Locus Technology Validation

Locus Technology Validation
The first step in the validation of the Locus Technology was to determine whether the algorithms function accurately, reliably and reproducibly. The following are results from some of our validation experiments.

Confirmation of Known Protein Binding Sites
A basic validation of the Locus Technology is the ability to identify of known binding sites on proteins and to predict binding modes for known ligands. In the examples below, protein binding sites are defined by superimposing binding modes for multiple ligands or for multiple fragments, obtained experimentally via co-crystal structures or via Locus’ calculated fragment-protein interactions, respectively. In most cases, richer information about viable binding interactions is obtained using Locus Technology compared to experiment.

Calculated ligand binding modes are obtained by decomposing ligands into fragments, calculating the binding interactions with lowest free energy for each fragment, and reassembling the fragments to give back the structure of the parent molecule. Good agreement between experimental and calculated modes is expected for fragment-protein interactions with low entropies of interaction.

Elastase
Human neutrophil elastase is implicated in pulmonary inflammatory diseases such as emphysema and asthma. Locus Technology correctly profiled the elastase binding site after only 3 weeks of analysis, compared to 10 years of synthetic chemistry and crystallography required to identify the site experimentally.


Dihydrofolate reductase
Dihydrofolate reductase is a target for cancer chemotherapy. The binding Df methotrexate, a well-known anti-cancer agent, to dihydrofolate reductase is an intensively studied test case in structural biology. Locus Technology identified key fragments of methotrexate in the correct binding positions on the protein and rebuilt the whole molecule in the experimentally observed binding mode. It is also known that the tail region of methotrexate does not bind to the surface of the protein, and does not add any potency to the drug. Locus algorithms correctly predicted that the tail region of the molecule does not contribute to binding affinity:


HIV-1 Protease
HIV-1 protease is an important protein target for AIDS therapy. Locus algorithms predicted the binding site occupied by Crixivan, a commercially successful protease inhibitor, and correctly predicted the binding modes of both high affinity and lower affinity fragments this ligand.


Calculation of Fragment Binding Free Energies
The distinguishing capability of the Locus Technology is the ability to rapidly and accurately calculate relative binding free energies for a series of fragments. The simplest test of this capability is to compare calculated vs. experimental binding free energies for a congeneric series of molecules. Since only a single fragment position is varied in a congeneric series, this essentially tests the ability to evaluate binding energies for individual fragments. We provide data below for binding affinities of known molecules in two different types of protein binding sites, a relatively inflexible protease active site and a structurally flexible kinase allosteric site.

A more demanding test is to vary fragments at multiple positions across a molecule simultaneously, which is more sensitive to the effects of protein flexibility than is variation at a single position. Thus, while our calculations are based on a snapshot of a protein structure frozen in time, proteins are flexible and the structure of protein-ligand complexes can differ from that of native proteins. Although our technology cannot accommodate major reorganizations of protein structure, an example below shows that fragment-based molecule building is relatively insensitive to protein flexibility.

In evaluating the accuracy of our free energy predictions, one can use the accuracy of free energy perturbation calculations as a comparator. Although free energy perturbation calculations are too slow to be practical for de novo inhibitor design, they are the most accurate and rigorous alternative to Locus Technology. The accuracy of free energy perturbation calculations is on the order of 1 kcal/mol, which translates to 0.7 pIC50 units for binding data. In the examples below, Locus predicted free energies are relative and not absolute. A difference of 6 Locus energy units corresponds to approximately 1 log difference in IC50 values.

Protease Inhibition
The data below is for a series of protease inhibitors. The experimental results are still proprietary so that we cannot provide background on the protease. Nonetheless, we consider this protease active site to be an example of relatively inflexible binding sites, so that the prediction of fragment binding energies can be evaluated independent of protein flexibility with this data.


p38 Map Kinase
p38 Map kinase is a key protein in the signaling cascade for inflammatory factors such as IL-1 and is implicated in inflammatory diseases such as rheumatoid arthritis. This kinase has two substrate binding sites and a regulatory allosteric site. Allosteric sites such as that for p38 represent, by the nature of their biological function, highly flexible regions of the protein. Predictions of activity at this site, therefore, test the validity of fragment binding free energy calculations in the face of protein flexibility. Activity and crystallographic data used in these evaluations are from published structure activity data on compounds developed by Boehringer-Ingelheim. (Regan, J.; Breitfelder, S. et. al. J. Med. Chem. 2002, 45, 2994-3008.)




Free energy based predictions
The locus technology is based on a novel method to compute accurate free energies of binding of small molecules to proteins. These are then connected using proprietary de novo design software that computes the free energy of the overall molecule. The method has been validated by directing the de novo software to reproduce known ligands and their predicted binding. The rankings compare well to the experimental bindings, and the results are relatively insensitive to modest changes in side chain positions.

Comparison to docking methods
Current docking methods provide a score for different poses of a ligand in the binding site. While these are useful tools, the Locus method computes accurate free energies, which are more closely related to the measured binding.


Summary
The novel Locus technology, which includes free energy computation and de novo design, is able to accurately predict binding sites as well as map out the energetic nature of the binding site. The system provides a rich diversity of tightly binding molecules, and ranks their binding affinity. This provides a powerful guide for Locus and its partners to develop novel therapeutics.

VidLogo 3.2

VidLogo 3.2
Do you want to add Your or your Company Logo to any video? Logo is a small graphic sign usually put in one of the corners. Use VidLogo to modify video files and add logos and video watermarks. You can use animated logo or avi logo for videos like TV sign!

more »»

HIV/AIDS in Malawi

HIV/AIDS in Malawi
Photos by Jenny Matthews



Christian Aid visited Malawi to see how our partner organisations help people living with HIV. The Christian Health Association of Malawi (CHAM) funds hospitals and trains nurses but resources are tight - latex gloves are sometimes reused not thrown away. Nurses are overworked and many look after nieces and nephews orphaned by HIV.

the moon(next)

In the name of Allah, the Beneficent the Merciful.

1. The hour drew nigh and the moon did rend asunder.

2. And if they see a miracle they turn aside and say:
Transient magic.

3. And they call (it) a lie, and follow their low desires;
and every affair has its appointed term.

4. And certainly some narratives have come to them wherein
is prevention--

5. Consummate wisdom-- but warnings do not avail;

6. So turn (your) back on them (for) the day when the
inviter shall invite them to a hard task,

7. Their eyes cast down, going forth from their graves as if
they were scattered locusts,

8. Hastening to the inviter. The unbelievers shall say: This
is a hard day.

9. Before them the people of Nuh rejected, so they rejected
Our servant and called (him) mad, and he was driven away.

10. Therefore he called upon his Lord: I am overcome, come
Thou then to help.

11. So We opened the gates of the cloud with water pouring

12. And We made water to flow forth in the land in springs,
so the water gathered together according to a measure
already ordained.

13. And We bore him on that which was made of planks and
nails

14. Sailing, before Our eyes, a reward for him who was
denied.

15. And certainly We left it as a sign, but is there anyone
who

16. How (great) was then My punishment and My warning!

17. And certainly We have made the Quran easy for
remembrance, but is there anyone who will mind?

18. Ad treated (the truth) as a lie, so how (great) was My
punishment and My warning!

19. Surely We sent on them a tornado in a day of bitter
ill-luck

20. Tearing men away as if they were the trunks of
palm-trees torn up.

21. How (great) was then My punishment and My warning!

22. And certainly We have made the Quran easy for
remembrance, but is there anyone who will mind?

23. Samood rejected the warning.

24. So they said: What! a single mortal from among us! Shall
we follow him? Most surely we shall in that case be in sure
error and distress:

25. Has the reminder been made to light upon him from among
us? Nay! he is an insolent liar!

26. Tomorrow shall they know who is the liar, the insolent
one.

27. Surely We are going to send the she-camel as a trial for
them; therefore watch them and have patience.

28. And inform them that the water is shared between them;
every share of the water shall be regulated.

29. But they called their companion, so he took (the sword)
and slew (her).

30. How (great) was then My punishment and My warning!

31. Surely We sent upon them a single cry, so they were like
the dry fragments of trees which the maker of an enclosure
collects.

32. And certainly We have made the Quran easy for
remembrance, but is there anyone who will mind?

33. The people of Lut treated the warning. as a lie.

34. Surely We sent upon them a stonestorm, except Lut's
followers; We saved them a little before daybreak,

35. A favor from Us; thus do We reward him who gives thanks.

36. And certainly he warned them of Our violent seizure, but
they obstinately disputed the warning.

37. And certainly they endeavored to turn him from his
guests, but We blinded their eyes; so taste My chastisement
and My warning.

38. And certainly a lasting chastisement overtook them in
the morning.

39. So taste My chastisement and My warning.

40. And certainly We have made the Quran easy for
remembrance, but is there anyone who will mind?

41. And certainly the warning came to Firon's people.

42. They rejected all Our communications, so We overtook
them after.the manner of a Mighty, Powerful One.

43. Are the unbelievers of yours better than these, or is
there an exemption for you in the scriptures?

44. Or do they say: We are a host allied together to help
each other?

45. Soon shall the hosts be routed, and they shall turn
(their) backs.

46. Nay, the hour is their promised time, and the hour shall
be most grievous and bitter.

47. Surely the guilty are in error and distress.

48. On the day when they shall be dragged upon their faces
into the fire; taste the touch of hell.

49. Surely We have created everything according to a
measure.

50. And Our command is but one, as the twinkling of an eye.

51. And certainly We have already destroyed the likes of
you, but is there anyone who will mind?

52. And everything they have done is in the writings.

53. And everything small and great is written down.

54. Surely those who guard (against evil) shall be in
gardens and rivers,

55. In the seat of honor with a most Powerful King.

the moon

The Physical Moon and its History
(Moon Phases)
The light we see emanating from the Moon on any moonlit night is the result of reflected sunlight being reflected back to Earth. The familiar phases the Moon passes through in a month are caused by the different angles from which we see the sunlit side. The dark far side of the Moon only reflects 7% of the incoming light. Because of the Moon's reflective properties and since it illuminates no light of its own, it is said by mystics to be "receptive" (borrowing its light from the sun).
The Moon moves in an elliptical path tilted at a 5 degree angle to the ecliptic, and moves westward in its orbit across the sky. It takes about 12 hours for the Moon to travel from horizon to horizon. As it crosses the sunrise terminator, where the Sun is rising, the Moon is in its waxing phase, where it is increasing in light. As it crosses the sunset terminator, where the Sun is setting, the Moon is in its waning phase and is decreasing in light. If you look up in the sky on any given night and the Moon's right side is brightly lit, it is in its waxing phase. If the Moon's left side is brightly lit, then it is in its waning phase.

It takes 27.3 days to complete one orbit. It also rotates on its axis in 27.3 days, so it always keeps the same face pointing toward earth. A complete cycle from one new Moon to the next takes 29.5 days or one synodic month to complete.
Because the Moon's period of rotation is locked in phase with its orbit around the Earth, we usually only see the one side. However, the Moon has a slightly non-circular orbit, moving quicker when closest the Earth and more slowly when far away. This causes the Moon to appear to wobble a bit, and the usual face we see drifts sideways, to one side or the other, so that a few degrees of the far side can occasionally be seen. While its speed in orbit changes rhythmically, its axis rotation remains uniform. When the Moon is closest to Earth, the axial rotation falls behind a bit, turning its face to the East. While at a greater distance, the Moon moves ahead, turning its face to the West.

The tides, eclipses, and phases of the Moon are in direct correlation to the movement of the sun, moon, and earth. When the Moon is full, earthquakes, storms and floods increase in frequency.
The Moon begins its rhythmic cycle as the Moon's nearside crosses the sunrise terminator, from east to west, to produce a crescent Moon. The Moon waxes to a 1st quarter Moon, where the Moon is half illuminated, to a gibbous Moon. Then when the Earth, Sun and Moon are nearly in a straight line, with the Earth in the middle, a Full Moon occurs. This occurs when the Moon is in opposition to the position of the Sun. From the Full Moon, the Moon begins its waning cycle, crossing the sunset terminator to produce a waning gibbous Moon, to the last quarter. Finally we see only a waning crescent.


The complicated motion of the Moon results in a difference in the way we measure the monthly cycles. Time is measured by a reference point in the sky. We measure the time between one New Moon and the time it takes to return to the same position with respect to a fixed position in the sky. This is called a Sidereal month and is equal to 27.32166 days. The time between the transits of the Moon through one of its nodes is called the Draconic month; this equals 27.212220 days. The time between one New Moon to the next New Moon is called the Synodic month; this equals 29.530589 days and the time the Moon moves from equinox to equinox is called a Tropical month; this equals 27.321.58 days.
The Moon's perigee and nodes also move east and west with periods of 8.85 years and 18.61 years respectably. This results in different lengths of the Sidereal and Draconic months.