge Rule of 3 Fragment Library was distributed over 9 plates (80 compounds per plate) providing space for adequate positive and negative controls, allowing Z-factors to be determined. This was done for each plate for both the choline kinase assay (+) and thermal shift analysis (x). A Z-factor above 0.4 is acceptable and validates the data on that plate as being reliable.

The ~630 compounds from the MayBridge Rule of 3 Fragment Library were assessed for their ability to inhibit the *Tb*CK coupled enzyme activity assay at a single concentration of 0.5 mM (Figure 4A). At this relative high concentration only 9 of the compounds (1.4%) showed > 70% inhibition. These primary hits were retested in triplicate at 0.5 mM (Table 1), 2 of the 9(compounds 320 and 635) were confirmed as being false positives, while the re‐ maining 7 were confirmed to show good inhibition (80-100%) against the *Tb*CK coupled en‐ zyme activity assay. These 7 compounds were then tested against just the coupled enzymes, some inhibition was observed for some of the compounds, but this was insufficient to ac‐ count for the strong inhibition against the *Tb*CK, thus these 7 compounds were believed to show true *Tb*CK inhibition.

**Figure 3.** Quality control (Z factors) for the TbCK thermal shift analysis and coupled enzyme activity assay.

It is worth noting the lack of false positives arising from significant inhibition of either of the coupling enzymes, pyruvate kinase and lactate dehydrogenase this is encouraging when screening other ATP utilizing enzymes.

Thermal shift analysis of *Tb*CK with the ~630 compounds from the MayBridge Rule of 3 Fragment Library showed that the vast majority of compounds had little or no affect on the Tm of *Tb*CK (Figure 4B). Relatively few compounds showed an increase in Tm (stabilisation), and only a handful of these showing an increase in Tm> 1°C, i.e. compound 269 (Figure 2, dotted line), this was rather surprising given that ATP stabilises *Tb*CK by > 3°C. Significant‐ ly more compounds showed a destabilisation affect, with 3 compounds having > 10°C de‐ crease in the Tm, i.e. compound 242 (Figure 2, solid light line). Most of the compounds observed in this screen that show significant destabilisation of *Tb*CK, do not cause similar destabilisation affects with other enzymes that we have screened in a similar manner, the only exceptions are compounds 68 (2-aminothiophene-3-carbonitrile) and 565 (4-(2-ami‐ no-1,3-thiazol-4-yl)phenol) (Table 1).

Several drug discovery style studies have shown that an increase in the thermal stability of a protein is proportional to the concentration and affinity of the ligand to the protein in keeping with the equilibrium associated with ligand-protein binding [38, 41-44]. On those occasions where this interaction destabilizes a protein, i.e. lowering Tm, a thermo‐ dynamic model has been proposed which explains the how the same ligand can stabilise and destabilise different proteins [42]. While the same protein may be stabilized and de‐ stabilized by very similar ligands, this was exquisitely demonstrated by the changes in thermal stability of Acyl-CoA thioesterase, upon incubation with either CoA (destabilise) or Acyl-CoA (stabilise) [45].

**Figure 4.** A) Percentage inhibition of TbCK enzyme activity assay for each of the compounds tested. B) Observed ther‐ mal shifts of TbCK for each of the compounds tested

a Arbitrary library number

b CAS numbers are unique identifiers assigned by the "Chemical Abstracts Service" to describe every chemical descri‐ bed in open access scientific literature.

c Tm shift in °C, observed for TbCK in the presence of compound (1mM), value is mean ± SD from the Boltzman curve fitting, see Experimental for details.

d Cytotoxicity studies, see Major and Smith 2011 for details, values are percentage of controls in the absence of com‐ pound, either mean ± SD (n=3) or mean ± SE (n=2), the latter being in bold.

**Table 1.** The compounds from the Maybridge Rule of 3 library that show >70% inhibition of TbCK.

All of the compounds in the two data sets (the coupled enzyme activity assay and the ther‐ mal shift analysis), were compared to assess any correlation between the two very different methods. In other words looking for compounds that showed a significant change in Tm and a significant inhibition in *Tb*CK enzyme activity (Figure 5A). The vast majority of com‐ pounds showed little or no inhibition and little or no shift in Tm. Compounds showing < 40% inhibition of the enzyme activity were removed for clarification (Figure 5B), this highlighted that the majority of compounds that show *Tb*CK enzyme inhibition do not significantly alter the Tm of *Tb*CK. The exceptions are compound 565 with a decrease in Tm~9°C, and com‐ pound 68 and 95 with a decrease in Tm~2°C respectively, all show complete inhibition at 0.5 mM (Table 1). Twenty-one compounds showed > 40% enzyme inhibition, 9 of these (43%) displayed > 1°C change in *Tb*CK Tm. This is a substantial enrichment compared to the 7% of compounds with Tm shifts > 1°C observed for the entire library.

**Figure 5.** Scatter graph representation showing the correlation between the observed thermal shifts and percentage inhibition of TbCK enzyme activity. A) All of the compounds from the MayBridge Rule of 3 Fragment Library tested. B) For those compounds with > 40% (dotted line) inhibition of TbCK enzyme activity.

Of the compounds identified that alter the Tm of *Tb*CK by > 1°C, ~20% of them inhibit *Tb*CK enzyme activity by > 40%. This suggests that for *Tb*CK thermal shift analysis has al‐ lowed significant enrichment, but not total capture of the potential inhibitors of *Tb*CK. However, if a direct assay for a potential drug target was very problematic, prior thermal shift analysis could significantly streamline the number of compounds to be screened, thereby increasing the potential to identify lead compounds. Thermal shift analysis has the disadvantage that good inhibitors could be missed if they do not significantly alter the Tm of the protein.

This raises an interesting question, is it a viable option to target compounds that specifi‐ cally destabilise an enzyme, causing a decrease in enzyme activity? One could argue this is exactly what pharmaceutical companies are focusing their research efforts upon, but with a slightly different approach. Some of their therapeutic targets rely on finding com‐ pounds that disrupt various interactions; hetero- or homo-oligomeric protein-protein, DNA-binding protein and RNA polymerases, many of these are associated with signaling events. Success stories include the identification of HDM2 antagonists associated with P53 activation [46], the identification of anti-cancer agent for the BCL-XL protein-protein com‐ plex and several others, reviewed by Wells and McClendon [47] and more recently by Coyne and colleagues [48].

The techniques utilized to study the formation / disruption of protein-protein complexes are driven by high throughput drug discovery, including fragment based approaches, these in‐ clude X-ray crystallography, NMR, dynamic light scattering, differential static light scatter‐ ing, differential scanning fluorimetry [42-50].

In summary, destabilisation by a ligand could affect the oligomeric state of a protein, or in the case of a monomer disrupt intra-molecular interactions, i.e. between stacking α-helixes or β-sheets, causing partial unfolding and thus destabilisation.In the case of *Tb*CK, which we know exists as a dimer, one of several potential mechanisms of destabilisation could be dis‐ ruption of the dimer interface, whereby a ligand is able to bind to freshly exposed hydro‐ phobic surfaces on the protein, and this interaction allows further destabilisation of the monomer structure.

As it was clear that compounds that inhibit *Tb*CK enzyme activity do not necessarily show a significant increase or decrease in Tm, it was decided to compare inhibition of *Tb*CK enzyme activity with previously determined trypanocidal activities for the com‐ pounds [51]. From this comparison (Figure 6) a group of compounds above a threshold of > 70% inhibition of *Tb*CK showed significant trypanocidal activity (circled), suggesting a direct correlation.

Compounds from the May Bridge Rule of 3 Fragment Library with greater than 70% inhibi‐ tion (dotted line) of the TbCK enzyme activity are circled and numbered. Numbers corre‐ spond to arbitrary compound library numbers; see Table 1 for chemical structures and extra data. *T. brucei* survival data was previously determined [51].

Coupled Enzyme Activity and Thermal Shift Screening of the Maybridge Rule of 3 Fragment Library... http://dx.doi.org/10.5772/52668 425

**Figure 6.** Correlation of the percentage inhibition of TbCK enzyme activity assay and T. brucei survival.

**Figure 7.** IC50 curves of the promising compounds (278 and 346) that show significant TbCK inhibition.

IC50 values were determined for these compounds (Table 1, Figure 7) ranging from 100s of μM to low μM. For example, compound 278 (1-methyl-(1H-indol-6-yl)methanol) has an IC50 value of 25.45 ± 1.16 μM however the selectivity index is not very good, while compound 346 (3-pyridin-3-ylaniline) has an IC50 value of 12.35 ± 0.64 μM with a high selectivity index.

One of the strengths of the Maybridge Rule of 3 Fragment Library is the chemical diversity, additionally a range of analogous structures can normally be found within it allowing initial structure activity relationships to be formulated. There are several close analogues of com‐ pound 278 (1-methyl-(1H-indol-6-yl)methanol), which highlight that the N-methyl indole moiety seems necessary to have any *Tb*CK inhibition and the methanol portion of the mole‐ cule can not be replaced by a carboxylic acid. Investigation of the CHEMBL database for similar compounds identified 1-Methyl-1H-pyrrolo[2,3-c]pyridine (CHEMBL594467) which was screened as one of a library of tricyclic and bicyclic analogues of indoloquinoline alka‐ loids against a variety of protozoan parasites. The analogue mentioned here showed weak trypanocidal activity (624 μM) against *Trypanosoma brucei rhodesiense*, but significantly better (37 μM) against *Plasmodium falciparum* [52].

Another analogue, 1-Methyl-1H-indole (CHEMBL19912) has been shown to interact with human intracellular adhesion molecules and highlights the importance of selectivity [53]. 1H-indol-5-yl-methanol (CHEMBL1650258) has previously been screened against *Leishmania* as a potential PTR1 inhibitor but was shown to be inactive at 500 μM [54]

For the relatively simple compound 346 (3-pyridin-3-ylaniline), there are several analo‐ gous structures in the library, including compound 262 (2-(1H-imidazo-1-yl)aniline) which shows ~55% *Tb*CK enzyme inhibition and is trypanocidal. Compound 347 (4-pyri‐ din-3-ylaniline) is a structural isomer of 346 but shows no *Tb*CK enzyme inhibition and is not trypanocidal. The only related structure in the CHEMBL database was 3-(pyri‐ din-3-yl)benzenaminium (CHEMBL1778131) which was shown to be a weakinhibitor of metallo-β−lactamase IMP-1 [55].

### **4. Conclusions**

In this study, screening of a comparatively small fragment library by two different screening methods has allowed identification of several compounds that interact with and inhibit *Tb*CK, a genetically validated drug target against African sleeping sickness. Some of the in‐ hibitory fragments were also selectively trypanocidal, considering these are relatively sim‐ ple molecules with no optimization, finding low μΜ inhibitors is very encouraging. Moreover some of the morphological phenotypes of these trypanocidal compounds include cell-cycle arrests similar to those observed for the *Tb*CK conditional knockout grown under permissive conditions.

This study highlights that if faced with a drug target that is problematic to screen, prior ther‐ mal shift analysis could significantly triage the number of compounds to be screened, there‐ by significantly increasing the potential to identify lead compounds. This approach obviously has the limitation that potential inhibitors could be missed if they do not signifi‐ cantly alter the Tm of the protein.

Future follow up work with *Tb*CK will include expanding the structure activity relationship of our most promising hits identified by this study. Their trypanocidal mode of action will be investigated by undertaking various *in vivo* biochemical phenotyping experiments to as‐ certain if they are inhibiting *Tb*CK, thus causing a lack of *de novo* PC synthesis, known to be essential for the parasite.

The ultimate goal is to identify new easy to make, affordable, easy to administer, drugs in the fight against African sleeping sickness and other closely related protozoan transmitted Third World diseases.

# **Acknowledgements**

This work was supported in part by a Wellcome Trust Senior Research Fellowship (067441) and Wellcome Trust project grants 086658 and 093228. We thank the late and sorely missed Dr Rupert Russell (St Andrews), supported by SUSLA, for access to the May Bridge Rule of 3 Fragment Library.

## **Author details**

Louise L. Major, Helen Denton and Terry K. Smith

Biomedical Sciences Research Centre, The North Haugh, The University, St. Andrews, Fife, Scotland, U.K.

### **References**

A Quantitative Model of Thermal Stabilization and Destabilization of Proteins by Li‐ gands Biophysical Journal 2008 (Vol. 95, Issue 7, pp. 3222-3231)


*Combinational antiviral therapies for HIV*

Given that viral pathogens are absolutely dependent on the host for propagation, even more so than bacterial pathogens, research in host-directed anti-virals has advanced at a faster pace than that for anti-bacterial agents. Human Immunodeficiency virus type 1 (HIV-1), a lentivirus of the retroviral family and the causative agent of AIDS, is the most-widely stud‐ ied viral pathogen to date. HIV-1 infection causes a dramatic decline in host CD4+ T cell numbers and a progressive failure of the immune response, which makes the host suscepti‐ ble to opportunistic infections and cancer. The highly glycosylated HIV-1 envelope, in com‐ bination with the extreme diversity of circulating viral strains, have presented daunting challenges for development of an effective vaccine. Furthermore, the virus establishes chron‐ ic infection that resists the highly active antiretroviral therapy (HAART). Conventional HAART for HIV-1 infection combines three main classes of anti-viral drugs:

HAART is usually patient-specific, and its formulation is determined by the viral load and drug resistance. A traditional HAART consists of two NRTIs and a NNRTI or a PI [25]. More advanced combination therapies include a fourth class of antiretroviral drugs, HIV entry in‐ hibitors. HIV-1 entry into human cells is dependent on several sequential steps that include binding of viral envelope protein gp120 to the CD4 receptor, and conformational change in gp120 that increases its affinity to the chemokine co-receptors (CCR5 or CXCR4) and expos‐ es gp41, an HIV envelope protein that executes the fusion of HIV and host cell membranes.

Currently, there are two approved inhibitors of HIV-1 entry:

The β-chemokine receptor CCR5 was found to act as a major co-receptor for the macro‐ phage-tropic HIV-1 R5 strains, predominant in the early asymptomatic stages of virus infec‐ tion, whereas the T-cell-tropic strains (using the CXCR4 co-receptor) become prevalent in the symptomatic stages concomitant with the decline of CD4+ T-cells [26]. CCR5 is an attrac‐ tive target for development of HIV-1 entry inhibitors, given the discovery that HIV-1 nonprogressors, individuals homozygous for a 32-bp deletion in the coding region of CCR5 gene (CCR5Δ32) were naturally resistant to infection with R5 HIV-1 [27]. Natural and syn‐ thetic CCR5 ligands such as RANTES, AOP-RANTES, Mip-1α, Mip-1β,and Met-RANTES were found to efficiently protect against R5 HIV-1 infection [28, 29]. Thus, the first published high throughput screen (HTS) for discovery of non- peptide inhibitors of HIV-1 entry was performed in a virus-free cell-based system using [125I]-labeled RANTES. A strong inhibitor of RANTES binding to CCR5 stably expressed on the surface of CHO cells was identified from the library of Takeda Chemical Industries. Further chemical modifications of the lead compound designated TAK-779 produced a potent (IC50 1.4 nM in CHO/CCR cells) and se‐ lective CCR5 antagonist capable of blocking R5 HIV-1 infection *in vitro* [30].

*ana* cotyledon seedlings, which signifies a loss of chlorophyll from plant tissues and is indicative of bacterial pathogenesis [23]. A screen of ~200 small molecules active in *Arabidop‐ sis* (LATCA, Library of Active Compounds in Arabidopsis) identified several sulfanilamide compounds, including sulfamethoxazole, sulfadiazine, and sulfapyridine, that prevented co‐ tyledon bleaching upon *P. syringae* infection. The most potent compound, sulfamethoxazole, also inhibited *P. syringae* growth in mature soil-grown plants. A similar assay was used to implicate the same compound, sulfamethoxazole, and the indole alkaloid gramine as inhibi‐ tors of *Fusarium graminearum* fungal infection in *Arabidopsis* and wheat, indicating that this strategy represents a relevant surrogate system for identification of compounds that can pre‐

Given that viral pathogens are absolutely dependent on the host for propagation, even more so than bacterial pathogens, research in host-directed anti-virals has advanced at a faster pace than that for anti-bacterial agents. Human Immunodeficiency virus type 1 (HIV-1), a lentivirus of the retroviral family and the causative agent of AIDS, is the most-widely stud‐ ied viral pathogen to date. HIV-1 infection causes a dramatic decline in host CD4+ T cell numbers and a progressive failure of the immune response, which makes the host suscepti‐ ble to opportunistic infections and cancer. The highly glycosylated HIV-1 envelope, in com‐ bination with the extreme diversity of circulating viral strains, have presented daunting challenges for development of an effective vaccine. Furthermore, the virus establishes chron‐ ic infection that resists the highly active antiretroviral therapy (HAART). Conventional

**2.** non-nucleoside RT inhibitors (NNRTIs), which target the non-catalytic domain of RT,

HAART is usually patient-specific, and its formulation is determined by the viral load and drug resistance. A traditional HAART consists of two NRTIs and a NNRTI or a PI [25]. More advanced combination therapies include a fourth class of antiretroviral drugs, HIV entry in‐ hibitors. HIV-1 entry into human cells is dependent on several sequential steps that include binding of viral envelope protein gp120 to the CD4 receptor, and conformational change in gp120 that increases its affinity to the chemokine co-receptors (CCR5 or CXCR4) and expos‐ es gp41, an HIV envelope protein that executes the fusion of HIV and host cell membranes.

**2.** maraviroc, a small molecule entry inhibitor that prevents interaction between gp120

The β-chemokine receptor CCR5 was found to act as a major co-receptor for the macro‐ phage-tropic HIV-1 R5 strains, predominant in the early asymptomatic stages of virus infec‐

HAART for HIV-1 infection combines three main classes of anti-viral drugs:

vent agriculturally-important infectious disease [24].

**1.** nucleoside reverse transcriptase inhibitors (NRTIs),

Currently, there are two approved inhibitors of HIV-1 entry:

**1.** enfuvirtide, a peptide fusion inhibitor that binds to gp41 and

*Combinational antiviral therapies for HIV*

and

164 Drug Discovery

and CCR5.

**3.** protease inhibitors (PIs).

The number of CCR5 inhibitors has significantly grown since the discovery of TAK-779, but very few compounds have entered clinical trials, and only maraviroc has been approved for clinical use [31]. A radiolabeled-chemokine binding assay similar to one applied for the identification of TAK-779 was used in a HTS of a small molecule library at Pfizer for the dis‐ covery of UK-107,543, which had become a scaffold for intensive medicinal chemistry, pro‐ ducing ~1,000 analog compounds, from which maraviroc (UK-427,857) was selected for its excellent preclinical pharmacokinetics (90% inhibitory concentration of 2 nM in pool of PBMCs from various donors) [32]. Despite its proven efficacy against HIV-1 R5 infection, maraviroc is vulnerable to gp120 escape mutations [33]. Site-directed mutagenesis and mo‐ lecular modeling studies have identified a common binding pocket on CCR5 that is shared by various small-molecule CCR5 inhibitors [34, 35, 36]. Emerging details on gp120 and CCR5 points of interaction and binding thermodynamics provide valuable information that can be applied in developing tools for rational design of novel HIV-1 entry inhibitors [37, 38]. Efficient block of HIV entry into host cells is essential to curtail virus dissemination and is a key step towards eradication of HIV infection. The current HAART regiment can reduce HIV replication to very low levels (below 50 copies/ml plasma) and can lead to recovery of CD4+ T-cell counts but not cure the infection. Patients that have been successfully treated with HAART for years have experienced a rapid virus rebound upon termination of the therapeutic regiment [39, 40]. Such clinical cases present evidence that HIV establishes a chronic infection that resists current HAART designed to target actively replicating virus. A deliberate and controllable induction of HIV-1 replication from its latent reservoirs in com‐ bination with HAART is a novel and actively pursued approach that aims to eliminate both active and latent viral pools [41].

Researchers often seek new anti-infective agents amongst small molecules that have previ‐ ously been approved for the treatment of cancer and neurological diseases, since they have well-established pharmacokinetics and in most cases, known molecular mechanisms of ac‐ tion. One example of this is the histone deacetylase (HDAC) inhibitor, valproic acid (VA), which had previously been approved for treatment of neurological and psychiatric disor‐ ders. HIV-1 has been shown to enter dormancy using epigenetic silencing via deaceylation of histones in the vicinity of the integrated viral genome [42]. Thus, VA was tested as a po‐ tential agent to disrupt HIV-1 latent infection. However, years of VA treatment in combina‐ tion with HAART showed no clearance of the latent HIV reservoir [43]. A more potent HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA), approved for treatment of cuta‐ neous T-cell lymphoma, was subsequently tested as a potential agent that could 'flush out' HIV-1 from latently infected cells, based on its superior effect to VA in cell culture models [44, 45]. A substantial effort has also been invested in the design and synthesis of bryostatin chemical analogs, small molecules that activate protein kinase C (PKC) with single nanomo‐ lar concentration [46]. PKC activation leads to phosphorylation of nuclear factor κB (NFκB), a key transcription factor regulator of HIV-1 gene expression [47]. However, modulation of NFκB activity requires great caution, since abnormal NFκB signaling has been related to the pathophysiology of inflammatory diseases and neurodegenerative disorders [48].

*C. elegans*, a hermaphroditic nematode normally found in soil, is a versatile, more ethicallyacceptable whole animal system for high-throughput analysis of host response to pathogen infection. *C. elegans* contains a fully sequenced genome that facilitates both genetic and ge‐ nomic analysis, offering an ideal compromise between organismal complexity and experi‐ mental tractability. *C. elegans* offers other experimental advantages, including a rapid 2-3 week life span, simple growth conditions, target-selected gene inactivation, and a relatively low cost of maintenance compared to other whole animal systems. A wealth of experimental data has demonstrated that many developmental, neurological, and biochemical processes have been highly conserved between *C. elegans* and mammals. For example, cellular func‐ tions as diverse as innate immunity, the first line of defense against pathogen infection, and RNA interference to downregulate gene expression via double-stranded RNA, are found in both *C. elegans* and higher eukaryotes, suggesting the existence of a common ancestor of these diverse species. Thus, anti-infective compounds identified using a *C. elegans* infection

Small Molecule Screens to Identify Inhibitors of Infectious Disease

http://dx.doi.org/10.5772/52502

167

*C. elegans* as a model host system has been well-studied for numerous bacterial pathogens, in‐ cluding the Gram-positive *S. aureus*, *S. pneumoniae*, and *B. thuringiensis*, and the Gram-nega‐ tive *B. pseudomallei*, *P. aeruginosa*, and *S. marcescens*. In general, different types of bacteria are fed to *C. elegans* in place of their normal *E. coli* food source to provoke detectable symptoms of

A small manual screen of 6000 synthetic compounds and 1136 natural extracts were ana‐ lyzed in an immunocompromised mutant of *C. elegans* infected with *Enterococcus faecalis* to identify compounds that promoted host survival. [51]. A total of 16 compounds and 9 ex‐ tracts were identified that either modulated bacterial growth *in vitro*, impaired pathogen vir‐ ulence, or boosted host innate immunity. Furthermore, 15 out the 16 compounds did not kill

The development of automated sorting and handling of *C. elegans* rapidly enabled highthroughput screening of small chemical libraries to identify compounds that enhanced sur‐ vival of *C. elegans* in response to bacterial infection. This methodology was enabled by the Complex Object Parametric Analyzer and Sorter (COPAS) BioSort worm sorter (Union Bio‐ metrica) to dispense a defined number of living worms into multi-well plates, which were then imaged using automated microscopy to quantify worm survival. A library of 37,200 compounds and natural product extracts was screened using the same *C. elegans*-*E. faecalis* infection system described above [52]. Twenty-eight compounds and extracts were identi‐ fied that enhanced survival of infected *C. elegans*. Six structural classes of identified com‐ pounds did not affect the growth of *E. faecalis* itself, suggesting that the small molecules inhibited a specific aspect of the host-pathogen interaction. Interestingly, two structural classes are similar to compounds previously identified in a high-throughput screen to iden‐ tify inhibitors of *P. aeruginosa* biofilm development, indicating the presence of common mo‐

A *P. aeruginosa* infection model of *C. elegans* has also been developed to screen for novel antiinfective compounds. The high-throughput assay was based on *P. aeruginosa*-induced slow

illness, such as locomotion dysregulation, intestinal cell lysis, and shortened life span.

*C. elegans* or mammalian erythrocytes, indicating that the compounds are not toxic.

lecular targets across multiple bacterial species for drug discovery [53].

model may also be translatable in humans.

*Small molecule inhibitors of bacterial infection*

A HTS of a small molecule library recently identified novel HIV latency activators [49]. The screen was performed using a lymphoma CD4+ T-cell line (SupT1) harboring latent recombi‐ nant HIV-1 and two reporters that reflect early and late virus gene expression incorporated in the HIV-1 genome [50]. A luminescent assay based on secreted alkaline phosphatase (SEAP) activity, incorporated in the late virus gene transcripts, was applied to screen a chemical library of ~200,000 compounds. Validation of 27 hits with diverse chemical struc‐ tures demonstrated induction of latent virus from various cell models. Compounds with a selective index (CC50/EC50) above 25 were chosen for downstream medicinal chemistry mod‐ ifications. Moreover, the lead compounds were shown to reactivate latent HIV from primary resting CD4+ T-cells with no induction of cell proliferation. Small molecule activators of la‐ tent HIV that act in concert using different mechanisms have a better chance of purging the virus out of infected cells [49]. Such pre-clinical data strongly suggests that successful treat‐ ment of HIV infection can be achieved only through combinational therapy consisting of di‐ verse class of antiviral drugs.


*3.5. Self-Heating*

During cyclic loading, self-heating of the specimens may occur, depending on the stress amplitudes and strain rates imposed during each cycle, which may introduce a high amount of plastic deformation. The temperature measurements showed that the self-heating was homogeneous in the middle of the gage section of the run-out samples. For the broken samples; however, a localized and sharp increase of temperature was observed before the final breakdown. Typical chronograms from these measurements are plotted in Figure 7a, and a temperature variation of almost 50 ◦C can be observed before rupture (see red curve). The maximum homogeneous temperature values as a function of the applied stress were measured for samples tested in TC (see Figure 7b). The maximum surface temperature evolution with the applied stress amplitude followed an almost linear trend for both polished and 60 min of SMAT at RT conditions. In the fatigue limit stress range, the polished samples (~205 MPa) exhibited a maximum measured temperature of approximately 50 ◦C, whereas, for 60 min RT SMAT (~245 MPa), this value was higher than 100 ◦C. Nevertheless, the measured temperature values for the highest stress amplitudes were comparable: 320 ◦C for the initial state loaded at 260 MPa and 280 ◦C for the

treated samples loaded at 275 MPa. After SMAT, in order to obtain the same level of self-heating, 15% higher stress amplitudes were required.

**Figure 6.** Secondary electron SEM pictures of surface initiation sites of (**a**) sample tested in rotating–bending after 20 min of cryogenic SMAT (corresponding to Figure 5c), and (**b**) sample tested in tension–compression after 60 min of SMAT at room temperature (corresponding to Figure 5e).

**Figure 7.** (**a**) Typical self-heating chronograms measured during TC loading, for breaking samples (in red) and run-out samples (in blue). (**b**) Maximum stabilized temperature as a function of σa.

### **4. Discussion**

Consistently with the results from the literature [13,15,23], the use of SMAT was found to be an efficient way to increase the fatigue resistance of the 304L stainless steel. Enhancements of 28% and 17% were observed in rotating–bending and tension–compression, respectively. Nevertheless, self-heating of the specimens during testing was not anticipated. The temperature evolutions with applied load presented in Figure 7b show almost linear trends with similar positive slopes for both polished and SMAT samples. In the case of the polished samples, a good accordance with the fatigue results obtained on 316 LN stainless steel by Tian et al. [33] can be noted, especially around 260 MPa where the number of cycles to failure and self-heating values were very similar. Also, for the low stress amplitudes related to the endurance limit, such as 200 MPa and 207 MPa, the maximum surface temperature measured during the fatigue tests was less than 70 ◦C, representing a self-heating that did not exceed 50 ◦C. After SMAT, the same level of self-heating required a 15% higher applied stress amplitude, confirming the difference of cyclic plastic strain accumulation between the SMAT and the untreated samples for the same applied stress amplitude. Indeed, the surface strain-hardening generated by SMAT pushed the onset of the plasticity at the surface to a higher stress range and delayed the generation of significant self-heating. Regarding the thermal exposure during the tests, the work of Kakuichi et al. [23] showed that the exposure to 300 ◦C during rotating–bending fatigue at high and low stress amplitudes does not relax the surface residual stresses of a 304L sample ultrasonic shot-peened at room temperature. This would mean that the observed self-heating mainly behaves as a mechanical property reducer and that the key factor governing the residual stress relaxation is the introduction of supplementary plastic strains.

Meanwhile, the presence and magnitude of residual stresses also play a role in RB and TC fatigue behavior and, as shown in Figure 4, different residual stresses values were measured before and after fatigue tests at the surface of specimens. Before fatigue, the different residual stress states are directly linked to the different studied conditions, whereas, after fatigue, relaxations depend on several testing parameters such as the type of loading or the applied stress amplitude. For example, it was shown in Figure 4 that the residual stress relaxation in austenite for the ground specimen after rotating–bending fatigue was only approximately −10% compared to −75% for the tension-compression testing condition. For the same level of stress amplitudes, due to the stress gradients during RB tests, plastic strains can mainly be introduced at the surface region of the specimen, whereas the whole volume of the TC sample can accommodate plastic strains during TC tests, resulting in more residual stress relaxation. The initial residual stresses can also favor the onset of plasticity, especially when significant compressive residual stresses are present in a part that is mechanically loaded at R = −1. The stress applied during the compressive stage of a fatigue test is added to the residual stress, reaching the material elastic limit at or below the surface, resulting in the relaxation of the residual stresses. Both elements can justify the specific behavior in the tension–compression testing condition. At this stage, the sequence of these different mechanisms is not known, as only surface residual stress relaxations were evaluated before and after the tests. Further investigations of the residual stress gradient evolution during fatigue loading are needed to better explain the behavior of the processed part during fatigue tests. Such an investigation will contribute to understanding when, how, and in which way the residual stress gradients relax during fatigue testing, finally providing a better idea of the role of the residual stresses in the fatigue behavior of 304L stainless steel treated by SMAT.

Concerning the comparison of untreated RB with untreated fully reversed (R = −1) TC fatigue tests, the gradient in the applied load within the specimens in RB is commonly used to explain a lower fatigue limit (approximately −10%) in TC [34]; however, in this study, a decrease of −18% of the fatigue limit was observed. The volume effect of the tested samples can be considered to explain this difference. Indeed, the samples tested in RB had a gauge of 6 mm in diameter and 25 mm in length, whereas the TC ones had a diameter of 9 mm with a length of 12.5 mm. Pogorestskii et al. [35] showed that, for 40Kh steel loaded in four-point rotating–bending, larger gauge length and diameter had a detrimental effect on the fatigue limit, and they defined two different coefficients: −0.18 MPa/mm for the length

and −4.4 MPa/mm for the radius. If similar behavior can be considered for the 304L stainless steel, an increase of 3 mm in diameter and a decrease of 12.5 mm in length would result in an overall decrease of approximately 5 MPa for the RB fatigue limit. The difference in fatigue limit between RB and TC would consequently be reduced to −15%, which is very similar to the −14.9% or the −11.9% proposed by Palin-Luc et al. [36] on 30NCD16 and XC18 steels, respectively.

For the SMAT conditions, a decrease of approximately −25% of the fatigue limit in TC compared to the RB one was obtained in Table 2 (from 320 to 240 MPa). This difference was significantly larger than for the initial state and could be due to the difference in treatment duration and resulting microstructure and mechanical property gradients. Similarly to the work of Sun et al. [37], one can consider that, after SMAT, the obtained functionally grade material behaves like a composite material composed of a SMAT-affected layer and a bulk core. Considering that the mechanical properties are proportional to the hardness, and by using a simple mixing law, the fatigue limit enhancement by SMAT can be determined as follows:

$$Enhuman\ \left[\%\right] = \left(\frac{f\_{\text{SMAT}} \times HV\_{\text{SMAT}} + f\_{\text{bulk}} \times HV\_{\text{bulk}}}{HV\_{\text{bulk}}} - 1\right) \times 100,\tag{2}$$

where *fSMAT* is the volume fraction of the SMAT-affected layer and *HVSMAT* is its mean hardness, where *fbulk* = (1 − *fSMAT*) and *HVbulk* = 210 (as shown in Figure 2a). The data used for the calculation and the obtained estimations are summarized in Table 3. The differences in diameter and hardened depth led to a higher fraction of SMAT-affected layer in the case of RB\_20 min RT (16%) than the two other conditions (11.3% for RB\_20 min CT and 11.8% for TC\_60 min RT). Nevertheless, the difference of mean hardness in the SMAT layer resulted in similar fatigue limit estimations (~267 MPa) for both RB conditions and a lower value for TC (~220 MPa). The estimated fatigue limits were compared to the experimental ones, and the respective errors were calculated.

**Table 3.** Different SMAT conditions with their respective affected volume fraction, mean hardness, and corresponding measured and estimated fatigue limits.

It is worth remembering that the effect of residual stresses and their variation during fatigue tests were not taken into account here. This approximation underestimates the fatigue limit enhancement by SMAT for all tested conditions. As shown in Table 3, the error of approximation was two-fold higher in RB than in TC. Even by considering the differences in sample geometry and hardening state resulting from the treatment conditions, a difference remains which can only be explained by the loading condition difference. An explanation can be that the most solicited area in RB is the SMAT-affected layer that has enhanced mechanical properties and is under significant compressive residual stress state. These facts suggest that the effect of SMAT would be more significant in RB.

In the case of rotating–bending, the use of cryogenic SMAT provided the same effect in terms of fatigue resistance as the SMAT at room temperature. Thus, despite potential beneficial modifications such as a lower roughness, a higher martensite fraction, and a slightly higher compressive residual stresses, the use of SMAT at cryogenic temperature did not bring the desired additional improvement. It is likely that the beneficial modifications induced by CT SMAT were counter-balanced by other factors linked to the sub-surface or to the surface modifications such as the lower hardness below 200 μm (see Figure 2a) compared to the room temperature treatment. Indeed, as shown in Table 3 for CT SMAT, the high surface hardness (~520 HV) together with the limited affected depth (~350 μm) led to

a very similar estimation of the fatigue limit compared to the RT SMAT that was characterized by a lower surface hardness (~450 HV) and a significantly higher affected depth (~500 μm).