Biophysical and computational fragment-based approaches to targeting protein-protein interactions: applications in structure-guided drug discovery.

Drug discovery has classically targeted the active sites of enzymes or ligand-binding sites of receptors and ion channels. In an attempt to improve selectivity of drug candidates, modulation of protein-protein interfaces (PPIs) of multiprotein complexes that mediate conformation or colocation of components of cell-regulatory pathways has become a focus of interest. However, PPIs in multiprotein systems continue to pose significant challenges, as they are generally large, flat and poor in distinguishing features, making the design of small molecule antagonists a difficult task. Nevertheless, encouragement has come from the recognition that a few amino acids - so-called hotspots - may contribute the majority of interaction-free energy. The challenges posed by protein-protein interactions have led to a wellspring of creative approaches, including proteomimetics, stapled α-helical peptides and a plethora of antibody inspired molecular designs. Here, we review a more generic approach: fragment-based drug discovery. Fragments allow novel areas of chemical space to be explored more efficiently, but the initial hits have low affinity. This means that they will not normally disrupt PPIs, unless they are tethered, an approach that has been pioneered by Wells and co-workers. An alternative fragment-based approach is to stabilise the uncomplexed components of the multiprotein system in solution and employ conventional fragment-based screening. Here, we describe the current knowledge of the structures and properties of protein-protein interactions and the small molecules that can modulate them. We then describe the use of sensitive biophysical methods - nuclear magnetic resonance, X-ray crystallography, surface plasmon resonance, differential scanning fluorimetry or isothermal calorimetry - to screen and validate fragment binding. Fragment hits can subsequently be evolved into larger molecules with higher affinity and potency. These may provide new leads for drug candidates that target protein-protein interactions and have therapeutic value.

Validity and Responsiveness of a Modified Balance Error Scoring System Assessment Using a Mobile Device Application in Patients Recovering from Ankle Sprain.

Background: Static balance is often impaired in patients after ankle sprains. The ability to identify static balance impairments is dependent on an effective balance assessment tool. The Sway Balance Mobile Application (SWAY App) (Sway Medical, Tulsa, OK) uses a smart phone or tablet to assess postural sway during a modified Balance Error Scoring System (mBESS) assessment and shows promise as an accessible method to quantify changes in static balance after injury. Purpose: The primary purposes of this study were to determine the ability to differentiate between those with ankle sprain versus controls (construct validity) and ability to detect change over time (responsiveness) of a mBESS assessment using a mobile device application to evaluate static balance after an acute ankle sprain. Study Design: Case-control study. Methods: Twenty-two military academy Cadets with an acute ankle sprain and 20 healthy Cadets were enrolled in the study. All participants completed an assessment measuring self-reported function, ankle dorsiflexion range of motion (via the weightbearing lunge), dynamic balance, and static balance. Static balance measured with the mBESS using the SWAY App was validated against laboratory-based measures. Cadets with ankle sprains completed their assessment twice: once within two weeks of injury (baseline) and again after four weeks of rehabilitation that included balance training. Independent and paired t-tests were utilized to analyze differences over time and between groups. Effect sizes were calculated and relationships explored using Pearson's correlation coefficients. Results: The mBESS scores measured by the SWAY App were lower in participants with acute ankle sprains than healthy Cadets (t = 3.15, p = 0.004). Injured participants improved their mBESS score measured by SWAY at four weeks following their initial assessments (t = 3.31, p = 0.004; Baseline: 74.2 +/- 16.1, 4-weeks: 82.7 +/- 9.5). The mBESS measured by the SWAY App demonstrated moderate to good correlation with a laboratory measure of static balance (r = -0.59, p \< 0.001). Conclusion: The mBESS assessed with a mobile device application is a valid and responsive clinical tool for evaluating static balance. The tool demonstrated construct (known groups) validity detecting balance differences between a healthy and injured group, concurrent validity demonstrating moderate to good correlation with established laboratory measures, and responsiveness to changes in static balance in military Cadets during recovery from an acute ankle sprain. Level of Evidence: Level 3.

Psychological Factors Are Related to Neuromuscular Asymmetries After Anterior Cruciate Ligament Reconstruction.

BACKGROUND: After an anterior cruciate ligament reconstruction (ACLR), only 47% of military members return to full duty, possibly due to persistent neuromuscular asymmetries. Psychological factors may also contribute to reduced return to duty in military members. HYPOTHESIS: Psychological factors and time since surgery would be associated negatively with neuromuscular asymmetries, asymmetries would be greater in cadets postsurgery when compared with healthy controls, and asymmetries would be greater at earlier timepoints after ACLR. STUDY DESIGN: Case control. LEVEL OF EVIDENCE: Level 4. METHODS: This study examined the relationship between psychological factors and time since surgery with neuromuscular asymmetry, compared neuromuscular asymmetries between cadets with and without a history of ACLR, and explored differences in neuromuscular asymmetries at different timepoints in cadets with a history of ACLR. A total of 37 cadets post-ACLR (18.3 ± 9 months) and 28 controls participated. Psychological factors were assessed using the Tampa Scale of Kinesiophobia and Anterior Cruciate Ligament-Return to Sport after Injury scale (ACL-RSI). Participants performed a drop-jump landing, joint positioning sense (JPS), and isometric quadriceps strength testing. Peak vertical ground-reaction forces (vGRF), absolute angle of replication, peak quadriceps torque, rate of torque development (RTD), and RTD time torque interval 200 ms (TTI200) were analyzed. RESULTS: The ACL-RSI score was significantly related to limb symmetry index (LSI) peak quadriceps torque (r = 0.617, P < 0.01), LSI RTD (r = 0.367, P = 0.05), and LSI TTI200 (r = 0.0489, P < 0.01), but not time since surgery, JPS, or LSI peak vGRF. Cadets with a history of ACLR had significantly lesser ACL-RSI scores and greater asymmetries compared with controls. CONCLUSION: Reduced psychological readiness was associated with increased neuromuscular asymmetries after ACLR. CLINICAL RELEVANCE: Clinicians should assess psychological readiness during rehabilitation after ACLR.

The Convergent Validity of the SWAY Balance Application to Assess Postural Stability in Military Cadets Recovering from Concussion.

Background: Concussions are often accompanied by balance disturbances. Clinically accurate evaluation systems are often expensive, large, and inaccessible to most clinicians. The Sway Balance Mobile Application (SWAY) is an accessible method to quantify balance changes. Purpose: To determine the known groups and convergent validity of the SWAY to assess balance after a concussion. Study Design: Case-Control Study. Methods: Twenty participants with acute concussion and twenty controls were recruited. At initial, one-week, and final return to activity (RTA) evaluations, all participants completed the Sports Concussion Assessment Tool (SCAT-5), and balance control measured by SWAY mBESS and NeuroCom Balance Master Sensory Organization Test (SOT). Mixed model ANOVAs were used to detect differences in SWAY mBESS and NeuroCom SOT scores with time (initial, one-week, final RTA) as the within-subjects factor and group (concussed, healthy) as the between-subjects factor. Spearman's Rho correlations explored the associations between NeuroCom SOT scores, SWAY scores, SCAT-5 symptom scores, and time in days to final RTA. Results: The sampled population was predominantly male and age (20 ± 1), and BMI differences were insignificant between groups. The SWAY did not detect differences between healthy and concussed participants and did not detect change over time [F(2,40) = .114, p = 0.89; F(2,40)= .276, p =0.60]. When assessing the relationship between the SWAY and the SOT, no correlation was found at any time point (r = -0.317 to -0.062, p > 0.05). Time to RTA demonstrated a moderate correlation with both SCAT-5 symptom severity score (r = .693, p < 0.01) and SCAT-5 total symptom score (r = .611, p < 0.01) at the one-week follow-up. Conclusion: The SWAY mBESS does not appear to be a valid balance assessment for the concussed patient. The SWAY mBESS in patients with concussion failed to demonstrate convergent validity and did not demonstrate an ability to validate known groups. When assessing the time to final RTA, the one-week post-initial assessment SCAT-5 symptom severity and total scores may help determine the length of recovery in this population. Level of Evidence: Level 3.

Inter- and Intra-Rater Reliabilities of the Army Combat Fitness Test Three-Repetition Maximum Deadlift Event Among Raters of Varying Professional Experience.

INTRODUCTION: The U.S. Army is updating the physical fitness assessment for soldiers to the six-event Army Combat Fitness Test (ACFT). A paucity of data regarding the ACFT maximum deadlift (MDL) event, especially in military populations, has increased concern over the objectivity of the test. The reliability of scoring the MDL has not been established. It is unknown if grader professional experience impacts the reliability of scoring, and if so, what level of experience is required for reliable assessment. Performance and assessment of the MDL could impact military occupational selection, promotion, and retention within the Army. The purposes of this study were to determine the inter- and intra-rater reliabilities of raters with varying degrees of professional experience on scoring the MDL and to determine the relationships between load lifted, overall lift success, sex, and body mass index (BMI). METHODS: The design is a reliability study. Approval was granted by the Naval Medical Center-Portsmouth Institutional Review Board. Fifty-five healthy soldiers and cadets from the U.S. Military Academy were recruited. Participants completed one data collection session, performing one MDL attempt. The attempt was video recorded using three devices: two handheld tablets placed perpendicular to the sagittal and frontal planes recording at 240 Hz and one digital camera positioned at a 45° angle recording at 30 Hz. A reference standard was established through slow-motion analysis of the sagittal and frontal plane recordings. Six raters with varying degrees of professional experience viewed the 45° camera recordings at real-time speed independently, in a random order, on two separate occasions. Lift success was dichotomously assessed as successful or unsuccessful according to the MDL standards. Cohen's kappa was computed to determine inter- and intra-rater reliabilities among raters. Bivariate correlation was used to assess associations among load lifted, BMI, and sex. A chi-squared test of independence assessed the relationship between sex and overall lift success. RESULTS: Inter-rater reliability between the six raters ranged from 0.29 to 0.69. Inter-rater reliability between the raters to the reference standard ranged from 0.28 to 0.61. Intra-rater reliability ranged from 0.51 to 0.84. Inter-rater reliability of raters who had attended a Training and Doctrine Command-approved ACFT certification course ranged from 0.51 to 0.66, while those who had not ranged from 0.34 to 0.46. BMI and sex were associated with load lifted (r = 0.405, P = .002; r = -0.727, P < .001, respectively). Overall lift success was not associated with load lifted (r = -0.047, P = .731). Overall lift success was not related to sex (χ2 = 0.271, P = .602). CONCLUSION: Inter-rater reliability of the six raters ranged from poor to substantial, while intra-rater reliability ranged from moderate to excellent. Compared to a reference standard, inter-rater reliability ranged from poor to substantial. The wide range in consistency demonstrated in this study, both between and within raters, brings into question the current subjective methods used to grade the MDL. More research is needed to understand the most feasible, valid, and reliable way to assess performance standards like the MDL that may affect a soldier's career progression.

The Effect of Blood Flow Restriction Therapy on Shoulder Function Following Shoulder Stabilization Surgery: A Case Series.

Background: Traumatic shoulder instability is a common injury in athletes and military personnel. Surgical stabilization reduces recurrence, but athletes often return to sport before recovering upper extremity rotational strength and sport-specific abilities. Blood flow restriction (BFR) may stimulate muscle growth without the need for heavy resistance training post-surgically. Hypothesis/Purpose: To observe changes in shoulder strength, self-reported function, upper extremity performance, and range of motion (ROM) in military cadets recovering from shoulder stabilization surgery who completed a standard rehabilitation program with six weeks of BFR training. Study Design: Prospective case series. Methods: Military cadets who underwent shoulder stabilization surgery completed six weeks of upper extremity BFR training, beginning post-op week six. Primary outcomes were shoulder isometric strength and patient-reported function assessed at 6-weeks, 12-weeks, and 6-months postoperatively. Secondary outcomes included shoulder ROM assessed at each timepoint and the Closed Kinetic Chain Upper Extremity Stability Test (CKCUEST), the Upper Extremity Y-Balance Test (UQYBT), and the Unilateral Seated Shotput Test (USPT) assessed at the six-month follow-up. Results: Twenty cadets performed an average 10.9 BFR training sessions over six weeks. Statistically significant and clinically meaningful increases in surgical extremity external rotation strength (p < 0.001; mean difference, .049; 95% CI: .021, .077), abduction strength (p < 0.001; mean difference, .079; 95% CI: .050, .108), and internal rotation strength (p < 0.001; mean difference, .060; CI: .028, .093) occurred from six to 12 weeks postoperatively. Statistically significant and clinically meaningful improvements were reported on the Single Assessment Numeric Evaluation (p < 0.001; mean difference, 17.7; CI: 9.4, 25.9) and Shoulder Pain and Disability Index (p < 0.001; mean difference, -31.1; CI: -44.2, -18.0) from six to 12 weeks postoperatively. Additionally, over 70 percent of participants met reference values on two to three performance tests at 6-months. Conclusion: While the degree of improvement attributable to the addition of BFR is unknown, the clinically meaningful improvements in shoulder strength, self-reported function, and upper extremity performance warrant further exploration of BFR during upper extremity rehabilitation. Level of Evidence: 4, Case Series.

Comprehensive Rehabilitation of the Athlete: A Specific and Purposeful Approach.

There is a need to improve the quality of rehabilitation in sports medicine to return athletes to optimal function safely and quickly, reducing the risk of reinjury. This paper describes a planning paradigm to guide clinical reasoning during individual treatment sessions and a model for planning the rehabilitation program from acute injury to return to play. The design of a rehabilitation program should be a collaborative, team effort, and accounting for the specific needs of the athlete. As the athlete progresses from acute injury management all the way back to full competition, the rehabilitation professional emphasizes the components of pain management, motion, motor control, and force production in varying degrees based upon phases of tissue healing and the athlete's response. Utilizing high-value, evidence-based treatments maximize both the effectiveness and efficiency of rehabilitation to restore and improve upon preinjury levels of physical performance.

A case-control study of gait balance control in veterans with chronic symptoms following mTBI.

BACKGROUND: Numerous investigations suggest mild traumatic brain injury (mTBI) may result in persistent gait balance control deficits. Furthermore, military Veterans with symptoms of chronic mTBI often present with physical symptoms that may be associated with impaired gait balance control which may lead to prolonged recovery, difficulty performing activities of daily living, and increased disability. It is therefore important to objectively quantify gait balance deficits in Veterans with chronic mTBI. RESEARCH QUESTION: Is gait balance control impaired in a group of Veterans with chronic symptoms of mTBI when compared to healthy matched Veterans? METHODS: Eight Veterans with symptoms of chronic mTBI (1 F/7 M) and eight healthy matched Veterans (1 F/7 M) completed a gait balance assessment under single- (ST) and dual-task (DT) conditions. Gait balance control was quantified with whole body center of mass (COM) total medial-lateral (ML) displacement and peak ML velocity, which were calculated from camera-based motion capture. RESULTS: Veterans with chronic mTBI walked with greater ML COM displacement (approximately 25 % increase) in both ST and DT walking (main effect of group, p = 0.018) when compared to healthy Veterans. The peak ML COM velocity was affected for both groups by performing a concurrent cognitive task (interaction effect, p = .012). Slower ML COM velocities in healthy Veterans suggest the adoption of a conservative balance control strategy, while faster ML COM velocities for Veterans with chronic mTBI may indicate a diminished ability to control ML momentum. SIGNIFICANCE: Increased frontal plane COM motion under both ST and DT walking was observed in Veterans with chronic mTBI symptoms many years after injury. This suggests gait balance control may be adversely affected during divided attention gait and highlights the need for comprehensive gait analysis in the management of these individuals.

Using IMU-based kinematic markers to monitor dual-task gait balance control recovery in acutely concussed individuals.

BACKGROUND: Concussion may result in acutely impaired dynamic balance control that can persist up to two months post injury. Such impairment has been detected using sophisticated whole body center of mass kinematic metrics derived from camera-based motion analysis under a dual-task paradigm. However, wearable sensor kinematics for describing gait imbalance is lacking. METHODS: This study employed a longitudinal design. Gait balance control of acutely concussed and healthy matched control participants was assessed at five post-injury time points (within 72 h of injury, at one week, two weeks, one month, and two months). Tri-axial accelerations and angular velocities were collected with a dual-task gait protocol using an inertial measurement unit placed over the fifth lumbar vertebra. FINDINGS: Eight consistent gait event specific peak accelerations and six peak angular velocities measured by the inertial measurement unit were examined. Peak yaw and roll angular velocities at heel strike and peak roll angular velocities during early single-support, distinguished healthy from concussed participants across the two month post-injury period, while peak vertical acceleration at the end of terminal stance peak medial-lateral acceleration to the right during loading response showed promise. INTERPRETATION: Utilization of peak accelerations and angular velocities collected from a single inertial measurement unit placed over the fifth lumbar vertebra in a divided attention paradigm may offer a clinically feasible method for detecting subtle changes in gait balance control in concussed individuals.

The effectiveness of battlefield acupuncture in addition to standard physical therapy treatment after shoulder surgery: a protocol for a randomized clinical trial.

INTRODUCTION: There is a large incidence of shoulder instability among active young athletes and military personnel. Shoulder stabilization surgery is the commonly employed intervention for treating individuals with instability. Following surgery, a substantial proportion of individuals experience acute post-operative pain, which is usually managed with opioid pain medications. Unfortunately, the extended use of opioid medications can have adverse effects that impair function and reduce military operational readiness, but there are currently few alternatives. However, battlefield acupuncture (BFA) is a minimally invasive therapy demonstrating promise as a non-pharmaceutical intervention for managing acute post-operative pain. METHODS: This is a parallel, two-arm, single-blind randomized clinical trial. The two independent variables are intervention (2 levels, standard physical therapy and standard physical therapy plus battlefield acupuncture) and time (5 levels, 24 h, 48 h, 72 h, 1 week, and 4 weeks post shoulder stabilization surgery). The primary dependent variables are worst and average pain as measured on the visual analog scale. Secondary outcomes include medication usage, Profile of Mood States, and Global Rating of Change. DISCUSSION: The magnitude of the effect of BFA is uncertain; current studies report confidence intervals of between-group differences that include minimal clinically important differences between intervention and control groups. The results of this study may help determine if BFA is an effective adjunct to physical therapy in reducing pain and opioid usage in acute pain conditions. TRIAL REGISTRATION: ClinicalTrials.gov NCT04094246 . Registered on 16 September 2019.

Reliability and practical clinical application of an accelerometer-based dual-task gait balance control assessment.

BACKGROUND: Gait balance control assessment using whole body center of mass (COM) kinematic measures in concussed individuals reveals persistent balance deficits up to two months post-injury. A reliable and clinically practical gait balance control assessment leveraging similar kinematic measures is necessary to improve concussion assessment and management. RESEARCH QUESTION: Can peak accelerations collected during a dual-task (DT) gait assessment from a single low back placed accelerometer be measured reliably on different days, by different raters, in different environments, and be practically applied in a Division One (D1) athletics program? METHODS: A single accelerometer placed on the low back over the L5 vertebra was utilized with a DT gait analysis protocol. Twenty (10 F) healthy participants performed the assessment in a laboratory and non-laboratory environment, on two separate days, and with two different raters. Eight gait event specific peak accelerations along three orthogonal axes were collected. In addition, data were collected from a cohort of 14 D1 female soccer players during a single assessment to explore the practical clinical application. RESULTS: Cronbach's α values for the eight metrics ranged from 0.881 to 0.980 and ICC values from 0.868 to 0.987. Average assessment time for the 14 D1 female athletes was 8.50 ± 0.58 min, and significant differences between walking conditions were identified for Vert Accel 1 (p < .01), Vert Accel 2 (p = .01), and A-P Accel (p < .01). SIGNIFICANCE: High Cronbach's α and ICC values coupled with a short assessment time and sensitivity to differences in gait balance control indicate our testing apparatus and protocol are both reliable and clinically practical. Additionally, gait event specific peak accelerations from a single accelerometer can detect subtle changes in gait balance control and may facilitate improvements in sport-related concussion diagnosis and return to activity decision making.

Synthesis and structure-activity relationship of aminopyrimidine IKK2 inhibitors.

The synthesis and structure-activity relationship of a novel series of aminopyrimidines are exemplified. Results of key compounds from within this series in the E-selectin reporter cell assay are also reported.

Achieving multi-isoform PI3K inhibition in a series of substituted 3,4-dihydro-2H-benzo[1,4]oxazines.

The SAR and pharmacokinetic profiles of a series of multi-isoform PI3K inhibitors based on a 3,4-dihydro-2H-benzo[1,4]oxazine scaffold are disclosed.

Detecting gait abnormalities after concussion or mild traumatic brain injury: A systematic review of single-task, dual-task, and complex gait.

BACKGROUND: While a growing number of studies have investigated the effects of concussion or mild traumatic brain injury (mTBI) on gait, many studies use different experimental paradigms and outcome measures. The path for translating experimental studies for objective clinical assessments of gait is unclear. RESEARCH QUESTION: This review asked 2 questions: 1) is gait abnormal after concussion/mTBI, and 2) what gait paradigms (single-task, dual-task, complex gait) detect abnormalities after concussion. METHODS: Data sources included MEDLINE/PubMed, Scopus, Web of Science, and Cumulative Index to Nursing and Allied Health Literature (CINAHL) accessed on March 14, 2017. Original research articles reporting gait outcomes in people with concussion or mTBI were included. Studies of moderate, severe, or unspecified TBI, and studies without a comparator were excluded. RESULTS: After screening 233 articles, 38 studies were included and assigned to one or more sections based on the protocol and reported outcomes. Twenty-six articles reported single-task simple gait outcomes, 24 reported dual-task simple gait outcomes, 21 reported single-task complex gait outcomes, and 10 reported dual-task complex gait outcomes. SIGNIFICANCE: Overall, this review provides evidence for two conclusions: 1) gait is abnormal acutely after concussion/mTBI but generally resolves over time; and 2) the inconsistency of findings, small sample sizes, and small number of studies examining homogenous measures at the same time-period post-concussion highlight the need for replication across independent populations and investigators. Future research should concentrate on dual-task and complex gait tasks, as they showed promise for detecting abnormal locomotor function outside of the acute timeframe. Additionally, studies should provide detailed demographic and clinical characteristics to enable more refined comparisons across studies.

Discovery of a Potent, Orally Bioavailable PI4KIIIß Inhibitor (UCB9608) Able To Significantly Prolong Allogeneic Organ Engraftment in Vivo.

The primary target of a novel series of immunosuppressive 7-piperazin-1-ylthiazolo[5,4- d]pyrimidin-5-amines was identified as the lipid kinase, PI4KIIIß. Evaluation of the series highlighted their poor solubility and unwanted off-target activities. A medicinal chemistry strategy was put in place to optimize physicochemical properties within the series, while maintaining potency and improving selectivity over other lipid kinases. Compound 22 was initially identified and profiled in vivo, before further modifications led to the discovery of 44 (UCB9608), a vastly more soluble, selective compound with improved metabolic stability and excellent pharmacokinetic profile. A co-crystal structure of 44 with PI4KIIIß was solved, confirming the binding mode of this class of inhibitor. The much-improved in vivo profile of 44 positions it as an ideal tool compound to further establish the link between PI4KIIIß inhibition and prolonged allogeneic organ engraftment, and suppression of immune responses in vivo.

Arpeggio: A Web Server for Calculating and Visualising Interatomic Interactions in Protein Structures.

Interactions between proteins and their ligands, such as small molecules, other proteins, and DNA, depend on specific interatomic interactions that can be classified on the basis of atom type and distance and angle constraints. Visualisation of these interactions provides insights into the nature of molecular recognition events and has practical uses in guiding drug design and understanding the structural and functional impacts of mutations. We present Arpeggio, a web server for calculating interactions within and between proteins and protein, DNA, or small-molecule ligands, including van der Waals', ionic, carbonyl, metal, hydrophobic, and halogen bond contacts, and hydrogen bonds and specific atom-aromatic ring (cation-π, donor-π, halogen-π, and carbon-π) and aromatic ring-aromatic ring (π-π) interactions, within user-submitted macromolecule structures. PyMOL session files can be downloaded, allowing high-quality publication images of the interactions to be generated. Arpeggio is implemented in Python and available as a user-friendly web interface at http://structure.bioc.cam.ac.uk/arpeggio/ and as a downloadable package at https://bitbucket.org/harryjubb/arpeggio.

Identifying Interactions that Determine Fragment Binding at Protein Hotspots.

Locating a ligand-binding site is an important first step in structure-guided drug discovery, but current methods do little to suggest which interactions within a pocket are the most important for binding. Here we illustrate a method that samples atomic hotspots with simple molecular probes to produce fragment hotspot maps. These maps specifically highlight fragment-binding sites and their corresponding pharmacophores. For ligand-bound structures, they provide an intuitive visual guide within the binding site, directing medicinal chemists where to grow the molecule and alerting them to suboptimal interactions within the original hit. The fragment hotspot map calculation is validated using experimental binding positions of 21 fragments and subsequent lead molecules. The ligands are found in high scoring areas of the fragment hotspot maps, with fragment atoms having a median percentage rank of 97%. Protein kinase B and pantothenate synthetase are examined in detail. In each case, the fragment hotspot maps are able to rationalize a Free-Wilson analysis of SAR data from a fragment-based drug design project.

What can we learn from the evolution of protein-ligand interactions to aid the design of new therapeutics?

Efforts to increase affinity in the design of new therapeutic molecules have tended to lead to greater lipophilicity, a factor that is generally agreed to be contributing to the low success rate of new drug candidates. Our aim is to provide a structural perspective to the study of lipophilic efficiency and to compare molecular interactions created over evolutionary time with those designed by humans. We show that natural complexes typically engage in more polar contacts than synthetic molecules bound to proteins. The synthetic molecules also have a higher proportion of unmatched heteroatoms at the interface than the natural sets. These observations suggest that there are lessons to be learnt from Nature, which could help us to improve the characteristics of man-made molecules. In particular, it is possible to increase the density of polar contacts without increasing lipophilicity and this is best achieved early in discovery while molecules remain relatively small.

Extent of enthalpy-entropy compensation in protein-ligand interactions.

The extent of enthalpy-entropy compensation in protein-ligand interactions has long been disputed because negatively correlated enthalpy (ΔH) and entropy (TΔS) changes can arise from constraints imposed by experimental and analytical procedures as well as through a physical compensation mechanism. To distinguish these possibilities, we have created quantitative models of the effects of experimental constraints on isothermal titration calorimetry (ITC) measurements. These constraints are found to obscure any compensation that may be present in common data representations and regression analyses (e.g., in ΔH vs. -TΔS plots). However, transforming the thermodynamic data into ΔΔ-plots of the differences between all pairs of ligands that bind each protein diminishes the influence of experimental constraints and representational bias. Statistical analysis of data from 32 diverse proteins shows a significant and widespread tendency to compensation. ΔΔH versus ΔΔG plots reveal a wide variation in the extent of compensation for different ligand modifications. While strong compensation (ΔΔH and -TΔΔS opposed and differing by < 20% in magnitude) is observed for 22% of modifications (twice that expected without compensation), 15% of modifications result in reinforcement (ΔΔH and -TΔΔS of the same sign). Because both enthalpy and entropy changes arise from changes to the distribution of energy states on binding, there is a general theoretical expectation of compensated behavior. However, prior theoretical studies have focussed on explaining a stronger tendency to compensation than actually found here. These results, showing strong but imperfect compensation, will act as a benchmark for future theoretical models of the thermodynamic consequences of ligand modification.

Atomic interactions and profile of small molecules disrupting protein-protein interfaces: the TIMBAL database.

Growing evidence of the possibility of modulating protein-protein interactions with small molecules is opening the door to new approaches and concepts in drug discovery. In this paper, we describe the creation of TIMBAL, a hand-curated database holding an up to date collection of small molecules inhibiting multi-protein complexes. This database has been analysed and profiled in terms of molecular properties. Protein-protein modulators tend to be large lipophilic molecules with few hydrogen bond features. An analysis of TIMBAL's intersection with other structural databases, including CREDO (protein-small molecule from the PDB) and PICCOLO (protein-protein from the PDB) reveals that TIMBAL molecules tend to form mainly hydrophobic interactions with only a few hydrogen bonding contacts. With respect to potency, TIMBAL molecules are slightly less efficient than an average medicinal chemistry hit or lead. The database provides a resource that will allow further insights into the types of molecules favoured by protein interfaces and provide a background to continuing work in this area. Access at http://www-cryst.bioc.cam.ac.uk/timbal.